LECTURE 4/ PROTEIN CHEMISTRY

 Proteins – are complex, organic nitrogenous substances

OUTLINE
with very high molecular weights, found in all plant and
I. Protein Chemistry
A. Peptides
animal cells consisting largely or entirely chains of alpha-
II. Biological Functions amino acids united in peptide linkage (between the
III. Classification of Proteins nitrogen, the amino group, and carboxyl group of the
A. Classification based on composition, physical second amino acid).
and chemical properties.  Proteins contain Nitrogen in its basic structure
i. Simple proteins
ii. Conjugated proteins PEPTIDES
iii. Derived proteins  chain of amino acids; amino acid polymers with low
B. Classification based on the shape and certain molecular weights, typically consisting of less than 50 a. a.
physical characteristics
 “Oligopeptides” - consisting of 2 to 10 a. a.
C. Classification based on biologic functions
D. Classification of amino acids from a
 “Polypeptides” - have more than 10 a. a. (but less
Nutritional point of view than 50) residues
i. Synthesis of Non-essential amino  “Proteins” - more than 50 a. a. residues
acids
ii. Classification as Ketogenic or BIOLOGICAL FUNCTIONS
Glucogenic
IV. Levels of Structural Organization 1. Catalyst of chemical reactions [e.g., enzymes]
A. Primary Structure  Catalyst – hastens/speeds up a chemical reaction
B. Secondary structure  enzyme is called a catalyst. chemical reactions
i. Coils or Helices (Alpha-Helix) occur naturally, but enzymes make them faster.
ii. Sheets or Pleats (Beta Pleats)
iii. Super secondary Structures Or 2. Regulators of chemical reactions [e.g., hormones like
Motifs insulin and glucagon which are protein in nature]
C. Tertiary structure 3. Transport of substances [e.g., hemoglobin (blood) ion
i. Important Features channels (membrane)]
ii. Molecular Chaperones  Hemoglobin – oxygen transporter
D. Quarternary structure
 Ion channel – particular ions can be
i. Interactions That Hold Subunits
transported in and out of the cell
Together
V. Protein Denaturation
o example: sodium-potassium pump
A. Causes of Protein Denaturation
(sodium ions enter cell, potassium
VI. Protein Purification ions leave the cell)
i. Characteristics Of Proteins That 4. Storage function [e.g., apoferritin]
Are Used In The Various  Apoferritin – storage of iron
Separation Procedures  Myoglobin – storage of oxygen in muscles;
A. Chromatography and Types counterpart ng hemoglobin but in the muscles
i. Gel Filtration Chromatography 5. Mechanical support [e.g., collagen, elastin, fibrin and
ii. Affinity Chromatography keratin]
iii. Gel Electrophoresis  Collagen – makes skin smooth and elastic
B. Steps in Protein Analysis o when you're old, may decrease in
i. Determine Amino Acid Composition collagen production
of the Protein  fibrin – protein acting as mechanical support
ii. Determine the N And C Terminals found in the connective tissues
iii. Determine the Amino Acid
 keratin – protein acting as mechanical
Sequence
support sa hair
iv. Check Number of Polypeptide
6. Coordinated motion [e.g., actin and myosin]
Chains
v. Identification of Component Amino  the movement in the muscles is not an actual
Acid contraction but sliding action/movement
where myosin moves over actin
 actin – thin muscle filament or muscle protein
PROTEIN CHEMISTRY  myosin – thick muscle filament or muscle
protein
 Protein came from the word “Proteios” – primary, holding 7. Defense/Immune protection [e.g., antibodies]
first place, of first importance.  antibodies in blood called immunoglobulins
 Protein – most important macromolecule since it has a lot protect us
of functions

