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DOI: https://dx.doi.org/10.18535/jmscr/v4i12.14

Utility of Osazone Test to Identify Sugars

Authors
Dr Tejas Shah, MD, Dr Nikunj Modi, MD
(Biochemistry), Dept. of Biochemistry, Shri M P Shah Govt Medical College, Jamnagar, Gujarat (India)
Corresponding Author
Dr Tejas Shah
MD (Biochemistry), Dept. of Biochemistry,
Shri M P Shah Govt. Medical College, Jamnagar, Gujarat (India)
Email: tejas.1112@gmail.com, Mob: 9879061781
ABSTRACT
Carbohydrates (sugars) are the most abundant organic molecules in nature. Sugars having reducing property
react with phenylhydrazine hydrochloride to form characteristic osazones (crystals). These osazones can be
correlated with clinical conditions like arabinose in autism, xylose in small bowel disease and galactose in
galactosemia. The study aimed to evaluate utility of osazone test to identify sugars by demonstrating
characteristic osazone. Study was conducted at Biochemistry department, Shri M P Shah Govt. Medical
College, Jamnagar, Gujarat. We selected sugars like glucose, fructose, mannose, galactose, lactose, maltose,
arabinose and xylose to demonstrate their osazone. All sugars were made available commercially. 2 grams%
stock solution prepared of each sugars. Osazone test is performed for each sugar in the boiling water bath and
noted down the time for appearing of crystals. Then the shape of osazone of each sugar was examined under
microscope. We observed that crystals were appeared at specific interval of time during boiling and cooling
slowly after boiling. We found characteristic shape of osazone of sugars under microscope. Glucose, fructose,
galactose and mannose formed needle shape osazone. Maltose formed sun flower shape osazone. Lactose
formed cotton-ball shape osazone. Arabinose formed dense ball needle shape osazone. Xylose formed fine but
long needle shape osazone.The study demonstrated characteristic shape of osazone of sugars by performing
osazone test. This simple, cheap and less time consuming test can be used to identify and differentiate sugars
encountered in clinical practice.
Keywords: Carbohydrates, phenylhydrazine, osazone.

INTRODUCTION intercellular communication.[2] Sugars are

Carbohydrates (Sugars) are defined chemically as
aldehyde or ketone derivatives of the higher
polyhydric alcohols, or compounds which yield
these derivatives on hydrolysis.[1] They have a
wide range of functions, including providing a
significant fraction of the energy in the diet of
most organisms, acting as a storage form of
energy in the body, and serving as cell membrane
components that mediate some forms of
classified as simple and complex. Simple sugars
are called monosaccharides which cannot be
hydrolyzed into smaller compound. Complex
sugars are disaccharides/oligosaccharides/
polysaccharides which can be hydrolyzed to
monosaccharides. Sugars having free carbonyl
group (either aldehyde or ketone) are called
reducing sugars. The osazone reaction was
developed and used by Emil Fischer to identify

Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016 Page 14361

JMSCR Vol||04||Issue||12||Page 14361-14365||December
aldose sugars differing in configuration only at the
alpha carbon.[3] All reducing sugars will form
osazones with excess of phenylhydrazine when
kept at boiling temperature. Osazones are
insoluble. Each sugar will have characteristic
crystal form of osazones which can be used to
identify and differentiate sugars in biological fluid
like urine.[4] By detecting such sugars, correlation
can be made with clinical conditions like (a)
glucosuria in diabetes mellitus, fructosuria in
fructose intolerance/hereditary fructosuria, lactose
in lactose intolerance, galactose in galactosemia[5]
(b) arabinose in autism/candidiasis[6] and (c)
xylose in small bowel disease/malabsorption
syndrome.[7, 8] Recent study was done to evaluate
characteristic osazones of uncommonly
[9]
encountered sugars. The present study was
aimed to evaluate utility of osazone test to identify
sugars by demonstrating characteristic osazone.
MATERIAL AND METHODS
The present observational study was carried out at
Biochemistry department during July 2016 to
August 2016. As this study did not involve any
kind of intervention to living animals, Institutional
Ethics Committee permission was not taken. We
used commercially available monosaccharides and
disaccharides of analytical grade. We selected
commonly encountered sugars like glucose,
fructose, mannose, galactose, maltose, lactose
along with uncommonly encountered sugars like
arabinose and xylose for study.
Principle of osazone test:[9, 10]
Reducing sugar reacts with one molecule of
phenylhydrazine hydrochloride (C6H5-NH-NH2)
to form phenylhydrazone hydrochloride. This
complex reacts once again with another molecule
of phenylhydrazine hydrochloride to give an
intermediate keto derivative. This again reacts
with one more molecule of phenylhydrazine
hydrochloride to give corresponding osazone.
This reaction is complete in 3 steps and consumes
3 moles of phenylhydrazine. During reaction with
monosaccharides, additional phenyl hydrazine is
consumed in oxidizing the adjacent OH-group to
Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016
2016
carbonyl group which then forms a second phenyl
hydrazone. Such bisphenyl hydrazones are called
osazones.
Sugar + phenylhydrazine hydrochloride  (sugar-
phenylhydrazone) + H2O
sugar-phenylhydrazone + 2 (phenylhydrazine
hydrochloride)  osazone+C6H5NH2+NH3+ H2O
Procedure:[11, 12]
1. Preparation of stock solution; We
weighed 2 grams (gm) of each sugar and
took it into sterile glass beaker of 150 ml
capacity. Then added distilled water to
make total volume 100 ml. Thus each su-
gar stock solution (2 gm%) was prepared.
2. Preparation of phenylhydrazine
mixture: 1 part of phenylhydrazine
hydrochloride mixed with 2 parts of
sodium acetate in a mortar.
3. Osazone test: We took 5 ml of sugar stock
solution in sterile test tube. Then we added
1 gm of phenylhydrazine hydrochloride
mixture and 2 drops glacial acetic acid (to
maintain pH of the solution) into it. Then
after mixing well, we put test tube into
boiling water bath and noted down the
time for appearance of osazone crystal.
4. Microscopic examination: 2 drops of
sugar crystals were taken by sterile glass
pipette and carefully placed on the glass
slide. Then cover slip was placed above it
and examined under high resolution
microscope. After clear vision of crystals,
snapshots were taken for record.
RESULTS
The present study was performed on 6
monosaccharides (glucose, fructose, mannose,
galactose, arabinose, xylose) and 2 disaccharides
(maltose, lactose). When observation made,
crystals were formed after specific time interval
either by boiling or by cooling slowly after
boiling. When examining under microscope,
characteristic crystals were found. They were
shown from figure 1 to 8. Table 1 showed
characteristics of osazone studied.
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JMSCR Vol||04||Issue||12||Page 14361-14365||December 2016
Table 1. Characteristics of osazone studied
Name of sugar Shape of crystal Time required for crystal Physical conditions
formation (min) Boiling Cooling slowly after boiling
Glucose Needle 6 + ++
Fructose Needle 4 + ++
Mannose Needle 10 + ++
Galactose Balls with thorny edge 10 + ++
Arabinose Dense ball needle 15 + ++
Xylose Fine-long needle 15 + ++
Maltose Sun flower 30 - +
Lactose Cotton-ball 30 - +
+ = appearance of crystal, ++ = intense appearance of crystal, - = no appearance of crystal.

Figure 1. Needle shaped crystals (Glucose) Figure 5. Dense ball needle shaped crystals
(Arabinose)

Figure 2. Needle shaped crystals (Fructose)

Figure 6. Fine-long needle shaped crystals
(Xylose)

Figure 3. Needle shaped crystals (Mannose)

Figure 7. Sun flower shaped crystals (Maltose)

Figure 4. Balls with thorny edge shaped crystals

(Galactose) Figure 8. Cotton ball shaped crystals (Lactose)
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JMSCR Vol||04||Issue||12||Page 14361-14365||December
DISCUSSION
Carbohydrates (sugars) are the most abundant
biological molecules.[13] Glucose is the major
metabolic fuel of mammals (except ruminants)
and a universal fuel of the fetus. It is the precursor
for synthesis of all the other carbohydrates in the
body. Biologically, the most important epimers of
glucose are mannose (epimerized at carbon 2) and
galactose (epimerized at carbon 4). Fructose has
the same molecular formula as glucose but differs
in that there is a potential keto group in position 2.
Chemically, sugars are reducing compounds if
they have free carbonyl group, and are sometimes
known as reducing sugars. This provides the basis
for a simple chemical test for glucose in urine in
poorly controlled diabetes mellitus.[14, 15] One of
these tests is osazone test.[3] Osazone test is the
simplest test to identify and differentiate reducing
sugars in biological fluid like urine. It is routinely
performed for glucose, fructose, mannose, maltose
and lactose. Monosaccharides are powerful
reducing agent in hot solution.[4, 9] We found
yellow, needle shaped crystals of glucose, fructose
and mannose while balls with thorny edge shaped
crystals of galactose (refer figure 1 to 4). Glucose,
mannose and fructose due to similarities of
structures form the same osazones. But since the
structure of galactose differs on C-4, that part of
the molecule unaffected in osazone formation, it
would form a different osazone.[1, 16] Because of
forming same shaped osazones, it is difficult to
differentiate glucose, fructose, and mannose. But
time of appearance of crystals may have some
utility in diagnosing diabetes and fructose
intolerance. Lactose and maltose are reducing
disaccharides because beta (14) and alpha
(14) glycosidic linkages seen respectively spare
carbonyl carbon so that both form osazones.[4]
Lactose formed cotton-ball shaped crystals and
maltose formed sun flower shaped crystals only
after cooling slowly in present study (refer figure
5 and 6). Osazones of reducing disaccharides are
more soluble in hot solution and don’t appear
during boiling. Hence they formed crystals slowly
on cooling the solution.[1, 16] We have also
examined uncommonly encountered monosaccha-
Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016
2016
rides like arabinose and xylose. Both are five
carbon sugars found in plant gums, proteoglycans
and glycosaminoglycans. They are constituent of
glycoproteins.[14] We observed dense ball needle
shaped and fine-long needle shaped crystals of
Arabinose and xylose respectively (refer figure 7
and 8). It is suspected that the arabitol produced
by the yeasts in the gastrointestinal tract is
absorbed in the portal circulation, and is then
converted into arabinose by the liver. It is not
metabolised endogenously and is eliminated by
the urine. Arabinose is often found to be raised in
the presence of intestinal candidiasis, and is
commonly found in autistic children. It is
suspected that autistic children may have
deficiencies of one or more enzymes that are
involved in the metabolism of pentoses. High
levels of arabinose have been found linked to
proteins in serum glycoproteins of serum of
schizophrenic patients and in children with
behavioural disorders.[6] Xylose is absorbed
unchanged by the duodenum and jejunum. Its
incomplete absorption allows for its possible use
as a malabsorption test or to identify small bowel
mucosal disease.[7, 8, 17] The findings of this study
were supported by Vinod BS et al study.[9] For
more evaluation high pressure liquid chromatogra-
phy (HPLC) is used nowadays which not only
differentiate but quantify such sugars.[18] In the
absence of HPLC setup osazone testing can be
utilized.
CONCLUSION
We demonstrated characteristic osazone of
commonly and uncommonly encountered
reducing sugars. Osazone test have great utility to
identify and differentiate such sugars. Moreover,
it is a simple, less time consuming and easily
performed in laboratory by technician as
compared to HPLC.
LIMITATIONS
We did not performed osazone test on urine
samples of patients. So it is difficult to evaluate
the factors which can affect the crystal formation
in actual diseased condition.
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JMSCR Vol||04||Issue||12||Page 14361-14365||December
ACKNOWLEDGEMENTS
We are thankful to Mr. M H Rajapara and Mr.
Manish Pandya, laboratory technicians,
Biochemistry department, Shri M P Shah Govt.
Medical College, Bhavnagar for their help and
support during this study.
Source of support: None
REFERENCES
1. Chatterjee MN, Shinde R, editors.
Textbook of medical biochemistry. 8th ed.
Ajanta offset & Packagings
Ltd:Jaypee;2012:p.23-8.
2. Ferrier DR, editor. Lippincott’s illustrated
reviews Biochemistry. 6th ed. Lippincott
William & Wlikins: Wolters Kluwer;
2014:p.83-5.
3. Internet source:
http://www2.chemistry.msu.edu
4. Vasudevan DM, Shreekumari S,
Vaidyanathan K, editors. Textbook of
biochemistry for medical students. 7th ed.
Ajanta offset & Packagings Ltd, Jaypee;
2013:p.69-74.
5. Dennis LK, Eugene B, Anthony SF,
Stephen LH, Dan LL, Larry J, editors.
Harrison’s principles of internal medicine.
16thed. McGraw-Hill; 2005:p.2319-23.
6. http://www.labco.es/media/pdf/en_downlo
ad_13.pdf. Arabinose test for the
Diagnosis of intestinal Candidiasis: Labco
quality diagnostics.
7. Christie DL. Use of the one-hour blood
xylose test as an indicator of small bowel
mucosal disease. The Journal of Pediatrics.
1978; 92(5):p.725–728
8. Buts JP. One-hour blood xylose test: A
reliable index of small bowel function. The
Journal of Pediatrics.1978; 92(5):p.729-33.
9. Vinod BS, Santhi S, Krithika. Osazones of
the Uncommonly Encountered Reducing
Sugars. International Journal of Interdisc-
iplinary and Multidisciplinary Studies.
2015 ;(2)9:p.24-29.
Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016
2016
10. Rafi MD. Textbook of biochemistry for
medical students. 2nded. Universities press
(India) Pvt. Ltd; 2014:p.30.
11. Chary TM, Sharma HO, editors. Practical
biochemistry for medical and dental
students. 1sted. Noida: Jaypee;2004:p.6-7.
12. Chawla R. Practical clinical biochemistry:
Methods and interpretations. 3rded.
Noida:Jaypee; 2006:p.35.
13. Voet D, Voet JG, Pratt CW, editors.
Fundamentals of biochemistry: Life at the
molecular level. 3rded. Wiley;2008:p.219.
14. Rodwell VW, Bender DA, Botham KM,
Kennelly PJ, Weil PA, editors. Harper’s
illustrated biochemistry. 30th ed. Lange:
McGraw Hill; 2015:p.152-55.
15. Ngugi MP, Njagi JM, Kibiti CM,
Ngeranwa JJN, Njagi ENM. Diagnosis of
Diabetes Mellitus. International Journal of
Diabetes Research 2012;1(2):p.24-27.
DOI: 10.5923/j.diabetes.20120102.01
16. Gupta SK, Ghalaut VS, Jain A, editors.
Manual of practical biochemistry for
MBBS. 2nded. Arya publishing company;
2013:p.23.
17. Robert MC, Arthur IA. D-Xylose Testing:
A Review. Gastroenterology 1988;
95:p.223-31.
18. Wilson AM, Work TM, Bushway AA.
HPLC Determination of Fructose,
Glucose, and Sucrose in Potatoes. Journal
of Food Science 1981;46(1):p.300-01.
DOI: 10.1111/j.1365-2621.
Page 14365
Carbohydrates and Lipids

Chapters 10 and 11,

Biochemistry – A short course – 3rd ed.

