BIOCHEMISTRY

Protein and Amino Acid Chemistry Part 1

Dr. Alcantara
Proteins are complex organic nitrogenous substances with very high o CLASSIFICATION OF PROTEINS:
molecular weights, found in all plant and animal cells, consisting largely A. Classification based on Composition, Physical and Chemical
or entirely of alpha-amino acids united in peptide linkages. Properties of the Protein
1. SIMPLE PROTEINS- made up of AMINO ACIDS ONLY
 FUNCTIONS OF PROTEINS: a. Albumin
1. Catalyst of chemical reactions - Soluble in water
Enzymes are mainly proteins in nature, thereby proteins - Dilute aqueous salt solution
could serve as catalysts - Heat coagulable
b. Globulin
2. Transport and Storage - Insoluble in water
Example of transport would be hemoglobin → transports - Soluble in aqueous salt solution
oxygen - Heat coagulable
Example of storage would be ferritin → storage form of c. Glutelin
iron - Soluble in dilute acid and alkalies
- Heat coagulable
3. Coordinated Motion It is a plant protein. Example of it can be
The relaxation and contraction of muscles, we need found in wheat which is called glutenin.
proteins there which are actin and myosin Glutenin is the glutelin found in wheat.
There is also a glutelin found in rice which
4. Mechanical Support is called Oryzenin.
Examples are Collagen and Keratin
d. Prolamine
5. Immune Protection - Alcohol soluble protein
Antibodies are derived from gamma globulins, which are It is a seed protein. An example of it can be
proteins. found in corns which is called Zein. There is
also a seed protein found in wheat called
6. Generation and Transmissions of Nerve Impulses Gliadin.
There are neurotransmitters that are derived from
different amino acids: e. Albuminoid or Scleroprotein
 Epinephrine - Least soluble
 Norepinephrine It is any animal protein. Example would be
collagen and keratin.
7. Control of Growth and Differentiation
f. Protamine
Example would be the suppressor or repressor proteins
- Simplest protein
which are important control elements that silence a
- Basic
specific DNA of a cell This is essential for the orderly
- Soluble in water
growth and differentiation of cells.
- Dilute ammonia, acid and alkali
- Found in spermatozoa
8. Cell Signaling
g. Histone
Example would be membrane receptors such as insulin
- Basic protein
receptors
- Soluble in water
- Dilute acid and alkali
9. Hormones
- Found in combination with DNA
Protein Hormones includes Insulin, Thyrotropin, 2. CONJUGATED PROTEINS- made up of AMINO ACIDS +
Somatotropin (Growth Hormone), Luteinizing Hormone, OTHER SUBSTANCES (PROSTHETIC GROUP)
and Follicle Stimulating Hormone
Prosthetic Group Example
a. Nucleoprotein Nucleic Acid
10. Proteins are one of the major source of biological
b. Glycoprotein &
membranes Carbohydrates
Mucoprotein
Example would be transmembrane proteins (integral
Phosphoric Acid Casein
proteins) or peripheral proteins embedded on cell c. Phosphoprotein
residues
membrane
Prosthetic group Hemoglobin
d. Chromoprotein
that gives color
e. Lipoprotein Lipids LDL,HDL
Metals or Cytochrome
f. Metalloprotein
Minerals

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Protein and Amino Acid Chemistry Part 1
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3. DERIVED PROTEINS 6. Some amino acids or their derivatives act as chemical
a. Primary Derived messengers
b. Secondary Derived o GABA, serotonin

B. Classification Based on Shape and Certain Physical Characteristics
of the Protein
1. FIBROUS PROTEINS- elongated
- Tough
- Insoluble in water
- Arranged around a single axis to form a fiber
- Involved in structural functions
- E.g. Collagen- most abundant, 25% protein of the body
Keratin- hair, scales, horns, wool, nails and feathers
2. GLOBULAR PROTEINS- circular
- Involved in mobile and dynamic functions
- For transport purposes
- E.g. Enzymes, hemoglobin, plasma proteins
7. Several amino acids act as metabolic intermediates
o Arg, citrulline, ornithine – urea cycle
o BASIC STRUCTURE OF AMINO ACIDS
o Each amino acid has a central carbon, called the alpha
carbon, to which four different groups are attached:
1. a basic amino group (-NH2)
2. an acidic carboxyl group (-COOH)
3. a hydrogen atom (-H)
4. a distinctive side chain (-R)