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 8. Cell signaling [e.g., membrane receptors (which are
protein in nature) such as insulin receptor]
 insulin promotes glucose utilization or entry
ng glucose sa cell
9. Control of growth and differentiation [e.g., repressor
proteins]
CLASSIFICATION OF PROTEINS
CLASSIFICATION BASED ON COMPOSITION,
PHYSICAL, AND CHEMICAL PROPERTIES
 most proteins are heat coagulable like egg na pag may
heat, magsosolidify pag niluto
SIMPLE PROTEINS
1. Albumin – soluble in water and dilute aqueous salt
solution; heat coagulable (will denature when heated,
magiging solid)
 Albumin – main protein oncotic pressure
(pressure that holds water in the blood
vessel).
 pag kulang ka sa albumin, water will be
moving out of the blood vessel into the
connective tissue. kaya people suffer from
edema.
 Hypoalbuminemia - decrease in albumin
2. Globulin – insoluble in water; soluble in aqueous salt
solution; heat coagulable
3. Glutelin – soluble in dilute acids and alkalines; heat
coagulable [plant proteins – glutenin (found in wheat);
oryzenin(found in rice)]
 Glutenin - counterpart of glutelin in plants,
found in wheat
4. Prolamine – alcohol-soluble protein [e.g., seed
proteins – zein (corn), gliadin (wheat)]
5. Albuminoid – least soluble [e.g., animal proteins –
keratin, collagen]
6. Histone – basic protein (means composed of basic
amino acids); soluble in water, dilute acid and alkali;
found in combination with DNA (histone plays a major
role in dna packaging ‒ how u arrange dna to
chromosomes inside the nucleus)
 dna is negatively-charged so may association
with dna and a basic histone
7. Protamine – simplest; basic; soluble in water, dilute
ammonia, acid and alkali; found in spermatozoa
CONJUGATED PROTEINS (conjugated with another
macromolecule, ex: protein + nucleic acid)
1. Nucleoproteins – proteins that contain nucleic acid as
the prosthetic group [e.g., nucleohistone]
2. Glycoproteins & proteoglycans – contain
carbohydrates
 glycoprotein ay carbohydrate (glyco); more of
protein, less of carbo
 proteoglycan - mas maraming carbohydrate,
less protein
 # kung ano yung nahuli, ayun ang mas
marami
3. Phosphoproteins – proteins which have phosphoric
acid residues
4. Chromoproteins – proteins that contain prosthetic
groups that give color [e.g., hemoglobin-red]
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5. Lipoproteins – proteins associated with lipids/fats
[LDL, HDL]
6. Metalloproteins – proteins that contain minerals
[cytochrome, ceruloplasmin]
 Cytochrome – protein containing copper and
iron
 Ceruloplasmin – copper-containing protein
DERIVED PROTEINS
 partially digested proteins
 include artificially synthesized proteins
1. Primary derived
 Proteins
 Metaproteins
 Coagulated derived
2. Secondary derived
 Proteoses
 Peptones
 Peptides
CLASSIFICATION BASED ON THE SHAPE AND
CERTAIN PHYSICAL CHARACTERISTICS
A. Fibrous proteins – tough; insoluble in aqueous sol’n
involved in structural functions (e.g., collagen, keratin)
B. Globular proteins – soluble; diffuse readily; involved
in mobile and dynamic functions (e.g., enzymes,
plasma proteins, hemoglobin)
CLASSIFICATION BASED ON BIOLOGICAL
FUNCTIONS
A. Enzymes – dehydrogenases, kinases, etc.
B. Storage proteins – ferritin, myoglobin
C. Regulatory proteins – myoglobin, DNA- binding
proteins
D. Structural proteins – elastin, reticulin, collagen
E. Protective proteins – Ig(immunoglobulin or
antibodies), blood clotting factors (pag kulang dito,
mahirap magstop bleeding. pag naman sobra, can
cause clots or thrombosis)
 blood clotting factor 12 for hemophilia
(matagal magstop bleeding)
F. Transport proteins – plasma lipoproteins
G. Contractile or motile proteins – actin, myosin
 # sa exam daw magbibigay ng function tapos
magbibigay tayo ng example
CLASSIFICATION BASED ON AMINO ACIDS FROM A
NUTRTITIONAL POINT OF VIEW
 Essential or indispensable amino acids – cannot be
synthesized by the body so they must be provided in the
diet
o Phe Leu Lys His Val Trp Ile Arg Thr Met #
imemorize daw
o ^ kailangan complete sa diet mo.
o Arg considered as semi-essential kasi
napproduce siya sa body pero nagagamit din
agad sa urea cycle so you need other sources
of Arg
o negative nitrogen balance – (u lack protein
intake or kulang ka sa essential AA. pag may
sakit ka raw di ka agad makaka-recover
o positive nitrogen balance – (complete
essential AA sa diet mo)
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 Nonessential or dispensable amino acids – can be COILS OR HELICES (ALPHA-HELIX)
synthesized by the body so they need not be provided in  Coils or helices – intrachain hydrogen bonding
the diet.  Alpha-helix – discovered by Linus Pauling in 1951
 Stabilized by inter-residue H-bonds formed bet. the H atom
SYNTHESIS OF NON-ESSENTIAL AMINO ACIDS attached to a peptide N and the carbonyl O atom.
 Glycine ↔ Serine (HYDROGEN BONDING WITHIN PEPTIDE BOND)
 Proline ↔ Glutamic acid  Each peptide bond participates in H-bonding.
 Arginine ↔ Glutamic acid  alpha-helix forms spontaneously as it is the lowest energy,
 Histidine ↔ Glutamic acid most stable conformation for a polypeptide chain.
 Tryptophan ↔ Alanine  There are 3.6 amino acid residues per turn with a pitch
 Phenylalanine ↔ Tyrosine (distance bet. corresponding points per turn) of .54 nm (5.4
 Threonine ↔ Glycine A); spacing per residue is .15 nm (1.5 A).
 Methionine ↔ Cysteine  Amino acid R groups extend outward from the helix.
 Aspartic acid ↔ Asparagine
 Glutamic acid ↔ Glutamine
 Pyruvate ↔ Alanine
 Oxaloacetate ↔ Aspartic acid
 a-ketoglutarate ↔ Glutamic acid
 3-phosphoglycerate ↔ Serine