1
 Be punctual for practicals and workshops

 When you write down your protein ID, make

sure
you differentiate 1 vs I, 5 vs S, 0 vs O, etc

 Able to do dilutions
 Able to prepare a proper standard curve
The Biochemistry of Carbohydrates and Lipids

O H
C H3 (CH2)n C O C H
O
C H3 C H C H (CH2)n C O C H
O
H C O P O
O
H
 Carbohydrates: Biological functions

Monosaccharides (e.g. glucose)

Energy Disaccharides (e.g. maltose) Glycolysis and TCA cycle (Krebs cycle)
Polysaccharides (Starch, Glycogen)

Biosynthesis Basic building blocks (metabolites) for many molecules

Ribose DNA

Cell membrane Cellulose, Chitin

Cell Structure (Cell wall of plants; exoskeleton of arthropods)

Glycoproteins
Cell communication

4
 Lipids: Biological functions

Energy • Fats Fatty acid catabolism (β-oxidation)

storage for fatty acids

Biosynthesis Glycolipids
Sugar + lipid glycolipid

Lipid bi-layer
Cell Structure Cell membranes

Cell communication Phospholipids Steroid hormones (e.g. cholesterol)

5
 Mono-, di-, oligo-, polysaccharides
Monosaccharides - Simple sugars
Glucose
(β-D-Glucopyranose)
Function: Energy and biosynthesis
Disaccharides – Two monosaccharides (e.g. sucrose,
lactose. Maltose and cellobiose)
Oligosaccharides - containing typically three to ten
monosaccharides (multiple varieties such as the ones in
glycoproteins)
Polysaccharides – Complex molecules made up
of more than 10 monosaccharides bound together
(e.g. starch, glycogen and chitin)
6
 Monosaccharides - the Simplest Carbohydrates

Monosaccharides are the most simple carbohydrates, (CH2O)n, that contain

two or more hydroxyl groups and an aldehyde or ketone (e.g. glucose,
fructose, galactose, mannose, ribose)

Types of monosaccharides
No. of carbons Example
Triose 3 Dihydroxyacetone and glyceraldehyde

Pentose 5 ribose

Hexose 6 glucose

Note that Tetroses (four-carbon sugars) and Heptose (a seven-carbon sugar)

also exist, but are less common

Check out this link for a good breakdown of monosaccharides - http://www.genome.jp/kegg/catalog/codes2.html 7

 Triose

Ketotriose Aldotriose Aldotriose

Such depiction of sugars is by Fischer Projection, showing

sugars in their open chain form

8
 Functional Groups Present in Sugars

Monosaccharides contain two or more hydroxyl groups and an aldehyde or ketone

OH
C hydroxyl group (also called an alcohol group)

O
C Ketone group
O
C
H
Aldehyde group
R
 Asymmetric (chiral) carbon

Asymmetric carbon
(Chiral carbon)

(C3H6O3) (C3H6O3) (C3H6O3)

Symmetrical Asymmetrical molecules

Mirror images which are not superimposable
(enantiomers)

An asymmetric (chiral) carbon is asymmetric, has 4 different

groups attached to it
 D-sugar, L-sugar
Dextrorotary - compounds which rotate light to the right (clockwise)
Levorotary - compounds which rotate light to the left (anticlockwise)
Asymmetric carbons
Asymmetric carbon

(C3H6O3) (C3H6O3) (C3H6O3)

(C6H12O6) (C6H12O6)

‘OH’ on the RIGHT ‘OH’ on the LEFT Asymmetric carbon farthest away
D-sugar L-sugar from aldehyde (or ketone)
determines whether it is D or L

11
12
 Know your Carbohydrates

How do we identify Monosaccharides?

Aldehyde (aldose) , Hexose Ketone (ketose) , Hexose

Aldohexoses Ketohexose

Numbering of
carbons begins
closest to
aldehyde
or
ketone

D- configuration of
sugars is most common
(C6H12O6) (C6H12O6) (C6H12O6) (C6H12O6) in nature
(in our body all are D- ,
none are L-)
Isomers

Epimers
13
 Draw your carbohydrates in Fischer
Projection

Draw structures of: D-Glucose

D-Fructose

(Point out aldehyde or ketone)

14
 D-Glucose D-Fructose

Aldehyde ketone

Any sugars with an aldehyde or a ketone are reducing sugars, because they can
donate electrons

15
 Monosaccharides in Cyclic Form
(Haworth Projection)
A linear monosaccharide that closes to form a six-atom ring is
called a pyranose because of its resemblance to pyran.

Pyran
D-Glucose Cyclic Form
Linear chain (pyranose)
Six-C ring

A linear monosaccharide that closes to form a five-atom ring is

called a furanose because of its resemblance to furan.

D-Fructose Cyclic Form

Linear chain (furanose)
Five-C ring

16
 Monosaccharides in Cyclic Form

35%

64%

<1%

17
 Monosaccharides in Cyclic Form

α and β cyclic forms of sugars are anomers

The α form means that the hydroxyl

at C-1 is below the plane of the ring.

New
Asymmetric carbon
(anomeric carbon)
D-Glucose

The β form means that the hydroxyl

at C-1 is above the plane of the ring.

18
https://www.edinformatics.com/interactive_molecules/a_b_glucose_differences.htm

19
 Draw your sugars in cyclic form

Draw structures of: α-D-Glucopyranose

β-D-Glucopyranose

20
 6

5 For
α-D-Glucopyranose
4 1
DDUD
3 2 DownDownUpDown

Remember
β’s are Good, Thumbs Up

For
β-D-Glucopyranose

UDUD
UpDownUpDown

21
 Mutarotation

Mutarotation is the change in ‘optical rotation’ of a sugar solution, because α and

β anomers usually have different specific optical rotations.

The equilibrium mixture of D-glucose is about:

64% β-D-glucopyranose
35% α-D-glucopyranose
1% linear chain

A solution of D-Glucose will be made up of β-D-glucopyranose, α-D-glucopyranose, but also trace

amounts of linear chain D-glucose.

22
 Glycosidic Bond, Phosphoester bond

A covalent bond formed between an anomeric carbon and a hydroxyl group of another
molecule is called an O-glycosidic bond, and the product is called a glycoside.

A bond formed between glucosamine and acetate, via N-glycosidic bond.

Carbohydrates also form ester linkages to phosphates (Phosphoester).
O
HO P OH
O-glycosidic bond N-glycosidic bond Phosphoester OH

maltose

23
 N-glycosidic and Phosphoester Bond

CH2OH CH2OH
O O
H H OH H H OH

HO OH H H HO OH H H
H2O

H NH2 HO C CH3 H NH C CH3

O O N-glycosidic bond

O H2O O
HO P OH + HO R HO P O R
Hydroxyl
OH OH
Phosphate
Phosphoester

24
Glycosidic Bond, Phosphoester bond

Phosphoester

N-glycosidic bond
Adenosine triphosphate (ATP)

Phosphoester bond
O

HO P O CH2 O OH
OH H H
H H

HO OH
β-D-ribofuranose 5-phosphate
25
 Deoxyribose and DNA

N-glycosidic bond

Phosphoester bonds

5’
4’ 1’
3’ 2’
Carbons numbered
on Ribose

26
Sugar Metabolites

27
 Disaccharides

H H

3 2

Lactose

Maltose
Sucrase cleaves sucrose (table sugar), lactase cleaves lactose (milk sugar), and maltase cleaves maltose.

28
 Polysaccharides
Large polymeric oligosaccharides are called polysaccharides. If all of the monosaccharides in
the polysaccharide are the same, the polysaccharide is called a homopolymer.

Polysaccharide of the same Polysaccharide of different

= homopolymer = heteropolymer
monosaccharide monosaccharides

Polysaccharides Linkages Presence Monomeric unit

α-1,4-glycosidic bonds
Glycogen Animals, humans α-D-glucose
α-1,6-glycosidic bond

Starch α-1,4-glycosidic bonds Plants α-D-glucose

Cellulose β-1,4-glycosidic bonds Plants β-D-glucose

29
 Starch
Amylose is a linear polymer of glucose units linked by α-1, 4-glycosidic bonds.

Amylopectin is a branched polymer, with an α-1, 6-glycosidic bond for every 30 α-1,
4-glycosidic bonds.

Starch (amylose)

α-1,4-glycosidic bonds

30
 Glycogen
Two chains of glucose molecules joined by α-1,4-glycosidic bonds are linked by an α-1,6-
glycosidic bond to create a branch point.

Such an α-1,6-glycosidic bond forms at approximately every 10 glucose units, making

glycogen a highly branched molecule.

Glycogen:

α-1,4-glycosidic bonds
α-1,6-glycosidic bond
(branch point)

31
 Maltose & Cellobiose

Cellobiose is a disaccharide in which two glucose molecules are linked by a β(1→4) bond

1 4

Cellobiose

β(1→4) glycosidic bond

Cellobiose is the basic disaccharide of cellulose

32
 Glycoproteins

Proteins with carbohydrates attached are called glycoproteins. There are

three main classes of glycoproteins:

Glycoproteins: The protein is the largest component.

Proteoglycans: Proteoglycans are mainly carbohydrates which are attached

to a protein. Proteoglycans play structural roles or act as lubricants.

Mucins or mucoproteins: Like proteoglycans, mucins are predominantly

carbohydrate. The protein is characteristically attached to the carbohydrate
by N-acetylgalactosamine. Mucins are often lubricants.

33
In all classes of glycoproteins, carbohydrates are attached to the nitrogen atom
in the side chain of asparagine ( via N-glycosidic bond) or to the oxygen atom of
the side chain of serine or threonine (via O-glycosidic bond).

asparagine (N-linkage)
serine or threonine (O-linkage)

34
In mucins, the protein component is extensively glycosylated to serine and threonine
residues beginning with N-acetylgalactosamine.

A region of the protein backbone rich in serines and threonines is the site of
glycosylation.

Mucins serve as lubricants in the gut.

35
 Lectins - sugar-binding proteins
Sugar-binding proteins bind to specific oligosaccharides on the cell surface.

Lectins are sugar-binding proteins.

The lectins are involved in cell-cell interactions and viral infection.

cell-cell interaction

Influenza virus (hemagglutinin, a lectin) binds to sialic acid

lymphocytes adhering to the endothelial
sugars located at the termini of oligosaccharides of
lining of a lymph node
glycoproteins and glycolipids on cell surface
36
 Lipids
Long hydrocarbon chain
acyl

Fatty Acid

Fatty acids: Used for energy and synthesis of more complex lipids
Triacylglycerols: Storage of fatty acids (x3) with glycerol as backbone
Phospholipids: Membrane lipids
Glycolipids: Membrane lipids bound to carbohydrates
Steroids: Polycyclic hydrocarbons (e.g. cholesterol)

37
 Fatty Acids

Fatty acids are chains of hydrogen-bearing carbon atoms (hydrocarbons) that have
a carboxylic acid at one end and a methyl group at the other end, hydrocarbons in
between.
H3C methyl group carboxylic acid
ω
α
β
Palmitic acid (C16)
C16:0
Nomenclature: carboxylic carbon as C1, followed by C2, C3 etc. C2 also called α,
and C3 as β, the last carbon as ω
Shorthand notation (number of carbon: number of double bonds
(configuration and location). For example: C20:4 all cis Δ6,9,12,15
20 carbons, 4 double bonds, all in cis configuration, the positions
the 4 double bonds at 5th carbon, 8th carbon, …)
38
 β
Ѡ
α
C18:4 all cis Δ6,9,12,15

39
 (pKa) 4.78

Physiological pH about 7. This pH is much above the pKa (pH 4.78) of Palmitic acid, the acid is
completely deprotonated

40
 trans- and cis- Fatty acids
Fatty acids may be saturated or unsaturated (monosaturated and
polyunsaturated cis fatty acids, trans fatty acids)

trans

cis cis gives rise to bend or kink

(altered membrane fluidity)
41
Natural unsaturated fatty acids are in cis configuration
Fatty acids in biological systems contain an even number of carbon atoms (why?), with the
16 and 18 carbon atom chains the most common.

In polyunsaturated fatty acids, the double bonds are separated by at least one methylene
group.