C. Classification Based on Biologic Functions

1. Enzymes- dehydrogenases, kinases
2. Storage- ferritin, myoglobin
3. Regulatory- DNA-binding proteins
4. Structural- collagen, proteoglycans, elastin
5. Protective- blood clotting factors, Immunoglobulins, interferon
6. Transport- hgb, lipoproteins, transferrin o All amino acids will have carboxyl, amino group and
7. Contractile or Motile- actin, myosin hydrogen but will differ in their side chain (-R)
o If there are four different atoms attached to a carbon, that
o AMINO ACIDS carbon is named as a chiral carbon.
FUNCTIONS OF AMINO ACIDS: o All amino acids will have a chiral carbon with the exception
1. Building blocks of proteins of GLYCINE
o A protein can only be called protein if that polypeptide
The side chain of GLYCINE is
chain is made up of 50 or more amino acids. Less than
hydrogen. There are four atoms to
50, it is only a polypeptide
the carbon but THEY ARE NOT
o Amino acids make up a protein, therefore it is their DIFFERENT FROM EACH OTHER.
building blocks Given this, the alpha carbon of
glycine IS NOT A CHIRAL CARBON
2. Precursor of various substances
o Glycine- heme, purine, creatine, glutathione, hippuric acid o Alpha carbon is ASYMMETRIC except for glycine.
o Phe and Tyr- thyroxine, epinephrine, melanin o Both D- amino and non- α amino acids occur in nature
o Tryptophan- niacin, serotonin, melatonin, indole, skatole o Only L- α amino acids are present in proteins
o Histidine- histamine o Genetic code specifies 20 L alpha amino acids
o Lysine and Methionine- carnitine

3. Source of energy
o 4 cals/gram of protein

4. Special amino acids act as components of certain types of

proteins
o Hydroxyproline & hydroxylysine- collagen
o Gamma-carboxyglutamic acid prothrombin
o Desmosine (derivative of lysine)- elastin

5. Phosphorylation and dephosphorylation of Serine, Threonine

and Tyrosine play major role in activation and inactivation of
enzymes.

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Protein and Amino Acid Chemistry Part 1
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o ABBREVIATION OF AMINO ACIDS Aliphatic Unbranched

Glycine

Alanine

NOTES: For Glycine, its side chain is hydrogen only while Alanine’s
side chain is only the methyl group. Glycine is the simplest and
smallest amino acid.

Aliphatic Branched (Branched-Chain Amino Acids)

Valine
There are 20 known amino acids in nature. They could be
abbreviated using 3 letter abbreviation or a 1 letter abbreviation.
1. 3-Letter-Abbreviation – usually the first 3 letters of
the amino acid
EXEMPTIONS: Ile = Isoleucine Asn = Asparagine
Gln = Glutamine Trp = Tryptophan
2. 1-Letter-Abbreviation – NO NEED TO MEMORIZE. This
is used when you want to take US MLE.
Leucine
o CLASSIFICATION OF AMINO ACIDS BASED ON POLARITY:
A. Amino Acids with NONPOLAR or HYDROPHOBIC R Groups
- Referred to as NEUTRAL amino acids containing hydrocarbon R
groups →The term NEUTRAL is used because these R groups do
not bear positive or negative charges
- Do not bind nor give off protons and do not participate in
hydrogen or ionic bonds
- Its side chains can be thought of as “oily”, or lipid like, a
property that promotes hydrophobic interactions Isoleucine
- Interact poorly with water → Play an important role
maintaining the 3-dimenstional structure of proteins.
2 Types of Hydrocarbon Side Chains:
1. AROMATIC- there is a ring
o Phenylalanine = Benzene Ring
o Tryptophan = Indole Ring (has a dual ring)
2. ALIPHATIC- a straight or linear chain Methionine
1. Aliphatic Unbranched
o Glycine
o Alanine
2. Aliphatic Branched
o Valine
o Leucine
o Isoleucine Proline
o Methionine
o Proline