CLASSIFICATION AS KETOGENIC OR GLUCOGENIC

 Ketogenic – can be converted to ketone bodies (like
acetoacetic acid)
o 2 purely ketogenic AA ↔ leucine and lysine
 Glucogenic or Glycogenic – used as susbtrate for
synthesis of glucose
 4 AA that are both keto and glucogenic – Phenylalanine,
isoleucine, tyrosine, tryptophan
o the rest are glucogenic

o Right-handed helix – if the twist is to the right

LEVELS OF STRUCTURAL ORGANIZATION
PRIMARY STRUCTURE
 Refers to quantitative amino acid composition; sequence of
amino acids; number of peptide chains. nagttwist
 stabilized by peptide bond
 the backbone of a protein refers to the atoms that
participate in the formation of peptide bonds (between
carboxyl group of one Aa interacTing with the amino group
of the other AA)
 Most abundant amino acid in proteins ↔ [Leu, Ala, Gly,
Ser, Val and Glu]
 Rarest in proteins are ↔ [Trp, Cys, Met and His]
SECONDARY STRUCTURE
 Due to the formation of hydrogen bonds between peptide
bonds.
 hydrogen bonding between peptide bonds ↔ reaction
that maintains the secondary structure of proteins
o Left-handed helix – twist to the left
 Factors that destabilize the alpha-helix:
o Presence of adjacent similarly charged a. a.
o Presence of adjacent bulky R groups.
o Presence of proline (proline ↔ aka helix breaker
bc it has rigid ring that prevents the nitrogen-
carbon bond from rotating)
 contains rigid ring that prevents the N-C
bond from rotating
 no N-H group available to form
intrachain hydrogen bonds
SHEETS OR PLEATS (BETA PLEATS)
 Sheets or pleats – interchain hydrogen bonding
 Beta-pleated Sheet – 2nd most commonly occurring
protein secondary structure
 Formed when 2 or more almost fully extended polypeptide
chains lie side by side.
 H-bonds are interchain.
 R grps lie above or below the zigzagging planes of the
pleated sheet and are nearly perpendicular to them.
 Amino acids with less bulky R groups are present.
 Forms of Beta-pleated Sheet

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 o Antiparallel beta-sheet – neighboring H-bonded
polypeptide chains; run in opposite directions;
more stable; if N-C and C-N
o Parallel beta-sheets – H-bonded chains extend
in the same direction; same N ang simula

TERTIARY STRUCTURE
 3-dimensional structure
 Protein conformation
 results from folding of a polypeptide
 indicates how 2o structural features – helices, sheets,
bends, turns and loops assemble to form domains.
 the 3-dimensional shape of a folded polypeptide
 is result of the interactions among the R groups
 functional form of protein for a single polypeptide chain or
for monomeric protein
 What is responsible for the formation of tertiary
structures? ↔ the interaction between r groups or R-R
interactions