42
Fatty acids

43
The properties of fatty acids are dependent on chain length and degree of unsaturation.

Short chain length and the presence of cis double bonds enhances the fluidity of fatty
acids.

Certain Cis polyunsaturated fatty acids are essential components of our diets
because we cannot synthesize them, such as the ω-3 fatty acids.

44
 Glycerol

Glycerol is a three carbon organic molecule (metabolite) with 3 hydroxyl groups

H2C OH
HC OH
H2C OH

Glycerol

45
 Glycerol backbone in Fat (triacylglycerol)

Glycerol is very important for lipid biosynthesis as it forms the backbone

Fatty acids are stored as triacylglycerols (triglyceride) in which three fatty acids are
esterified to glycerol.
carboxylic acid
Hydroxyl O fatty acid
c
H2C OH HO
Ester bond
HC OH H2 O
H2C OH

Glycerol

Triacylglycerol (fat molecule)

46
 Practise Drawing

Glycerol

Triacylglycerol

47
 Triacylglycerols
In mammals, the major site for triacylglycerol storage is adipose tissue. Each adipocyte
(adipose cell or fat cell) contains a large lipid droplet, in which the triacylglycerols are
housed.

48
 Membrane Lipids (phospholipids)
Common types of membrane lipids: Amphipathic:
Both hydrophilic and hydrophobic
Phospholipids
Cholesterol
Glycolipids (not the focus of this unit)

Phospholipid
Hydrophilic heads

Hydrophobic tails
Lipid bi-layer

Amphipathic Phospholipids

49
50
 Phosphatidate (phosphatidic acid)
Diacylglycerol 3 phosphate

Identify fatty acid, ester bond, phosphate, glycerol back bone

R1 and R2 are usually unsaturated

51
 H O

H C O C R1
O

H C O C R2 Phosphatidylserine
O O

CH C O
-
H C O P O CH2
O
-
H NH3+

H O

H C O C R1
O

H C O C R2 Phosphatidylethanolamine
O

H C O P O CH2 CH2 NH3+

O
-
H

52
 H O

H C O C R1
O

H C O
O
C R2 Phosphatidylcholine
CH3

H C O P O CH2 CH2 N+ CH3

O
-
H CH3

H O

H C O C R1
O

H C O C R2
O Phosphatidylinositol
H C O P O OH
-
O H H H OH
H
HO OH H H
H OH 53
 H O H O

H C O C R1 H C O C R1
O O

H C O C R2 H C O C R2
O O

H C O P O CH2 CH CH2 O P O C H
- O
-
H O OH H

Diphosphatidylglycerol (cardiolipin)
 Cholesterol

Steroids are built on a tetracyclic platform, consisting of three cyclohexane

rings and a cyclopentane ring fused together.

Cholesterol is the most common steroid and plays a role in maintaining

membrane fluidity.

Cholesterol is also a precursor to steroid hormones.

55
Next lecture on Amino acids
Read Chapter 3 in Biochemistry – A Short Course

56
 Week 3 FPG

Basic Biomolecules 2
Amino acids
Chapter 3 – Biochemistry – A short course
A/Prof Liza Cubeddu l.cubeddu@westernsydney.edu.au
 Lecture Learning Outcomes
• Understand structure of amino acids (AA)
• Understand the properties of AAs
• Be able to group AA according to “type”
• Be able to draw some key AA (e.g., alanine,
serine, threonine, asparagine, cysteine, glycine)
• Be able to recognise ALL AAs
• Know what a peptide bond (amide bond)
looks like
• Understand key terms e.g., zwitterion and
amphoteric
Red, yellow & blue blocks
Want to arrange in a row of four blocks
How many different combinations can you make?

3 ×3 ×3 ×3 =3 = 81
4
 Building blocks of proteins
= Amino Acids
Say you have a peptide (a very small protein) made up of
five amino acids.

If any of 20 amino acids can be used in any of the 5 positions,

how many possible peptides can be made?

20 ×20 ×20 ×20 ×20 =20 = 3 200 000

5
Proteins – the “workers” in the cell

Insulin (produced by pancreas)

 R-group (varied side-chain)

R
COOH
Amino

COOH
α

H 2N Hydrogen

Carboxyl

H Cα is chiral

Hydrogen Cα is asymmetric
 R-group (varied)

R
at pH 7.4

Amino Carboxyl

COO -
α

pKa > 9 pKa < 3

H Cα is chiral

Hydrogen Cα is asymmetric
 Most amino acids are chiral
Exist as 2 mirror image forms (enantiomers)
Only L isomers found in proteins of living organisms
 Key facts
• most α-amino acids are chiral
• The a-carbon always has 4 constituents
• All have:
– an acidic carboxyl group
– a basic amino group
– an a-hydrogen connected to a-carbon
• The 4th contituent (R) is unique
– In glycine, 4th constituent is a hydrogen – not
chiral
Glycine is achiral
 R-group (varied side chain)

Amino group is protonated

R
COO -
α

Carboxyl group is deprotonated

H
A free amino acid in solution at pH 7.4 exists as a dipolar ion,
with a positively charged amino group and a negatively
charged carboxyl group

This dipolar ion is called a zwitterion

Cation Zwitterion Anion

Physiological pH ~7.4
Cation à Zwitterion à Anion

pKa = pH at which
amino acid half
dissociated

pI = pH at which
amino acid has
no net charge
pKa – related to a particular ionising group

pI (isoelectric point ) – related to the whole molecule

Titration Curve
Glutamic acid

pK1 carboxylic acid = 2.2

pK2 R group = 4.3
pK3 amino group = 9.7
pI = (pK1+ pK2)/2
= (2.2+4.3)/2
= 3.25
Properties of AA: amphoteric

• Property of acting as either acid or

base in the context of solution pH à
called amphoteric

• All AA contain:
an amino group – proton acceptor
a carboxyl group – proton donor
in context of solution pH
• So amino acids are amphoteric
 If you add base (OH-) à AA behaves like an acid
H O
H2N C C OH
H

H O
H2N C C O- H+
+
Glycine solution H OH-

H2O
 If you add acid (H+) àAA behaves like a base
H O
H2N C C OH
H

H O
H2N C C OH + H+

Glycine solution H

H O
+H3N C C OH

H
Properties of AA: form polymers
H O CH3 O
H2N C C OH + H2N C C OH
H H

Condensation Reaction
H2O
H O CH3
H2N C C N C COOH
H H H
 Understanding AAs

LOOK at the R group for

• Size
• Charge
• Possibility of chemical reaction
 Classifying amino acids
• 4 groups based on chemical characteristics of R
group:

1. Non-polar (Hydrophobic) – non-polar R groups

2. Polar (Hydrophilic) – R groups with
electronegative atoms
3. Positively charged – R groups +ve at pH 7.4
4. Negatively charged – R groups –ve at pH 7.4
 1. Hydrophobic amino acids
• Have non-polar R-groups
R-group = side chain

• Mainly hydrocarbon side chains

• Different sizes & shapes of side chains
• Can pack together to form compact structures
• Often buried inside protein core away from
aqueous solvent

Two kinds of solvents we deal with in this unit:

water & lipid
1. Hydrophobic
amino acids
Increasing hydrophobicity
Branched chain amino acids
Increasing hydrophobicity
 Aliphatic side chains

thioether cyclic aromatic aromatic

(or sulfide) (pyrrolidine ring)
C–S–C
 Proline (P)

• Proline is different from all other aa

• Pyrrolidine ring structure

Proline can be hydroxylated

COOH
1
This happens at C4
Number the carbons and HN C H
2
hydroxylate the molecule
H 2C CH2
3
CH2
4
Collagen contains this OH
hydroxyproline
 Quiz
• Which amino acid has the least amount of
stearic hindrance?
• Which amino acids have aromatic R
groups which are hydrophobic in nature
and neutral at any pH?
• Name the hydrophobic amino acid that has
a thioether bond within the hydrocarbon
chain.
• Which amino acids have saturated
hydrocarbons important in hydrophobic
interactions?
 2. Polar amino acids

• Have polar R-groups

• Side chains have electronegative atom that

hoards e-

• May participate in H-bonding

• Hydrophilic – “water-loving”
2. Polar amino acids
• OH à hydroxyl group
• Makes these polar amino acids very
hydrophilic and reactive
• Can be phosphorylated
– SH sulfhydryl – carboxyamide
or thiol

2 Cys can form

Disulfide bond
S–S
Cysteines can be oxidised
 Quiz
• Name three amino acids that contain
hydroxyl groups.
• Which amino acid can form disulfide cross-
links between peptide chains?
 3. Positively charged amino acids

• Side chains have a positive charge

• Are highly hydrophilic
3. Positively
charged
amino acids

– long – long
sidechain + sidechain +
amino group guanidinium
group
Lysine is an essential amino acid

• Humans cannot synthesize it – obtain from

diet.
• In expts on animals – kept on cereal-based
diet – inadequate dietary lysine increased
stress-induced anxiety.
• Recent research in humans shows may be
true for us too.
3. Positively
charged
amino acids

– imidazole ring
 Positively Charged Amino Acids
are Hydrophilic
• Lys/Arg amino/guanidinium
group.
• Histidine’s imidazole side
chain can be uncharged or
positively charged at
neutral pH.
• Histidine found at active
sites of many enzymes that
require a proton donor or
proton acceptor.
 Histidine ionization
imidazole ring (pKa ~6)
4. Negatively charged amino acids

• Side chains have a negative charge

• Aspartic acid & Glutamic acid
• Often called aspartate & glutamate after
ionisation
 Quiz
• Which amino acids are positively charged
at neutral pH?
 -CH2OCH2COO¯ Na+ Lys+

Carboxymethyl cellulose – cation exchange resin

-CH2OCH2COO¯
-CH2OCH2COO¯ Na+ -CH2OCH2COO¯ Lys+
Elution Lys

Na+

Equilibration (at low pH) Sample binding at low pH Elution at high pH

pI of lysine is about pH 9.7

• By adjusting pH, you can change the
charge of a given analyte

• Remember:

If pH in solution > pI of a given analyte, the analyte is

negatively charged
If pH in solution < pI of a given analyte, the analyte
is positively charged
 Quiz

The isoelectric point (pI) of glutamic acid is

3.22, and the pI of arginine is 10.76.

At pH 8.5, which amino acid is positively

charged?
Quiz
Levels of structure in proteins
 Functions of AAs

• Are the basic structural unit of proteins

• Many, or modifications of them are
neurotransmitters à e.g glutamic acid
 Primary structure

• Formation of an amide bond between

two amino acids
• Also called a peptide bond
• Peptide bond is formed by condensing
an amine with a carboxylic acid
• A peptide bond is the basis of proteins
Where does this happen in the cell? Which enzyme catalyses the reaction?
 AAs as part of proteins

• The amino and carboxyl groups are not

functional as they form the peptide bond
• Each AA has a specific side chain
• The side chain gives its characteristic
properties and functions in a protein
 Nucleotides

Learning outcomes:
1. Explain the role of these molecules in Biochemistry
2. Draw basic molecules
3. Use nucleotide nomenclature
4. Explain using chemical diagrams why DNA and RNA has polarity
5. Explain using chemical terminology how DNA forms a double helix
6. Explain using chemical terminology how RNA can form multiple structural forms
7. Give examples of how the chemistry of DNA/RNA can be used in the laboratory to
understand features of the DNA
L.O.1: Important?
 Energy
 monomers make DNA and RNA

 2nd messengers

 Co-factors

 Cell signalling

factors Interesting fact:

Nucleotides can be synthesised
de novo or recycled via salvage
pathways
 NAD + (Nicotinamide adenine dinucleotide)

 Carry e-
 Ratio of NAD+/NADH is an
indicator or metabolic state
(resting state 700:1)

n.b. these will be revisited in the metabolism lectures

(FAD and NADP+ have similar structures)
Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence
Luis Rajman, 1Karolina Chwalek, 1David A.Sinclair (2018) Cell Metabolism Vol 27, p 529-547

https://doi.org/10.1016/j.cmet.2018.02.011

Summary
Nicotinamide adenine dinucleotide (NAD), the cell’s hydrogen carrier
for redox enzymes, is well known for its role in redox reactions. More
recently, it has emerged as a signalling molecule. By modulating NAD+-
sensing enzymes, NAD+ controls hundreds of key processes
from energy metabolism to cell survival, rising and falling depending
on food intake, exercise, and the time of day. NAD+ levels steadily
decline with age, resulting in altered metabolism and increased disease
susceptibility. Restoration of NAD+ levels in old or diseased animals
can promote health and extend lifespan, prompting a search for safe
and efficacious NAD-boosting molecules that hold the promise of
increasing the body’s resilience, not just to one disease, but to many,
thereby extending healthy human lifespan.

https://www.youtube.com/watch?v=Ng-Krb6602g
L.O. 2: What are they made up of?

 Base
 Ribose
 Phosphate
Bases

RNA only DNA only

Draw ribose
Draw ribose
L.O.3: A nucleoside (base + sugar)

Nucleosides can cross cell

membranes where as
nucleotides cannot.
Means that nucleoside
analogues are used as
antiviral or anti cancer
agents
Nucleotides

Disorders of
purine
metabolism
cause gout
(urate crystals
in joints)
The Gout (James Gillray, 1799) depicts the pain of the artist's
podagra as a demon or dragon.[
 cGMP

a common regulator of ion channels, glycogenolysis, and

cellular apoptosis, relaxes smooth muscle tissues. In
blood vessels, relaxation of vascular smooth muscles
lead to vasodilation and increased blood flow.
Convention : RNA monomers

 Nucleoside: adenosine
 Nucleotide: adenylate or
adenosine-5’-triphosphate
 Symbol: A or ATP
Convention: DNA monomers

 Nucleoside: deoxyadenosine
 Nucleotide: deoxyadenylate or
deoxyadenosine-5’-triphosphate
 Symbol: dATP dA or A
L.O. 4: Importance of 5’ and 3’ ends
L.O. 5: DNA structure
 Structure of DNA
•Antiparallel
•Major/minor grooves
•Sequence specific structure
•Linear or circular
•methylation
•Other forms
•A-DNA
•Z-DNA

One of the common themes of biology is

self-assembly.
Can you think of other examples of molecules self- B-DNA
assembling in biochemistry?
What drives self-
assembly?
L.O. 6: 2’ OH

 Complex structures
 ss
Transcription and Translation
Types of RNA

 tRNA
 mRNA
 rRNA
 snRNAs-spliceosome
 Hammerhead ribozymes
 siRNAs or RNAi
L.O.7: Observing DNA and RNA

dsDNA -> ssDNA

Increasing temp
Melting temperature-Tm
Melt temperature: Tm

 Is the temp where half the molecules are ss.