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Dr. Alcantara
NOTES: Methionine is the source of S-adenosylmethionine (SAM) Hydroxyl-containing Amino Acids
which is the methyl group donor in methylation reactions. o Contain polar hydroxyl groups → Enables them to participate in
Proline is referred to as the “helix breaker.” For all the amino hydrogen bonding, which is an important factor in protein
acids, the R-group is attached to the alpha carbon, but for PROLINE, structure
o -OH groups of Serine and Threonine = points for attaching
the R-group is not only attached to the alpha carbon but also to the
carbohydrates
amino group. That is why Proline is cyclic in appearance and o Sites of Phosphorylation reactions
structure. It is also the reason why Proline is sometimes referred to o Site of Glycosylation reactions
as an Imino Acid. o Important in formation of mucin

Aromatic
Serine

Phenylalanine

Threonine

Tryptophan

Tyrosine

MNEMONICS FOR NONPOLAR or HYDROPHOBIC R Groups

All Alanine Unbranched
Girls Glycine Unbranched
Pay Proline Branched Amide (CONH2) derivatives of Glutamate and Aspartate
Large Leucine Branched o Important in detoxification of ammonia
Value Valine Branched o Glutamine is the primary source of urinary ammonia
In Isoleucine Branched
Pretty Phenylalanine Aromatic
Thick Tryptophan Aromatic
Make up Methionine Branched Glutamine
NOTE: Some make ups are HYDROPHOBIC so always remember
make ups. Highlighted in red are aromatic.

3 Types of POLAR AMINO ACIDS:

1. Uncharged Polar- neutral or no charge
2. Positively Charged Polar- basic
3. Negatively Charged Polar- acidic
Asparagine
B. Amino Acids with UNCHARGED POLAR R Groups
- Have a zero net charge at neutral pH
- Have functional groups capable of hydrogen bonding with
water
- Important factor in protein structure Thiol Group containing (Thioether side chain)
Hydroxyl-containing Amino Acids o Contains the sulfhydryl (-SH) group
o Serine o When two cysteine residues combine a strong disulfide bond is
o Threonine formed and cystine is the resulting product
o Disulfide bond is also important in maintenance of protein
o Tyrosine
structure
Amide (CONH2) derivatives of Glutamate and Aspartate
o Glutamine
o Asparagine Cysteine
Others (Thiol Group containing)
o Cysteine

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o Selenocysteine- 21st L-α-amino acid

o A selenium atom replaces the sulfur of its structural analog,
cysteine
o It is a rare amino acid residue that is inserted into polypeptide Histidine
during translation rather that created through postsynthetic
modification
o Unlike the other 20 genetically encoded amino acids, it is not
specified by a simple 3-letter codon
o It is present at the active site of several human enzymes that MNEMONICS FOR POSITIVELY CHARGED POLAR R Groups
catalyse Red-Ox reactions Love Lysine
(Thioredoxin, Glutathione All Arginine
Peroxidase, Deiodinase) Humans Histidine
o Impairment in human
NOTE: Basically, in life we should always be positive so, LOVE ALL
selenoproteins have been
HUMANS
implicated in tumorigenesis and
atherosclerosis, and are
D. Amino Acids with NEGATIVELY CHARGED POLAR R Groups
associated with selenium
- Interactions between acidic and basic amino acids
deficiency cardiomyopathy
results in the formation of ionic bond ( also called salt
(Keshan Disease)
bond, electrostatic bond)
- The 2 amino acids have R groups with a net negative
MNEMONICS FOR UNCHARGED POLAR R Groups
charge at pH 7.0, each of which has a second carboxyl
Some Serine group
Times Threonine - Important in maintaining protein structure
Clingy Cysteine
Girls Glutamine
Negatively Charged Polar R Groups (Acidic Amino Acids)
Are Asparagine
Tenacious Tyrosine
NOTE: Uncharged amino acids tend to undergo hydrogen bonding
easily so they are “clingy” or “aggressive” Aspartic Acid
C. Amino Acids with POSITIVELY CHARGED POLAR R Groups
- Also called Basic amino acids
- Histidine contains the imidazole group which is
responsible for the buffering capacity of hemoglobin
- Arginine contains guanido group
Glutamic Acid
Positively Charged Polar R Groups (Basic Amino Acids)