IMPORTANT FEATURES
 Factors that destabilize the β-pleated sheets / the ff makes  Amino acid residues that are distant from each other in the
it not compatible with the secondary structures primary structure come into close proximity when the
o Bulky R groups. # imemorize daw yung R groups polypeptide folds.
o R groups with like charges.  When polypeptide folds, proteins become more compact;
most water molecules are excluded from the proteins’
SUPERSECONDARY STRUCTURES OR MOTIFS interior making interactions bet. polar (nasa outer to) and
 Combinations of secondary structures; Occur as part of a nonpolar AA (nasa loob)possible.
larger functional unit  Large globular proteins (> 200 AA) often contain several
 Have different functions in different proteins compact units called domains.
 Have a particular function o Domains are structurally independent segments
 Have roughly 10 to 40 a. a. residues each that have specific functions (e.g. binding an ion)
 Common Supersecondary Structures  highest form attained by monomeric proteins are tertiary
o Helix-loop-helix structures *
o Coiled-coil motif  Types of Interaction that stabilize Tertiary Structure:
o Beta-alpha-beta unit o Hydrophobic interactions (among non-polar AA)
o Hairpin o Electrostatic interactions (salt bridges)
o Zinc finger o Hydrogen bonds
o Leucine zipper o Covalent bonds (like disulfide bridges; example 2
o Greek key cysteine magiging cystine )

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MOLECULAR CHAPERONES
 Protein folding is assisted by Molecular Chaperones and
enzymes
 Proteins can fold by itself kasi may R group pero folding
could be incorrect or improper
 Molecular chaperones – proteins that have the net effect of
increasing the rate of correct folding by binding newly
synthesized polypeptides before they are completely
folded. (ito yung nag-aassist na protein para macorrect ang
folding) [e.g., heat-shock proteins, chaperonins]
 Enzymes involved in protein folding:
o Peptidyl prolyl cis-trans isomerase
o Protein-disulfide isomerase
QUARTERNARY STRUCTURE
 highest level
 exhibited only by proteins containing more than one
polypeptide chain
 most proteins with molecular masses > 100 kD, consist of
more than one polypeptide chain
 Each polypeptide component is called a subunit
o Subunits may be identical or different.
o Protomers are identical subunits.
 highest structure for oligomeric proteins – those with
more than one subunit or more than 2 polypeptide chains
 describes the characteristic manner in which the individual,
folded polypeptide chains fit each other or interact with one
another so that they can act as one single molecule
 Hemoglobin has 4 subunits ‒ 2 alpha-globin and 2 beta-
globin chains. highest structure ng hemoglobin ay
quaternary structure
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INTERACTIONS THAT HOLD SUBUNITS TOGETHER
 Hydrophobic interactions
 Electrostatic interactions
 Hydrogen bonds
 Interpolypeptide disulfide bonds
o Hydrophobic interactions are the principal forces
that hold the subunits together.
o Electrostatic forces contribute to the proper
alignment of the subunits.
PROTEIN DENATURATION
 Denaturation – refers to the loss of function of the protein
when it loses its 3-dimensional structure.
 under most conditions, denatured proteins exist in a set of
partially folded states.
 denaturing forces disrupt the weak interactions in a
protein, primarily hydrogen bonds.
 cleavage of noncovalent bonds results to disruption of the
2° (secondary), 3° (tertiary) and quaternary structures.
primary structure lang ang hindi masisira
CAUSES OF PROTEIN DENATURATION
 Chemical, physical & biological alterations result from
denaturation
 Denaturing agents include:
o Physical [heat, extremes of pH, UV, surface
action, high pressure...]
o Chemical [organic solvents (alcohol, acetone),
solutes like urea, or by detergents]
 Certain globular proteins regain their native structure and
biologic activity if returned to conditions in which the native
conformation is stable – a process known as renaturation.
PROTEIN PURIFICATION
 Precedes protein analysis
 Fractionation procedures – to eliminate selectively the
other components of the mixture so that only the
required protein remains
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CHARACTERISTICS OF PROTEINS THAT ARE USED GEL ELECTROPHORESIS
IN THE VARIOUS SEPARATION PROCEDURES