 Use spectroscopy
 .

Log MW
.
. .

Distance

https://www.youtube.com/watch?v=0jmB1SuK-Vo
Haematoxylin stains nuclei of cells blue because
they are acidic

Purple nuclei
DNA Chips
 https://www.youtube.com/watch?v=6ZzFihESjp0

Exploiting DNA hybridisation

https://www.youtube.com/watch?v=6ZzFihESjp0
Proteins as enzymes
Learning outcomes
1. Understand enzyme nomenclature
2. List and explain the roles of cofactors
3. Explain the role and factors effecting enzymes and their activity
4. Describe the important parameters of MM kinetics and derive
values for these terms using experimental data.
Proteins
• Structural
• Bioactive
• Transport
• Catalysts
• Recognition molecules

• Etc......
Proteins • Structural
• Bioactive
• Transport
• Catalysts
• Recognition
molecules

• Etc......
Enzymes as catalysts
• Affect rate
• Don’t affect equilibrium
• Bind to their substrates
However
• They are specific
• Work under mild conditions
Induced fit.

http://www.youtube.com/watch?v=Ms_ehUVvKKk

• Enzymes/proteins are dynamic/flexible

• Too much flexibility -> denaturation (see the prac)
Enzymes
• Macromolecules
▫ Proteins
▫ RNA
• Regulated
▫ More on this next week....
 L.O. 1: Types
(Enzyme commission numbers)
Enzyme example(s)
Group Reaction catalyzed Typical reaction
with trivial name
To catalyze oxidation/reduction
EC 1 reactions; transfer of H and O AH + B → A + BH (reduced)
Dehydrogenase, oxidase
Oxidoreductases atoms or electrons from one A + O → AO (oxidized)
substance to another

Transfer of a functional group

EC 2 from one substance to another.
AB + C → A + BC Transaminase, kinase
Transferases The group may be methyl-, acyl-
, amino- or phosphate group

EC 3 Formation of two products from

AB + H2O → AOH + BH Lipase, amylase, peptidase
Hydrolases a substrate by hydrolysis

Non-hydrolytic addition or
EC 4 removal of groups from RCOCOOH → RCOH + CO2 or
Decarboxylase
Lyases substrates. C-C, C-N, C-O or C-S [X-A-B-Y] → [A=B + X-Y]
bonds may be cleaved

EC 5 Intramolecule rearrangement,
i.e. isomerization changes AB → BA Isomerase, mutase
Isomerases within a single molecule
Join together two molecules by
EC 6 synthesis of new C-O, C-S, C-N
X + Y+ ATP → XY + ADP + Pi Synthetase
Ligases or C-C bonds with simultaneous
breakdown of ATP
L.O 1. Basic enzyme terminology

•Enzyme (often ends with ….ase)

•Substrate
•Product
•Co-factor
•Co-enzyme
L.O. 2: Cofactors
• Optimum activity can depend on the co-
operation of non-protein substances called
cofactors
• the protein-cofactor complex is a holoenzyme
• the protein without the cofactor is an apoenzyme
Important cofactors
• Metal ions
• Vitamins
▫ NADH and NADPH (Vit B3)
▫ Folic acid
• Non vitamins
▫ ATP
▫ Coenzyme Q
▫ Heme
What do they do?
Can
change the 3D shape of the enzyme
participate in the reaction as substrate
(coenzymes)
act as a donor or acceptor of a particular chemical
group
group transfer agent
NAD+
• Carry e-
• Ratio of NAD+/NADH
is an indicator or
metabolic state
ATP (Adenosine-5’-triphosphate)

•Source of chemical energy

•Large amount of energy released during hydrolysis
•Not an energy store
•Ratio of ATP/ADP is a measure of how much
energy is available.
ATP hydrolysis
Metabolism
L.O. 3: Enzymes as catalysts
 rate of a reaction
regenerated at the end of the reaction
alter rate not the equilibrium constant
are the most efficient catalysts known
 L.O 3: Enzyme Kinetics
-the study of the rates of enzyme-catalysed
reactions
pH
Temperature
concentrations of
– reactants, products and inhibitors
Cofactors

Enzyme Kinetics allows us to:

Compare enzymes
Understand how they work and regulated
What makes a good catalyst?
• Specific
• Can bind substrate at low concentration
• Fast
• Can release product
• Stable

How do we do experiments to determine these

things?
How fast?
• Measure the rate of the formation of product or
reduction of substrate.
• Units of rate (velocity)
▫ Concentration/time eg: mM/min
▫ Amount/reaction volume/time
eg: mmoles/mL/min

d [S ] d [P ]
v=− v=
dt dt
What happens when excess [S] is
placed with an enzyme?

1. Transition phase (usually less than 1 sec)

2. Product formed is linear with time – also called
the steady state
3. Product formation plateaus
Steady state Transition state

[E ] + [S ] [ES] [E] + [P]

During initial velocity (ie steady state)

•Rate of ES formation=rate of ES breakdown
•[ES] is a constant
Determining initial velocity of a
reaction for a known [S]

Rise = increase in [P]

Run=time

Rise/run = increase in [P] per unit time

= velocity
This is a special velocity called “initial velocity”
L.O.4: Michaelis Menten Kinetics
• Simplest model
• Assumptions include
▫ Free diffusion
▫ [S] >> [E]
▫ Very little spontaneous product formation
▫ No cooperatively (more on that later)
How can we describe how fast can a fixed concentration of
enzyme work?

Vmax

• Is the fastest initial velocity the enzyme will

display under a defined set of experimental
conditions.
• These graphs can only be used to estimate Vmax
 How do we describe how tight
something binds to an enzyme?
• Km is

▫ [S] where half the active sites of enzyme

are full
▫ [S] where the enzyme is working at ½
Vmax.
▫ used as a measure of the affinity of the
active site for the substrate.
(note: units are in concentration)
Michaelis Menten Kinetic Equation:
Vo =(Vmax [S])
(KM +[S])

Vmax v

½ Vmax

0 [S]
Km
 Key Point:
These are only estimates of Km and Vmax
• Can we turn a curve into a straight line???
• Equation of a line y=mx+b
How do we get an accurate value for Vmax and Km?
Line-Weaver Burk Plot
1/ v

1/V max

1/[S]
0
-1/K m
How do we get an accurate value for Vmax?
Line-Weaver Burk Plot

1/ v0

y = mx + b

 1 
= K
1/V 1 1
max m
 +
v V max  [ ]  V max
0
S

1/[S]
0
-1/K m
Summarise important parameters
• Vo (concentration/time)
• Vmax (concentration/time)
• Km (concentration)
• Kcat (sec-1)
(also known as the turn over number, number of
substrate molecules processed per enzyme molecule
per second)

Ratio Kcat/Km is used as measure of enzyme

effectiveness
Example how to use a LWB plot to
understand how an enzyme works
-eg mechanism of enzyme inhibition
1/ v
Substrate with 0.3mM inhibitor
Substrate with 0.2mM inhibitor
Substrate with 0.1mM inhibitor

Substrate alone

1/Vmax

1/[S]
0
-1/Km
Competitive vs non-Competitive Inhibition

Effect on Km Effect on Vmax

Competitive
Non-competitive
 1/ v
Substrate with 0.3mM inhibitor

Substrate with 0.2mM inhibitor

Substrate with 0.1mM inhibitor

1/V max Substrate alone

1/[S]
0
-1/K
m
Other factors effecting enzyme
activity?
pH
Ionic strength
Temperature

http://www.youtube.com/watch?v=D2j2K
GwJXJc
 Non-Michaelis-Menten kinetics:
eg 6-phosphofructokinase (EC 2.7.1.11)

D-fructose-6-phosphate + ATP -> D fructose-1,6-phosphate + ADP

Protein structure •Tetramer
Binds F-6 -P, ATP, ADP.
•When F-6-P binds to a subunit
the Km of the other binding site
decreases
•Cooperative binding-allosteric
binding

Non-MM kinetics-
get sigmodial LWB
plots
 Carbohydrates and glycobiology

Tymoczko et al. Biochemistry – A Short Course

Chapter 10
Glycobiology: the study of the structure, function
and biology of carbohydrates

• Carbohydrate structure and function

• Lectins
• Roles of glycobiology in human health and disease
 Carbohydrates: any carbohydrates (monomers,
oligomers, polymers or glycoconjugates)

Glycans: the carbohydrates attached to a wide

variety of biological molecules through glycosylation

Glycosylation: the process of attaching carbohydrates

to other molecules such as proteins and lipids
 Protein glycosylation is the enzyme-catalysed
covalent attachment of carbohydrates to a protein

Approximately half of all proteins typically expressed

in a cell undergo this modification
From genome to proteins, and protein glycosylation

Carbohydrates in proteins impart an additional level of

'information content' to underlying protein structures
 It is estimated that for most genomes about 1% of the
coding regions are glycosyltransferases and glycosidases

Glycosyltransferases: to add sugars on

Glycosidases: to cleave sugars off
Why glycosylation for proteins?
 Protein folding
 Conformation stabilisation
 Protein function
 Protein secretion and targeting
 Protein-protein interaction
 Disruption of glycosylation cause disease
such as congenital muscular dystrophy
(disruption of O-glycans)

 Cell to cell interaction

 Signal transduction
 Microbial infection
When to think about the involvement of sugars
in cell-cell interaction, we are talking about sugars
on the cell membrane.

The cells are decorated with sugars

When to think about the involvement of sugars

in protein transport, we are mainly talking about
Intracellular events
GlcNAc Man Gal Glc

Sialic acid

GalNAc
 Glycosylation reactions

Essentials of Glycobiology
Second Edition
asparagine (N-linkage)
serine or threonine (O-linkage)

11
Draw asparagine

Draw serine and threonine

Draw asparagine residue in a protein

Draw serine and threonine residues in a protein

Draw asparagine linked with a glucose

Draw serine and threonine linked with a glucose

Draw N-acetylglucosamine linked to asparagine in a protein

Draw N-acetylgalactosamine linked to serine in a protein

ABO blood type is determined by two
glycosyltransferases
O blood type

oligosaccharide chain attached

to either lipid or protein on RBC
A blood type
B blood type
 Glycoprotein = sugars + protein (predominated by the protein)

Proteoglycan = polysaccharide + protein (predominated by carbohydrates)

Mucin (proteins in mucus) = sugars + protein (predominated by carbohydrates)

N-linked protein glycosylation in the endoplasmic reticulum (ER)
is a conserved three-phase process in eukaryotic cells.
It involves the assembly of an oligosaccharide on a lipid carrier,
dolichylpyrophosphate and the transfer of the oligosaccharide to
selected asparagine residues of polypeptides
in the lumen of the ER

glycosyltransferases

oligosaccharyltransferase
 O-linked glycosylation

N-linked glycosylation
Nucleotide-activated sugars
 N-Linked Protein glycosylation
1. Assembly of the precursor
oligosaccharide…

IMPORTANT POINTS:
– Assembly takes place on the carrier
lipid dolichol, anchored in the ER
membane.
– A pyrophosphate bridge joins the
1st sugar (N-acetylglucosamine) to
the dolichol.
– Sugars are added singly and
sequentially.
– The glycan assembly flips from the
cytosolic side to the ER lumen.
– More sugar molecules are added to
the assembly.
 Now in the second step, attachment
2. En-bloc transfer of the
oligosaccharide to the
protein
• One step transfer, catalyzed by
oligosaccharyl transferase, which
is bound to the membrane at the
translocator.
• Covalently attached to certain
asparagines in the polypeptide
chain (said to be “N-linked”
glycosylation).
• Attaches to NH2 side chain of
Asn but only in the context:
Asn-x-Ser or Asn-x-Thr
 Finally modification of
oligosaccharide
3. Modification of the
oligosaccharide by
removal of sugars…
 Transport from the ER to Golgi

• The N-linked glycoproteins leave the ER and travel to the

Golgi Apparatus.
• They travel in membrane vesicles that arise from special
regions of membranes that are coated by proteins.
The Golgi Apparatus has two major functions:
1. Modifies the N-linked oligosaccharides and adds O-linked
oligosaccharides.
2. Sorts proteins so that when they exit the trans Golgi network,
they are delivered to the correct destination.
Modification of the N-linked oligosaccharides is done by
enzymes in the lumen of various Golgi compartments.
Ribosomes bind the ER
• Proteins sorted by secretory
pathway start synthesis on a
free ribosome in cytoplasm.
• Ribosome directed to rough
ER docks with ER
membrane & protein
synthesis continues.
• Newly synthesised protein
transported through
membrane to ER.
• How does the ribosome
interact with ER??
Getting proteins into the ER
 Signal sequence
• Machinery required to direct ribosome to ER & to
translocate nascent protein across ER membrane
consists of 4 components:

1. Signal sequence – sequence of 9 – 12

hydrophobic residues near amino terminus.
Can be cleaved by signal peptidases
2. Signal recognition particle – recognizes signal
sequence on nascent polypeptide chain as
emerges from ribosome. It binds this sequence &
ribosome & shepherds ribosome with nascent
polypeptide chain to ER
 Signal sequence
3. SRP receptor – Integral membrane protein. SRP
binds this receptor
4. Translocon – this is a channel across the
membrane.
Channel opens when translocon & ribosome bind to
each other
Once ribosome bound to ER, protein synthesis
reactivated – nascent protein now directed through
ER membrane
Where does Dolichol come from?
• Dolichol is an isoprenoid compound synthesized by the
same metabolic route as cholesterol. In vertebrate tissues,
dolichol contains 18-20 isoprenoid units (90-100 carbons
total). Dolichol is phosphorylated by a kinase that uses
CTP to form dolichol Phosphate. Dolichol phosphate is
the structure upon which the carbohydrate moieties of N-
linked glycoproteins are built. After assembly on dolichol
phosphate, the carbohydrate structure is transferred to an
asparagine residue of a target protein having the sequence
Asn-x-Ser/Thr, where X is any amino acid.
Dolichols function as a membrane anchor for
the formation of the oligosaccharide
 O-linked glycosylation

The O-glycosidic mechanism is not as complex as that

of N-glycosylation. Proteins trafficked into the Golgi
are most often O-glycosylated by N-acetylgalactosamine
(GalNAc) transferase, which transfers GalNAc
to the -OH group of serine or threonine,
and then may add more sugars in a step-wise manner.
O-linked glycosylation
 N-linked protein
glycosylation
Cotranslationally or
posttranslationally
Complex oligosaccharides
(assembly of oligosaccharides
first, then en bloc transfer to
Asn in proteins)
ER and Golgi
Attached to asparagine residue Attached to serine or threonine
Consensus sequence: NXS/T
Such as antibodies
O-linked protein
glycosylation
Posttranslationally
Simple oligosaccharides
(step-wise addition of
monosaccharides to Ser ot Thr
in proteins)
Mainly Golgi
residue
No consensus sequence
Such as mucins
 Lectin

Carbohydrates-binding proteins. Therefore it binds to

glycoproteins

They bind carbohydrates with specificity

and promote molecular recognition
Lectin in action
METABOLISM:
LIFE FROM CHEMICAL PROCESSES
 BIOCHEMISTRY

Lipids
Carbohydrates
Proteins Metabolism
Enzymes
Nucleic acids

LIFE
LO 1: METABOLISM

the sum of the physical and chemical processes necessary

for life

• Made up of both catabolism and anabolism

• Catabolism: breaking down molecules into smaller units
• Anabolism: constructing molecules from smaller units
WHY DO WE EAT?
Growth
Repair
Reproduction
Heat
etc.

All require energy and building blocks

WHY DO WE
BREATHE?

Maximize energy from metabolism

Remove by-products of metabolism
THESE LECTURES WILL SHOW:

why we breathe
why we eat
how biochemistry leads to life!!!
MECHANISMS OF
PATHWAY REGULATION-
FLUX
Regulation of gene expression
activation of enzymes
• Rate limiting step
• Typically beginning of pathway
• Eg allosteric enzymes, availability of substrate, inhibitors, pH
 LO2: METABOLISM
Typical textbook describe the breakdown of glucose, fats and amino
acids
But……
• Organisms have diverse metabolic processes
• Plants
convert fat into CHOs

• Some bacteria
• Autotrophs-fix CO2
• Entner-Doudoroff pathway
• Lack of citric acid cycle
LO 3: OVERVIEW OF
METABOLISM
LO 4&5 : GLYCOLYSIS
Anaerobic
Small amounts of ATP are made
Glucose broken down to two molecules of pyruvate
STARTING AND END PRODUCTS: LOTS
OF STEPS IN BETWEEN

O
6
CH 2OH
H 3C C C
5
O OH
O
H H OH
4 1
O
OH H H 3C C C
HO H
O OH
3 2

H OH
pyruvic acid
b -D-glucopyranose
 SO HOW DOES IT
HAPPEN?
Lots of enzyme steps-some regulated
Glucose in the blood needs to enter and stay in the cell….
hexokinase
glucose-6-phosphate
remove water
O O
H H
HO P OH HO H -
O P O H
C6 C6
OH -
O
C O C O
Phosphate 5 5
from ATP H H H H H H
4 C C1 4 C C1
OH H OH H
OH OH ADP OH OH
C3 C2 C3 C2

H OH H OH
LO 4&5: THE OVERALL
PATHWAY
 LO 5: REGULATION
Fructose 6-phosphate to fructose 1,6-bisphosphate (irreversible reaction)

To make it more symmetrical C1 must be phosphorylated

Irreversible enzymes are often found at the beginning of
pathways
O- O-
-
O P O CH 2 OH -
O P O CH2
6 O OH
OH 6 O O-
5 2
O H OH 5
H OH 2
H O
CH 2OH HO P OH H CH2 O P O-
4 3 1 4 3 1
ADP
OH H OH H
O O
Phosphofructokinase
fructose-6-phosphate Phosphate
from ATP
Fructose 1,6-bisphosphate

Allosteric –substrate
Activation- ADP, Inhibition- PEP
LO 4: ATP SYNTHESIS
OF GLYCOLYSIS
Used in synthesis of
• G-6-P
• F-1,6 bisP

Made in
• 3 phosphoglycerate x 2
• Pyruvate 2 x

Net synthesis = 2
LO 5: LOOK AT PEP TO
PYRUVATE
(irreversible reaction)
phosphoenolpyruvate
HO C1 O
HO C1 O ADP
C2 O pyruvic acid
C2 O P
ATP H C3 H Or
H C3 pyruvate
pyruvate kinase
H H
LO 5: NAD+
GLYCERALDEHYDE-3-
PHOSPHATE
DEHYDROGENASE
• H+ transferred to NAD+
• Phosphate added from inorganic phosphate so no energy lost

O
-
O P O-
O- 1,3-bisphosphoglycerate

H C1 O P O C1 O

H C2 OH H C2 OH

H C3 O P H C3 O P

H H

NAD+ NADH + H +
LO 4: SUMMARY OF
GLYCOLYSIS
•anaerobic

•1 glucose to 2 pyruvates
•net energy gain: 2 ATP and 2 NADH

Problem:
•NAD+ is limiting in the cell. Needs to
be recycled!
LO 4: RECYCLING
NAD+

When little O2 is around………

lactate dehydrogenase

HO C1 O HO C1 O
NADH + H+
C2 O H C2 OH lactic acid

H C3 H NAD+ H C3 H

H H
pyruvic acid
 IN SOME
ORGANISMS…..
Anaerobic conditions: remove CO2 from pyruvic acid and
then add hydrogens.

HO C1 O NADH + H+ NAD+ H
H
C2 O H C2 OH
C2 O
H C3 H H C3 H
CO2 H C3 H
H H
H
pyruvic acid ethanol
acetaldehyde

pyruvate decarboxylase alcohol dehydrogenase

LO 4: WHAT HAPPENS IF
OXYGEN IS AVAILABLE
Activation of other pathways to make more ATP out of
pyruvate
• Krebs cycle
• Respiratory chain
LO 5: COMMON INTERMEDIATE:
ACETYL-COA

 The merging of several energy generating

pathways

 Formed from
• pyruvate
• Fatty acid breakdown
• Amino acid breakdown
•Pyruvate decarboxylase
•Found in the inner mitochondria membrane
CITRIC ACID CYCLE-C2
UNITS FROM
CATABOLISM

AA
LO 5: TCA CYCLE MATRIX
OF THE MITOCHONDRIA
ENTRY OF ACETYL-COA

•Some carbon lost as

•CO2
•intermediates for other
pathways

•NADH/FADH2 formed

•GTP formed (not much)

Krebs Cycle
or
Citric Acid Cycle
or
the Tricarboxylic Acid Cycle
OVERALL SUMMARY
OF THE TCA

Acetyl-CoA +2H2O +3NAD++FAD +ADP +Pi

2CO2+3NADH + 2H+ +FADH2+CoASH +ATP

OxPhos OxPhos

nb: GTP = ATP

 Metabolism 2
WHY WE BREATHE OXYGEN AND HOW IT
ALL WORKS TOGETHER!

LO1: IDENTIFY THE PRODUCTS OF THE TCA CYCLE

AND PREDICT THE EFFECT OF COMPOUNDS ON ITS
CONTROL

LO2: IDENTIFY AND EXPLAIN HOW THE OUTCOMES

OF OXPHOS AND THE RC LEAD TO ATP SYNTHESIS

LO3: IDENTIFY KEY POINTS IN METABOLISM WHERE

AA AND FATTY ACID CATABOLISM MERGE WITH CHO

LO4: APPLY KNOWLEDGE OF CONTROL OF

METABOLISM TO HUMANS IN THE FED AND FASTED
STATE
LO1: Control of the citric acid cycle
Electron transport chain (Respiratory chain)

ETC
 LO2: Outcomes of the respiratory chain

 Converts NADH, FADH2 into NAD+, FAD

 Glycolysis, fatty acid breakdown, TCA cycle

• Uses O2 and H+ to make H2O

• Produces proton motive force (PMF)
 LO2: Overview

 uses protein complexes or small molecule e- carriers

 inner mitochondrial membrane
 Complex 4: transfers e-s to O2
 during transfer most complexes pump H+ out of matrix

Complex 1

Coenzyme Q10 -ubiquinone

LO2: The respiratory chain

FADH2
LO2: What can be done with the PMF

ATP synthase
 LO2: ATP synthase

H+ ions flooding back into matrix causes

conformational change which results in synthesis
of ATP
LO2 Oxidative Phosphorylation
 Total ATP synthesis from glucose

 Glycolysis: –2 + 4 + (2 NADH (5-2 for transport=3)) = total 5

 PDH: 2 NADH = total 5
 TCA: 2 + 6 NADH (15) + 2 FADH2 (3) = total 20

 Grand total approx 30

(one NADH makes approx 2.5 ATP, FADH2 makes 1.5 ATP)
 LO3: Oxidation of fatty acids

– mitochondria but first step in the cytoplasm

– Converts acyl chain into acyl-CoA

acyl-CoA synthetase

O O
R C + CoA-SH R C
OH S CoA
+
H2O
 Shuttle

Acyl-CoA CoA-SH cytoplasm

carnitine acyltransferase
I outer mitochondrial
membrane
acylcarnitine
carnitine acylcarnitine translocase
inner mitochondrial
membrane

carnitine acylcarnitine
mitochondrial
carnitine acyltransferase
matrix
I

CoA-SH Acyl-CoA
 β-oxidation

FAD FADH2
H H H O
O
R R C C C
C C C
S S CoA
CoA H
H H
 Reaction 2

H O H H O
R C C C R C C C
S CoA S CoA
H OH H
 Reaction 3

OH H O O H O
R R C C C
C C C
S S CoA
CoA H
H H
+
+ NADH2
 Reaction 4

O H O O O
+ H3 C C
R C C C R C
S CoA S CoA S CoA
H
Acetyl-CoA
 Quick look at aa degradation

 Need to deal with the

 N

 C

O
NH 2
HN NH
O C
N N O
NH O H
2 H
Urea Uric acid
 LO3: Carbon skeleton

phenylalanine
acetyl-CoA tyrosine
glucose asparagine tryptophan
tryptophan aspartate leucine
cysteine lysine
glycine
serine oxaloacetate
alanine
pyruvate
citric acid
fumarate cycle
phenylalanine glutamate
tyrosine glutamine
histidine
 - ketoglutarate arginine
proline
succinyl-CoA
valine
isoleucine
threonine
methionine
 Lo4: Integration of Metabolism

 Need to control these pathways

 Prevent futile cycles
 Two metabolic pathways running in opposite directions with no
overall effect other than to release heat.
 Respond to change in need
 Cellular diversity
 LO4: Eg fuel usage in humans

 [BG] controlled at 4-7 mM

 Low
 Brain and RBCs have an absolute requirement for some glucose
 High
 Kidneys excrete glucose
 Waste
 Pancreas

 Produces insulin
 β-cells - GLUT2 high Km for glucose

 Hexokinase -> glycolysis->ATP -> dec in K+ in the cell ->

Insulin release
 target: skeletal muscle, heart, fat tissue, liver
And
 Glucagon
 α- cells

 Made as a prohormone

 Released when [BG] is low

 Target liver-glucose release then gluconeogenesis (makes glucose)
Gluconeogenesis

Where does the C come from?

•some amino acids
• lactate
•glycerol

Not just the reversal of glycolysis

 Fed state

http://www.discoverbiotech.com/wiki/-
/wiki/Main/Hormonal+regulation+of+fuel+metabolism;jsessionid=564707113B089D3DD21D962C4A802655
Fasting state
 Fasted state

 Fed state
 Brain glucose

 Liver and muscle- FA

 Glucose converted to glycerol and condensation with fatty

acids in fat tissue

 Fasted state
 Lack of glucose

 Glucagon -> FA mobilisation for muscle and liver

 Activation of glucose synthesising pathways-liver and kidneys

Glucose cannot be synthesized from FA
 Fuel molecules during starvation

Title

8
6 Ketone bodies
concentration
in plasma 4 Glucose
mMol fatty acids
2
0
0 2 5 10 15 20 25 30 35 40
days of starvation
Long term starvation
What are ketone
bodies?

acetone

acetoacetate

Beta-hydroxybutyric acid
Cell membrane – Structure and
Function

Tymoczko et al. Biochemistry – A

Short Course
Chapter 12
 Cell Membrane

➢ Plasma membrane (cytoplasm membrane)

➢ Membranes for subcellular organelles (ER, mitochondrion, etc)
 Cell Membrane

http://biologypop.com/cell-membrane-lipid-bilayer-video/
3
 Membrane Lipids

Three common types of membrane lipids: Amphipathic:

Both hydrophilic and hydrophobic
Phospholipids
Glycolipids
Cholesterol

Phospholipid
Hydrophilic heads

Hydrophobic tails
Lipid bi-layer

Amphipathic Phospholipids

5
Phospholipids and glycolipids form lipid bilayers in aqueous solutions.

The formation of membranes is powered by the hydrophobic effect.