STEPS IN PROTEIN ANALYSIS

CHROMATOGRAPHY AND TYPES
I. Determine amino acid composition of the protein
 Chromatography – process/technique for
II. Determine the N and C terminals
fractionation, isolation, purification and identification of III. Determine the amino acid sequence
substances in a mixture based on the relative affinities IV. Check number of polypeptide chains
of component mixtures between 2 phases: mobile
V. Identification of component amino acid
phase and stationary phase
 e.g. Paper Chromatography
I. DETERMINE AMINO ACID COMPOSITION OF THE
o Mobile Phase: organic solvent (yung solution,
kasi mobile - nagalaw) PROTEIN
o Stationary phase : Filter paper  [Acid hydrolysis; Basic hydrolysis; Enzymatic hydrolysis]
o Principle: Like dissolve like A. Hydrolysis of proteins by acids:
 Types of Chromatography o Heat proteins at 100 – 120oC for 10-24 hrs in 6 N
o Paper chromatography HCl
o Ion exchange chromatography [based on o Disadvantages:
charge]  All the Trp & variable amounts of Ser &
o Gel filtration chromatography [based on size Thr are destroyed
exclusion or molecular sieve  Gln and Asn are deamidated to Glu and
chromatography] Asp
o Affinity chromatography [ligand]  Glu undergoes intramolecular
o Gel Electrophoresis [MW] dehydration to form pyrollidone-5-
carboxylic acid
 Other amino acids may undergo
intermolecular dehydration forming cyclic
GEL FILTRATION CHROMATOGRAPHY anhydrides

B. Basic Hydrolysis
o Principal use of basic hydrolysis – To estimate Trp
o Incubate proteins w/ 2 to 4 N NaOH at 100oC for
4-8 hrs
o Disadvantages:
 Cysteine, cystine, Ser, Thr and Arg are
decomposed in the process
 Other amino acids may be partially
destroyed by deamination
 Racemization of amino acids occur

C. Ezymatic Hydrolysis
AFFINITY CHROMATOGRAPHY o # sa exam doc will give a sequence of amino
acids, then we are to predict how many fragments
can be produced using these particular enzymes

1. Pepsin – cleaves off peptide bonds whose amino

function is contributed by Phe, Tyr, Trp, Leu, Glu,
Gln; pepsin is in our stomach for digestion
2. Trypsin – major pancreatic enzyme; cleaves off
peptide bonds whose carbonyl function is
contributed by Lys and Arg
3. Chymotyrpsin – cleaves off peptide bonds whose
carbonyl function is contributed by Phe, Trp and
Tyr
4. Carboxypeptidase – cleaves off C-terminal
amino acids

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 5. Aminopeptidase – cleaves off N-terminal AA
6. Thermolysin – heat-stable bacterial protease;
cleaves off peptide bonds whose carbonyl function
is contributed by Leu, Val and Ile
7. Papain – found in certain plants like papaya;
cleaves off peptide bonds whose carbonyl function
is contributed by Lys, Leu, Arg, Gly.
8. Bromelain – derived from pineapple kasi contains
enzymes to digest proteins; cleaves off peptide
bonds whose carbonyl function is contributed by
Lys, Ala, Tyr and Gly;
 highest content of bromelain is found sa
matigas na inner part ng pineapple

II. DETERMINE THE N AND C TERMINALS

 N-terminal:
o Sanger’s reaction
o Dansyl chloride reaction
o Edman’s reaction
o Reaction with aminopeptidase
 C-terminal:
o Reduction with lithium borohydride
o Hydrazinolysis
o Reaction with carboxypeptidase

III. DETERMINE THE AMINO ACID SEQUENCE

 Edman’s reaction:
o liberates amino acids one at a time from the N-
terminus of a polypeptide. The N-terminal residue
is removed as a phenyl-thiohydantoin derivative
o carried out on a programmed machine called
sequenator

IV. CHECK NUMBER OF POLYPEPTIDE CHAINS

 If there are 2 or more chains, separate them.
A. Disulfide bonds
o oxidation with performic acid
o reduction with mercaptans
B. Noncovalent bonds
o denaturing agents

V. IDENTIFICATION OF COMPONENT AMINO ACID

1. Paper chromatography
2. Thin layer chromatography
3. Ion exchange chromatography
o Cation exchanger - polysulfonic; low to high pH
o Anion exchanger – polyamino; high to low pH
4. Electrophoresis
5. High performance liquid chromatography

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