 Phospholipids

Phospholipids are composed of four components: fatty acids (2 or more), a

platform, a phosphate, and an alcohol.

Two common platforms are glycerol and sphingosine.

Phospholipids with a glycerol platform are called phosphoglycerides or phosphoglycerols.

The major phospholipids are derived from phosphatidate.

7
 Phospholipids

Ester bond
Fatty acid
Glycerol

Phosphate group

8
 Fatty acids

Lenoleate (Linoleic acid), C18:2 all cis ∆9,12

Shorthand notation, total No. carbons : No. double bonds, Δ double bond positions
9
 H O

H C O C R1
O

H C O C R2 Phosphatidylserine
O O

CH C
-
O
H C O P O CH2
O
- NH3+
H

H O

H C O C R1
O

H C O C R2 Phosphatidylethanolamine
O

H C O P O CH2 CH2 NH3+

O
-
H

10
 H O

H C O C R1
O

H C O C R2 Phosphatidylcholine
O CH3

H C O P O CH2 CH2 N+ CH3

-
H O CH3

H O

H C O C R1
O

H C O C R2
O Phosphatidylinositol
H C O P O OH
-
O H H H OH
H
HO OH H H
H OH 11
 H O H O

H C O C R1 H C O C R1
O O

H C O C R2 H C O C R2
O O

H C O P O CH2 CH CH2 O P O C H
- O
-
H O OH H

Diphosphatidylglycerol (cardiolipin)
 Phospholipid degradation
• Glycerophospholipid degradation occurs by phospholipases present in
tissues (membrane bound or free), pancreatic juice, and venoms
• Phospholipases are specific for ester bonds in the glycero-phospholipids:
phospholipases A1, A2, C, and D
 H O

H C O C
O

H C O C
O

H C O P OH
O
H
 H

H C OH
O

H C O C
O

H C O P OH

Lysophospholipid
 H O

H C O C
O

H C O C
O
H O P
H C O P O
OH H H
HO O
H
O H OH P
H H

Phosphatidylinositol 4,5-bisphosphate
These phospholipids are asymmetrically distributed between
the two halves of the membrane bilayer.
The outer leaflet of the plasma membrane consists mainly of
phosphatidylcholine, whereas

phosphatidylethanolamine and phosphatidylserine are

the predominant phospholipids of the inner leaflet.
 Cholesterol provides fluidity of membrane

Steroids are built on a tetracyclic platform, consisting of three

cyclohexane rings and a cyclopentane ring fused together.

Cholesterol is the most common steroid and plays a role in maintaining

membrane fluidity.

Cholesterol is also a precursor to steroid hormones.

19
Temperature
 Characteristics of membranes:

1. Membranes are lipid bilayer, that form closed boundaries. The membranes are
composed of lipids (phospholipids and cholesterol) and proteins, and are decorated
with carbohydrates.
2. Membranes are noncovalent assemblies and are asymmetric in that the outer surface is
always different from the inner surface.
3. Membranes are fluid structures.

4. Membrane is selectively permeable or impermeable. Small nonpolar compounds are

permeable across cell membrane, whilst polar or charged molecules are impermeable.
5. Proteins (channels and carriers) serve to mitigate the impermeability of membranes by
transporting molecules across the cell membrane.
6. Cell membrane is involved in signal transduction, enzymatic reaction and cell-cell
interaction.
 Lipids and Many Membrane Proteins Diffuse
Laterally in the Membrane

Lipids rapidly diffuse laterally in membranes, although transverse diffusion or flip-

flopping is very rare without the assistance of enzymes.

The prohibition of transverse diffusion accounts for the stability of membrane

asymmetry.
Membrane proteins
are mobile
 Membrane Function

The structure of phospholipids is responsible for the basic function

of membranes as barriers between two aqueous compartments.

The membrane proteins are responsible for transport,

Enzymatic reaction, signal transduction and cell-cell communication
The ability of small molecules to cross a membrane is a function of its
hydrophobicity.

Indole is more soluble than tryptophan in membranes because it is uncharged. Ions

cannot cross membranes because of the energy cost of shedding their associated
water molecules.
 Proteins Carry Out Most Membrane Processes

While membrane lipids establish a permeability barrier, membrane

proteins allow transport of molecules and information across the
membrane.

Membranes vary in protein content from as little as 18% to as much

as 75%.
Integral membrane proteins are embedded in the hydrocarbon core of the
membrane.

Peripheral membrane proteins are bound to the polar head groups of

membrane lipids or to the exposed surfaces of integral membrane proteins.

Some proteins are associated with membranes by attachment to a

hydrophobic moiety that is inserted into the membrane.
Proteins separated by SDS-PAGE
Membrane-spanning α helices are a common structural feature of integral
membrane proteins.

Other means of embedding integral membrane proteins is by using β strands to

form a pore in the membrane or by embedding part of the protein into the
membrane.
 A Major Role of Membrane Proteins Is to Function
as Transporters

A small molecule will spontaneously cross a membrane if two conditions are met:
1. The concentration of the molecule is higher on one side of the membrane than the
other.
2. The molecule is lipophilic or nonpolar.

Molecules meeting these criteria can simply diffuse across the cell membrane
(Diffusion, osmosis).

Polar molecules can diffuse across a membrane down their concentration gradient only
with the assistance of particular proteins called channel or carrier proteins. Such
movement is called facilitated diffusion or passive transport.

Movement of molecules against a concentration gradient requires a source of energy

and is called active transport.
 12.5 A Major Role of Membrane Proteins Is to
Function as Transporters

Ion channels are passive transport systems that allow specific and rapid transport of ions
down their concentration gradients.

Channels can be activated by changes in the voltage across a membrane (voltage-activated

channels) or by binding of specific molecules to the channels (ligand-activated channels).

Tetrodotoxin, produced by the puffer fish, is a lethal inhibitor of the Na+ channel.
 Channel Proteins

Permit the passive

movement of
molecules or ions of
appropriate size
through an aqueous
pore

© 2010 Paul Billiet ODWS

What kinds of amino acid residues facing inside of the pore or in touch of lipids?
 Carrier proteins

Bind to specific solutes to transport them

across a membrane

© 2010 Paul Billiet ODWS

 Glucose transporters
Transporters Location Km
Glut1 Heart, predominant in red blood cells ~1 mM
Glut2 Pancreas (β cells) ~15 mM
Liver
Glut3 Brain, neurons ~1 mM
Glut4 Muscles, adipose tissues (translocated to ~5 mM
plasma membrane upon insulin binding
to its receptor)
Glut5 Enterocytes (small intestine), transporting
fructose
 Active transport by protein pump

Protein pumps use energy to move a molecule against its concentration

gradient in the process of active transport.
The Na+-K+ ATPase or Na+-K+ pump uses the energy of ATP hydrolysis to
simultaneously pump three Na+ ions out of the cell and two K+ ions into the cell
against their concentration gradients.

Because the reaction includes an intermediate in which the enzyme is

phosphorylated, such pumps are called P-type ATPases.
Week 14
Final Lecture: Revision
300936 Functional Proteins and Genes sum21

Data File
Practical 2 – Chemistry of Carbohydrates: Monosaccharides and Disaccharides
Engagement with Prac 2 will develop your knowledge of carbohydrates, particularly the structures of
monosaccharides and disaccharides. Prac 2 also introduces some analytical techniques that can be
used to identify carbohydrates, that is Paper Chromatography, the Benedict’s test, and Osazone
crystal formation.

Information and data related to the Prac 2 demonstration videos is presented below. Please analyse
these results prior to the week 3 workshop and before attempting the Post-Lab quiz – Prac2.

Data can be found in sections: Part A - Paper Chromatography, Part B - Benedict’s test and Part C -
Osazone formation, and are related to the equivalent sections in the Practical Manual.

Part A - Paper Chromatography

The order of sugars and unknowns here matches that applied to the chromatography paper shown
in the Prac 2 demonstration video – paper chromatography. The order of sugars here also applies to
all Student data schematic paper chromatography images presented below.

Standard Sugars
1. Glucose
2. Galactose
3. Fructose
4. Sucrose
5. Lactose
6. Maltose

Unknown samples
7. Banana extract
8. Unknown sugar X (in student data this is the position of Unknown A, B, C, D)

The mobility of sugars can be presented and compared as Retention factor (Rf). This is a ratio of the
distance travelled by a sugar relative to the distance travelled by the solvent. It is given by the
following formula.

Retention factor = Distance travelled (sugar)/ Distance travelled (solvent)

1
Image - Schematic of paper chromatography of sugars. This image is a representation of the Paper
chromatography carried out in the Prac 2 demonstration video. Here the solvent front is represented
as a dashed line and each standard sugar or unknown sample is shown as a blue oval. Note that the
distance of each sugar spot is measured from the point of application to the centre of the spot. The
distance that the solvent moved is measured from the point of application to solvent front. When
analysing the unknown samples it is important to consider their mobility relative to the standard
sugars. Here Unknown sugar X (no. 8) moved a distance comparable to maltose (no. 6). As no other
standard sugar travelled the same distance, it is appropriate to suggest that unknown sample X
contains maltose. We cannot say this with certainty, however, and further confirmation via other
testing methods would be required.

Note that Glucose, Galactose, and Fructose (no. 1, 2, and 3) travelled a further distance relative to
Sucrose, Lactose, and maltose (no. 4, 5, and 6). Think about the structure of these sugars.

Also consider why sucrose is represented as a pale blue spot. Why would sucrose not stain as darkly
as the other standard sugars? Again, think about the structure of sucrose and what functional groups
are found or available on it at high temperature (more than 80 oC). Hint: think on the results of the
Benedict’s test.

2
Image – Paper chromatography of sugars after staining.

This is the actual Paper chromatography shown in the demonstration video, after staining.

Here the sugar spots can be seen following staining protocol with silver nitrate (you do not need to
know the chemistry of the staining in detail). Please note that the positions of the sugar spots are
approximate to the schematic shown above (in the schematic the separation has been exaggerated
slightly for ease of interpretation). The structure of the carbohydrates will contribute to their
interaction with the mobile/ stationary phase, and therefore their mobility. Note the location of the
monosaccharides and disaccharides. Sucrose has stained a light colour compared with the other
sugars, e.g. glucose.

Overall, separation has occurred between the sugars, and the unknown sample X can be tentatively
identified as maltose.

The Banana extract spot seems more smeared. Why would a complex food sample appear smeared
relative to a standard sugar? Note that in the schematic above the Banana extract is represented as
two spots that overlap. Which two sugars are shown to be present in banana?

Figure 1. Paper chromatography of carbohydrates as shown in the demonstration video.

3
Image – Paper chromatography – Separation of Pen Ink. Part A was extended to demonstrate the
separation of coloured dyes found within various inks. Figure 2 A – shows the pen types used. B –
application of inks to chromatography paper. C – clear separation of molecules can be seen following
chromatography. At the top of the separation the solvent front is clear. Note that some molecules
were present at the leading edge of the solvent, which indicates they had a very strong interaction
with the solvent (i.e. mobile phase). Other molecules did not move from the point of application,
which indicates they had a very strong interaction with the paper (i.e. stationary phase). This
highlights the separation power of paper chromatography and demonstrates that inks are made of
mixtures of dyes. For instance, the Green Marker separated into its blue and yellow component
dyes.

Given the separation of dyes in the blue pen (no. 2 position) would the structure of the dyes be the
same?

Figure 2 A

Figure 2 B

Figure 2 C

4
Student Data for Part A – Paper chromatography
Below are four schematic representations of paper chromatography. Each has a different Unknown
sample applied at position no. 8. The unknown samples are referred to as:

 Unknown sample A
 Unknown sample B
 Unknown sample C
 Unknown sample D

It is your objective to identify the sugar(s) present in each of these unknown samples. Note that you
will also receive Student data sets for the Benedict’s test and Osazone crystal formation carried out
on Unknown A,B,C,D. These samples will remain consistent between the three types of experiments
and you should consider if the results corroborate each other before coming to a final conclusion.

Note that the sugars in positions no. 1-7 are as stated on page 1 (i.e. Standard sugars).

Student Data – schematic of paper chromatography – Unknown sample A.

5
Student Data – schematic of paper chromatography – Unknown sample B.

6
Student Data – schematic of paper chromatography – Unknown sample C.

7
Student Data – schematic of paper chromatography – Unknown sample D.

8
Part B - Benedict's Test for reducing/non-reducing sugars
The Benedict’s test is a copper based test that indicates the presence of reducing sugars. It involves
a redox reaction that leads to the reduction of copper ions.

Please look at the images below and know why a Negative reaction returns a blue colour and a
positive reaction returns a red colour after heating with Benedict’s reagent. Some things to consider:
What makes the solution blue? What functional groups on carbohydrates are important for the
colour change to occur? Are all sugars reducing sugars?

Image – Benedict’s Test - A and B show a positive reaction of Benedict’s test with reducing sugar,
following heat treatment. Red precipitate can be seen. C – Negative reaction of Benedict’s test.
Colour of solution remains blue following heat treatment.

A B

9
Look at the results of the Benedict’s test carried out on the standard sugars shown in the table
immediately below. You should consider this table in conjunction with the table of unknown samples
which form part of the Student Data for Part B.

Table – Results of Benedict’s test on Standard sugars following heating.

Standard Sugar Observation (colour) Conclusion

Galactose Red Reducing sugar present
Fructose Red Reducing sugar present
Lactose Red Reducing sugar present
Sucrose Blue Reducing sugar absent
Glucose Red Reducing sugar present
Maltose Red Reducing sugar present

Negative control (water) Blue Reducing sugar absent (as

expected)

Student Data for Part B – Benedict’s test

Here are the results from a series of Benedict’s tests carried out on unknown samples. The method
used was as stated in the Practical Manual. Briefly, Benedict’s reagent was added to an aliquot of
each unknown sample and heated to 80 oC, 5 min.

You are to write a brief conclusion (as shown in the table above) regarding the presence or absence
of reducing sugar in each unknown sample (unknown sample A, B, C, D).

Note that unknown sample A, B, C, D are the same as those used in Part A – paper chromatography.

Table – Results of Benedict’s test on Unknown samples following heating.

Unknown samples Observation (colour) Conclusion

Unknown sample A Red
Unknown sample B Blue
Unknown sample C Red
Unknown sample D Red

Negative control (water) Blue Reducing sugar absent (as

expected)
Positive control (glucose) Red Reducing sugar present (as
expected)

10
Part C - Osazone Formation
Osazone crystals are formed when sugars react with phenylhydrazine under high temperature (100
o
C). The molecular structure of sugars is different enough to result in identifiable osazone crystals.

Look at the osazone crystal structure for each standard sugar. Then compare the osazone formations
shown for the Unknown sugar samples A,B,C,D to those of the Standards. Identify the sugar(s) found
within each Unknown sample A,B,C,D.

Standard Sugars
Glucose
Galactose
Fructose
Sucrose
Lactose
Maltose

Standard Osazone formations are as shown in the following reference, Shah and Modi (2016). Please
look at this paper as it will help you understand the structures of carbohydrates and the use of
osazone formations for identification. Note the different formations of monosaccharides relative to
disaccharides.

Shah, T, and Modi, N 2016, ‘Utility of Osazone Test to Identify Sugars’, JMSCR, Vol. 04, Issue no 12,
Page 14361-14365. DOI: https://dx.doi.org/10.18535/jmscr/v4i12.14

Student Data – Osazone formation

Below are the Osazone crystal formations associated with the Unknown samples A,B,C,D. Please
identify the sugar(s) present in each unknown sample.

Osazone formation of Unknown Sample A:

11
Osazone formation of Unknown Sample B:

Not applicable. Note, Osazone did not form from this sample.

Osazone formation of Unknown Sample C:

Osazone formation of Unknown Sample D:

12
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Prac 3 - Practical 3 report

Functional Proteins And Genes (Western Sydney University)

StuDocu is not sponsored or endorsed by any college or university

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https://www.khanacademy.org/test-prep/mcat/biomolecules/amino-acids-and-proteins1/v/
classification-amino-acids

Pre-work
1. In 300816 Cell Biology the amino acids were arranged into two broad groups. What
were these groups and what part of an amino acid defined the group it was placed in?
The two main groups are polar (hydropholic ) and non-polar (hydrophopic).
R-group = side chain defines the group of an amino acids.

An amino acid is an organic molecule that is made up of a basic amino group (−NH 2), an acidic
carboxyl group (−COOH), and an organic R group (or side chain) that is unique to each amino
acid. The term amino acid is short for α-amino [alpha-amino] carboxylic acid.
Each amino acid is bound to a unique chemical group at this position called its side chain. It is
this side chain that makes each amino acid different, giving each amino acid a unique set of
chemical properties. The side chain is often abbreviated as an R group and denoted with the
letter R for short.

Non-polar: alkyl and aromatic.

Alkyl side chains contain 7 different amino acids.
Aromatic amino acids made up of carbon and hydrogen only.

Polar: neutral, acidic and basic.

Neutral: 6 amino acids, have a side chain contain an oxygen or sulphur atom.
Acidic: 2 (aspirate and glutamate), have carboxylic acid as part of their side chain, hence very
strong hydrogen donor, and left in an anion form.
Basic: 3, contain nitrogen atom which is very willing proton acceptor,

There are 20 main amino acids different in their side chains (R).

2. What other ways could amino acids be grouped?

alkyl and aromatic (non-polar).
neutral, acidic and basic (polar).
Positively charged and negatively charged.

3. What is the definition of pH? (Do not use the words ‘acid’ or ‘acidity’ in your answer!)
pH is a measure of hydrogen ion concentration [H +]. The pH scale usually ranges from 0 to 14
4. What is an acid? What different ways can it be defined?
An acid is a chemical species that donates protons or hydrogen ions and/or accepts electrons.
Most acids contain a hydrogen atom bonded that can release (dissociate) to yield a
cation and an anion in water

5. What is a base?
a base is a chemical species that donates electrons, accepts protons, or releases hydroxide
(OH-) ions in aqueous solution

6. What is a zwitterion?
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A zwitterion is a molecule that contains both a positively-charged functional group and a

negatively-charged functional group. The molecule as a whole is uncharged.

7. What reaction joins amino acids together to form a protein? Can you draw the bond
that joins the amino acids?
The bond that holds together the two amino acids is a peptide bond, or a covalent chemical
bond between two compounds (in this case, two amino acids). It occurs when the carboxylic
group of one molecule reacts with the amino group of the other molecule, linking
the two molecules and releasing a water molecule

8. What are the four levels of protein structure?

primary, secondary, tertiary, and quaternary structure.

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9. What types of chemical bonds or intermolecular forces hold proteins together?

Peptide bonds, Ionic bonds, Disulfide bonds, Hydrogen bonds and Hydrophobic Interactions.

10. You did paper chromatography in practical 2 – what other types of chromatography
are there and how could they be used to separate mixtures of biomolecules?
There are four main types of chromatography. These are Liquid Chromatography, Gas
Chromatography, Thin-Layer Chromatography and Paper Chromatography.

Chromatography is based on the principle where molecules in mixture applied onto the
surface or into the solid, and fluid stationary phase (stable phase) is separating from each
other while moving with the aid of a mobile phase.

The isoelectric point (IP) is the pH at which the amino acid has an overall zero. charge. The
isoelectric points (IP) of amino acids range from 2.8 to 10.8.

The isoelectronic point or isoionic point is the pH at which the amino acid does not migrate in
an electric field. This means it is the pH at which the amino acid is neutral.

When the charge is zero at a certain pH, this pH is called the amino acid’s isoelectric point
(pI) and is defined as the pH at which there is no net charge (i.e net charge is zero)

The pH at which 50% of acetic acid CH3COOH is dissociated to CH3COO- is called the pKa of
acetic acid. The pKa of acetic acid is in the acidic region (pH<7).

Bases do the same thing, a base like an amine (-NH2) is protonated as N+H3 at low pH, and
N+H3 is deprotonated to NH2 + H+ at higher pH. It is just that, unlike acids, the pH at which this
dissociation occurs is in the basic region (pH >7).

Each individual group will have its own pKa which means that there can be different charges on
an amino acid at different pHs.

At different pHs an amino acid will have a different overall charge (+ve, 0 or –ve). This means we can purify an
amino acid using ion exchange chromatography.

In cation exchange chromatography we use a resin that has SO3- groups (anions) on its surface which can bind to
positively charged amino acids.

Because the pI is often different between amino acids then we can use cation exchange chromatography to
separate mixtures of amino acids.

Why pI is different between amino acids?

Because different proteins will have different combinations of amino acids and therefore different
isoelectric points, which allows proteins to be separated from one another and thus purified.

Part A. Cation exchange chromatography to separate amino acids

Discussion of procedure
 Why is the resin equilibrated with citrate buffer (pH 3.75)?
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Because the samples, amino acids, are dissolved in citrate buffer, so we should use the same
buffer to equilibrate the resin, so when the sample is loaded, the resin
and the sample are in the same buffer. It is important to have a sample compatible to analytital
procedure.

Before use, ion exchange columns are equilibrated at a pH using an equilibration buffer. ...
The pH needs to be maintained so that the analyte of interest is charged and will bind to the resin. For
proteins, the charge is based on the protein's isoelectric point, where the protein is neutrally charged,
and measured as pI.

Discussion
 Indicate which of the three amino acids has eluted in the citrate buffer, which amino acid
has eluted in the borate buffer, and which amino acid has eluted with NaOH.
 Describe in your own words and diagrams why the separation has occurred. Explain
how the structure of the amino acids (you may need to draw them) contributes to the
separation?

The resin medium is negatively charged and when the environmental pH is above the amino
acid’s PI, the amino acid will be negatively charged and will not bind to the sesin, and therefore
will flow out and be investigated in the test tube.
Ion exchange chromatography is based on the mutual attraction of oppositely
charged particles.

If you fill a chromatography column with a resin whose outside is covered with
negative charges, e.g. R-SO3- groups and you pass a liquid containing positive and
negative ions, the positive ions will be attracted by the R-SO3- groups and the
negative ions will be repelled. Thus the positive ions are retained in the column and
the negative ions will flow with the liquid and leave the column.

Alternatively, if you would fill the column with a resin whose outside is covered with
positive charges, e.g. R-NH3+ groups, these would retain the negatively charged
ions form the solution.

Amino acids have both amino-groups, that can be protonated to -NH3+ groups, and
carboxylic acid groups, that can be de-protonated to -COO- groups.

Wether or not amino acid particles have a net positive or negative charge depends
on the pH of the solution they’re in.
The pH at which the amino acid particles have a netcharge of 0 is called the
isoelectric point, or pI.

e.g. ALANINE has a pI = 6,11, that means that at a pH < 6,11 the particles will be
more and more protonated, leading to positively charged ALA-ions (ALA+), and at a
pH > 6,11 the ALA particles will be de-protonated, leading to negatively charged
ALA-ions (ALA-).

e.g. ARGININE has a pI = 10,76, so at pH< 10,76 there will be positively charged
ARG-ions (ARG+) and at pH> 10,76, there will be negatively charged ARG-ions
(ARG-)

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Now suppose we have a mixture of ALA and ARG, solved in a buffer solution of pH 5,
then there will be ALA+ and ARG+ ions in this solution.
When we introduce this mixture in a R-SO3- resin containing column, both the ALA+
and ARG+ will bind to the column.

If we now flush the column with a buffer solution of pH 8, the ALA+ will change to
ALA- (for pH 8 is above the pI 6,11 of ALA) and these negatively charged ions no
longer will bind to the negatively charged resin particles, so they will leave the
column along with the buffer solution.
The ARG+ ions will keep a positive charge (pH 8 < pI 10,76 for ARG) and will still
bind tot the negatively charged resin particles in the column.
Thus the ALA is separated from ARG.
When you now flush the column with a buffer pH 12, then the ARG- ions will also be
washed out of the column and you can collect them.

It’s a little simplistic, but this is the principle.

An amino acid is an organic molecule that is made up of a basic amino group (−NH2), an acidic
carboxyl group (−COOH), and an organic R group (or side chain) that is unique to each amino
acid. The term amino acid is short for α-amino [alpha-amino] carboxylic acid.

 Why did you collect flowthrough in step 3?

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To investigate if there is any amino acid has flowed out the column, this means has not
bonded to the resin.

 Why did you collect three fractions for each elution (buffer) ? What might happen if you
collected only one fraction? Can you collect more than 3 fractions?
To make sure that we have eluted out all amino acid molecules that detached the column
for each buffer elution, to get efficient volume from the buffer; to wash out all amino acid
molecules out of the column.
If I collected only one fraction, some molecules may still in the resin column and not
eluted out, and, as the result, the separation is not efficient.
And, of course, we can collect more than 3 fractions, all depending on the column, if the
amount in the column is big enough we need to collect more than 3.

Because we have three amino acids with different charge, and it is supposed that amino acids
with the biggest positive charge will flow out first, and if there are other amino acids with lower
positive charge will flow out.
if I collected only one fraction, I would not be able to determine which amino acid has flowed
out. I cannot collect more than 3 fractions, as the column will run dry.

 Are there any other separation techniques you could use to analyse a mixture of amino acids?
There several techniques for amino acids separation, such as high performance liquid
chromatography (HPLC)

Part B. Denaturation of an enzyme by heat and other factors

casein: the main protein present in milk and (in coagulated form) in cheese. It is used in
processed foods and in adhesives, paints, and other industrial products.

Discussion
 Make a table containing the treatments and your observations after each treatment.

 Explain your observations in terms of how heat, urea, and pH denaturation affect the
function of the rennin enzyme. It is not appropriate to just say that you have denatured
the enzyme. You need to specifically describe how each treatment has altered
intramolecular bonds that contribute to protein structure.

Each urea molecule has one oxygen atom and two nitrogen atoms, those oxygen and nitrogen
atoms are electronegative atoms that can denature the amino acids’ structure (disrupt the
hydrogen bonds in rennin).

The enzyme, which is a protein, contains several types of bonds like hydrogen
bonds, covalent bonds,ionic bonds etc. which maintains the 3-D structure of the
protein. These bonds vary in strength
.The bonds which are most sensitive to temperature change are Hydrogen
Bonds. .The bonds which are most sensitive to temperature change are Hydrogen
Bonds
Urea breaks the Hydrogen bonds in the secondary structure of proteins and alters the
conformation.

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Enzymes are proteins, and as such they are sensitive to changes in their
environment which could cause them to denature, or unravel, or sort of “melt” in a
way that the specific arrangement of their amino acids that is required for them to
function is lost.

RENNIN has an optimum pH of 3.4 for the proteolysis of bovine serum albumin 1 and 3.8 for
poly-L-glutamic acid2. At pH values between 5 and 7 it will coagulate milk and slowly attack
casein. It has maximum stability at pH 5.4, while at values above 7 it loses activity rapidly.

As the pH decreases below the optimum, enzyme activity also decreases. ... At extremely low
pH values, this interference causes the protein to unfold, the shape of the active site is no
longer complementary to the substrate molecule and the reaction can no longer be catalysed by
the enzyme.

In a less extreme sense, when the pH is lowered, there are more H+ ions in
solution, so anionic regions that are exposed on the enzyme surface - that may
have needed to remain anionic in order for the enzyme to function may become
protonated (begin to bind up covalently many of those H+ ions) and so lose their
ability to function.

The opposite may happen when the pH is raised - carboxylic acid and amine groups
may become deprotonated - that is - be stripped of H+ ions by the now more basic
solution, and similarly lose their native chemical reactivity.

Proteins exist on the knife-edge of stability - change their environmental conditions

much in any way and they turn to mush. This is why the concept of biological
homeostasis is so important: cells expend a tremendous amount of their energy
maintaining a constant internal environment because it is in these conditions they
are trying to maintain that their proteins/enzymes operate most effectively.

Hints: Do you know the structure of urea? To explain how urea works you might want to draw its
structure and compare to the peptide bond. How are they similar/different? You may also want
to think about what heat and acids do at the molecular level.

 In step 6 why do we bring the sample back to neutral pH before we add milk in step 7?

 In step 7, why do we do the incubation at 37 C?



 How can we improve step 6?

Step 6 is deficient and can be improved by splitting it into two steps into two another tubes, one
tube add HCl to rennin sample to low the ph, then add the milk to investigate the effect of low
pH on rennin. In the next tube add HCl to a rennin sample then add sodium hydroxide NaOH to
neutralize the sample pH, bringing the pH up, to see if neutralizing pH can restore the rennin
function if the low pH denature the rennin enzyme. In this way we can better understand the
effect of pH.

Part C. Estimation of Protein Concentration in Solution

Discussion

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 Plot your results (Recall – practical 1 to see how to plot this data) and explain how you
determined the concentration of your unknown Bovine Serum Albumin sample. How confident
are you that your answer is correct? Explain your reason for this.

More proteins leads to more reaction and more intensive colour.

 Do you have protein in your rennin sample, if so what is its concentration? How would you
quantify the amount of renin used in Part B?

 Based on your results of Part C, if you have to explain the BCA assay to someone what would
you describe is a possible limitation of the method?

The Bradford assay is linear over a short range, typically from 0 µg/mL to 2000 µg/mL, often making
dilutions of a sample necessary before analysis. In making these dilutions, error in one dilution is
compounded in further dilutions resulting in a linear relationship that may not always be accurate.

Disadvantages of the BCA protein assay

The reaction may be less sensitive to the type of amino acids present in the solution but the
reaction is influenced by cysteine, tyrosine and tryptophan residues. The presence of these
amino acids will produce color that may interfere with your results.

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 Week 4 FPG

Basic Biomolecules 3
Protein Structure: from primary to tertiary
Chapter 4 – Biochemistry - A short course
 Lecture Learning Outcomes
At end of lecture you should be able to:
– Explain and describe protein 1° structure
Amino acids form polypeptide chains
– Explain and describe protein 2° structure
Polypeptide chains can fold into regular structures
– Explain and describe protein 3° structure
Water-soluble proteins fold into compact structures
Levels of structure in proteins
 Primary Structure

• Polypeptide = amino acids linked by

peptide bonds

• Peptide bond also called amide bond

• Each amino acid in a protein can also be
referred to as a residue
 α-amino group

Condensation reaction

peptide bond
Components of a polypeptide chain

• Constant part – main chain – peptide backbone

• Variable part – distinctive side chains
Components of a polypeptide chain

• Constant part – main chain – peptide backbone

Components of a polypeptide chain

• Variable part – distinctive side chains

Components of a polypeptide chain

amino group carbonyl group

• Backbone rich in H-bonding potential

• Each residue has:
C=O – good H-bond acceptor
N–H (except proline) – good H-bond donor
 Polypeptide has directionality

Start End
 Polypeptide has directionality

NH3+ COOH-

Start End
 Polypeptide has directionality

Start End
Polypeptide chain properties

• Peptide – a few residues joined together

• Polypeptide chain ~ 50-2000 residues
joined together
• Polypeptide > 5 000 MW – commonly
referred to as a protein

• Nomenclature – Protein molecular weight

(MW) units is g mol-1
• e.g., 10 000 g mol-1 has mass of 10 000 Da
(Daltons) or 10 KDa (Kilodaltons)
Acid-base properties of peptides

One free α-amino group

Two ionizable
R groups

One free α-carboxyl group

 Question

H2N-Ala-Ala-Gly-Glu-Leu-Trp-Lys-Arg-Pro-Gly-COOH

What is the net charge of the above peptide at pH 7.4?

 Polypeptide chains are flexible
yet conformationally restricted
• Protein structure stabilized by multiple weak
interactions.
• BUT covalent bonds (S–S) in polypeptide
backbone place constraints on structure.
§ Peptide bond is locked in trans conformation
§ Peptide bond is rigid and planar
 Polypeptide in trans configuration
= less steric clashing
Sidechain of amino acid O
H
H
C N
Cα N
C Cα Cα Cα

O
Cysteine can form covalent bonds

disulfide bond
Cysteine side chains can form
covalent bonds with eachother
 Primary Structure
• Primary structure dictates 3D structure of protein
• 3D structure determines protein function
• Thus protein function depends on 1° amino acid
sequence
• All species produce thousands of different
proteins
• Each amino acid plays vital role in determining
3D structure of a protein
• Each protein has a specialized function in the cell

Structure is important for protein function

 Alterations in Primary Structure
Example: Sickle cell anaemia
• Haemoglobin – oxygen carrier protein
• 1 amino acid altered in β chain of haemoglobin from Glu
à Val
• Insoluble haemoglobin precipitates & red cells assume
sickle shape.
• Sickle cells block capillaries – are destroyed.
• Both copies of gene mutated – die young.
• Certain parts of Africa/Med – high incidence sickle cell
anaemia.
• Evolutionary advantage - only 1 mutant gene for
haemoglobin -> no severe symptoms of sickle cell
syndrome AND protects from malaria.
Levels of structure in proteins
Secondary Structure
 Secondary Structure
• Proteins will fold up into a stable 3D structure
during or shortly after synthesis.

• Initial “local” folding events

– amino acids adjacent in 1° sequence
– result in well-defined 2° structure elements

• Most prominent examples of 2° structures

(1) a helix
(2) b sheet
(3) turns and loops
 1. α helix
• Tightly coiled rod-like structure
• Side chains of amino acids (aa)
bristle out from axis of helix
• All backbone CO and NH
groups form H-bonds except
those at end of helix
 α helix
• Basically all a helices in proteins – Right-handed

Anti-clockwise Clockwise
Schematic views of α helix
 Complex views of α helix
Side view

End view

End view – ball & stick

 H-bonding in an α helix
H-bond 4 residues ahead in sequence

H-bond 4 residues ahead in sequence

• Amide H – H-bond donor

• Carbonyl O – H-bond acceptor
• All main chain CO & NH groups are H-bonded
except for amino acids at ends
Properties of α helix

• 3.6 residues/turn of a helix

• Amino acid spaced 3 & 4 apart are

close – on same side of helix

• Amino acids spaced 2 apart are on

opposite sides of helix
Which aa are found in a helix?
• Helix formers:
smaller & non-branched aa
Ala, Cys, Leu, Met, Glu, Gln, His, Lys
• Destabilize helix:
branched aa
Val, Thr, Ile à due to steric clashes
H-bond donor or acceptor not good
Ser, Asp & Asn à compete for main-
chain NH & CO groups
• Helix breaker:
Proline à Can’t be a H-bond donor
– can be 1st residue of helix
 Cyclic Proline disrupts structure
R group

carboxyl group
α-amino
Can’t be a H- Can be a H-bond
bond donor in a acceptor this
polypeptide side
chain
 Ferritin – largely α helical

75% of residues in ferritin are in α helices

~25% all soluble proteins are composed of α helices
PDB ID 1AEW
 Transmembrane proteins can use a
helices to span membrane
hydrophilic

hydrophobic

hydrophilic

Lipid environment of a cell

 2. β sheets
• β sheets formed by adjacent β strands
• Polypeptide in β strand is fully extended
3.5 Å between each aa instead of helix ~1.5 Å
• Side chains alternate above & below plane

C N

Cα
H
 β sheets are stabilized by H-
bonding between strands
• H bonds link strands in β sheets
• β sheets made up of 4 - 5 (up to 10) β strands
• Strands of a β sheet may be parallel, anti-
parallel or mixed
• β sheets can be flat or adopt twisted
conformation
 Anti-parallel β sheet

Cα
C N

H-bonds O
between
strands H

H-bonds connect
single amino acids on
each strand
 Topology diagram of an
antiparallel β sheet
loops anti-parallel
β sheet
 Parallel β sheet

H-bonds
between
strands

H-bonds connect
amino acid on 1 strand
with two aa on other
 Topology diagram of a
parallel β sheet

parallel
loops
β sheet
 Which residues in a β sheet?

• Large aromatic residues (Tyr, Trp, Phe) &

branched amino acids (Thr, Val, Ile) favoured in
β strands in middle of β sheets

• Different types of residues (e.g., Pro) found in

edge strands in β sheets
Schematic of a β sheet

Rotate
by 90°
Transmembrane proteins can use β
barrel to span membrane

PDB 1A0S
 Turns & loops
• Globular proteins are compact – require
reversal in polypeptide chain direction
• Accomplished by common structural
elements: turns & loops
• Often on surface of proteins – participate in
interactions b/n other proteins & environment
• Pro common in turns – H-bond acceptor
• Loops exposed to aq. environment usually
composed of hydrophilic R groups: Ser, Gly,
Asp, Asn
 Turns & loops
flexible loops
Reverse turn

CO NH

PDB 1FTP
 Proline disrupts structure

Can’t be a H- Be a H-bond
bond donor in a acceptor this
polypeptide side
chain
 Summary 2° structure
• 2° structure
– regular arrangement of amino acid residues
in a segment of a protein.
– each residue spatially related to its
neighbours in a similar way.
• Most common 2° structures are
– α helix
– β sheets
– Turns & Loops
 Higher order protein structure

Tertiary (3°) structure

• Overall 3D spatial arrangement of all atoms in

protein
• Longer-range interactions
• Residues far apart in protein sequence may be
close in space and interact
 Weak interactions – Structure

• Hydrophobic interactions – major contributors to

stabilizing globular form of most soluble proteins

• Hydrogen bonds & ionic interactions optimized

in structures that are thermodynamically most
stable

• Covalent bonds (disulfide bond) in polypeptide

backbone places constraints on structure
 Tertiary structure – two groups

Fibrous proteins – polypeptide chains arranged in

long strands or sheets e.g., α-keratin & collagen

Globular proteins – polypeptide chains folded into

spherical or globular shape
– water soluble compact
 Globular proteins
• Segments of polypeptide chain fold back on
themselves to generate compact structure
• Folding provides structural diversity for
function
• Water soluble – perform most of the chemical
transactions in cell
• Include enzymes, transport, motor, regulatory
proteins, immunoglobulins etc
 Globular proteins are compact
• Human serum albumin has 585 residues

Dimensions if it
was a fully Actual
extended α helix dimensions in
native form
 Structural Biology
• X-Ray Crystallography (1957 first structure)
• 1962 Nobel Prize in Chemistry – Perutz and
Kendrew – first atomic structure of protein
using X-ray crystallography
• Nuclear Magnetic Resonance Spectroscopy
(1982 first structure)
• 2002 Nobel Prize in Chemistry – Wütrich 3D
structures of proteins using NMR
• Reveal 3D structures of proteins and how
they work at the atomic level
http://www.rcsb.org/pdb/home/home.do
 Principles of 3° structure
• Globular proteins form complicated 3D
structures
• Not much empty space in interior
• Interior – mainly hydrophobic amino acids
• Exterior – charged & polar amino acids
 Myoglobin - example
• First protein to be seen in atomic detail
• Extremely compact
• Single polypeptide chain 153 amino acids
• O2 carrier in heart & skeletal muscle
• Capacity to bind O2 depends on heme
prosthetic (helper) group
 Myoglobin

Ribbon + side chains (blue) Space-filling model

PDB 1MBO
 Myoglobin

Ribbon representation Surface representation

PDB 1MBO
 Myoglobin

Ribbon + side chains (blue) Space-filling model

PDB 1MBO
 3° Structure – divided into
structural & functional units
• Structural unit: Motif or Supersecondary
structure
• Functional unit: Domain
 Structural unit – Motif

Motifs or Supersecondary structures

• Combinations of 2° structure found in
proteins
• Motifs may not be independently stable
– What does this mean?
• Look at some common motifs
Helix-turn-helix
1LMB
Motifs

β-α-β loop β barrel Coiled coil

7AHL
Helix-turn-helix Motif

Helix-turn-helix motif
DNA

2O49L
Motif – all α
Motif – all β
Motif – α+β
 Functional unit – Domain

• Some proteins contain compact structures

called domains
• Domains are connected by a flexible segment
of polypeptide - Like pearls on a string
• Domains are independently stable
• Domains can function independently
ssDNA binding protein + ssDNA
– this protein has one domain

2MNA
ssDNA binding protein + ssDNA
Proteins can contain 2 or more
of the same domain

Cell surface protein CD4

1WIO
Proteins can contain different
domains

DNA repair protein Nbs1

3HUE
Levels of structure in proteins