MNEMONICS FOR NEGATIVELY CHARGED POLAR R Groups

Lysine Angry Aspartic Acid
Girls Glutamic Acid
NOTE: Negative attitude= Anger so Angry Girls, lol

FAMILIARIZATION TIPS:
Amino Acids with Hydroxyl Group
o Serine, Threonine, Tyrosine
o Ser, Tara Tagay!
o –OH is Alcohol
Arginine
Aromatic Amino Acids (meaning with rings)
o Phe, Tyr, Trp, His
Ring names:
o Phenylalanine- phenyl ring ο Histidine- imidazole ring
o Tyrosine- phenol ring o Proline- pyrolidine ring
o Tryptophan- indole ring

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Protein and Amino Acid Chemistry Part 1
Dr. Alcantara
Branched Amino Acids: Exercise:
VLIMP=BLIMP 1. Lysine + Glutamic Acid
 Lysine = Basic Amino Acid (NH3+)
o Valine, Leucine, Isoleucine, Methionine, Proline  Glutamic Acid = Acidic Amino Acid (-COOH)
Nice to Know:  Ionic bonding
o Asparagine- from asparagus, 1st amino acid discovered 2. Alanine + Valine
o Glutamate- first isolated from wheat gluten  Alanine = Nonpolar
o Tyrosine- first isolated from cheese  Valine= Nonpolar
o Glycine- sweet taste  Hydrophobic bonding
3. Cysteine + Tyrosine
o TYPES OF INTERACTIONS ENTERED INTO BY THE DIFFERENT R  Cysteine = -SH containing
GROUPS (SIDE CHAINS) OF AMINO ACIDS  Tyrosine = -OH containing
 Hydrogen bonding
1. Hydrogen Bonding 4. Serine + Tyrosine
o R-group with an –OH VS an R-group with a –COOH:  Serine = -OH containing
Amino Acids with –OH in the R-group: SERINE,  Tyrosine = -OH containing
TYROSINE, and THREONINE  Hydrogen bonding
Amino Acids with COOH in the R-group: ASPARTIC ACID
and GLUTAMIC ACID
Example: Serine – Glutamic Acid o PHYSICAL PROPERTIES OF AMINO ACIDS
o R-group with an –OH VS another R-group with an –OH 1. Solubility
Example: Serine – Serine, Serine – Threonine - All amino acids are soluble in water
o R-group with an –SH VS an R-group with an –OH - R-groups of Polar, Uncharged amino acids = more
Amino Acids with –SH in the R-group: CYSTEINE soluble in water / more hydrophilic
Example: Cysteine – Serine POLARS (most soluble) →NONPOLARS with short R
groups → NONPOLARS with long R groups
2. Ionic Interaction
Arrange from most soluble to least soluble in water:
o R-group with a -COOH VS an R-group with an amino group
o Arginine, Alanine, Isoleucine
(NH3+)
Between an acidic (-COOH) amino acid and a basic
(NH3+) amino acid
Acidic Amino Acids: ASPARTIC ACID, GLUTAMIC ACID
Basic Amino Acids: LYSINE, ARGININE, and HISTIDINE
Example: Aspartic Acid – Arginine

3. Hydrophobic interaction of Nonpolar R Groups

o Interaction between Nonpolar R-groups
Nonpolar Amino Acids:
ALANINE, GLYCINE, VALINE, LEUCINE, ISOLEUCINE,
METHIONINE, PROLINE, PHENYLALANINE, and TRYPTOPHAN
Arrangement: Arg → Ala → Ile
Recall: All Girls Pay Large Value In Pretty Thick Make ups
2. Melting points
- Amino acids have a high melting point → Usually
4. Disulfide Bonds
200oC and above
o Between sulfur and sulfur
3. Taste
 Example: Cysteine – Cysteine
- Taste could either be sweet, bitter or tasteless
4. Appearance
If you are studying your amino acids, there are 4 things that you - Amino acids do not absorb visible light and thus are
must know: colorless. However tyrosine, phenylalanine, and
1. How to write the structure tryptophan absorb high-wavelength (250-290 nm)
2. How to abbreviate (3-letter-abbreviation) ultraviolet light. Tryptophan therefore makes the
3. How to classify : major contribution to the ability of most proteins to
 Is it Polar or Nonpolar? absorb light in the region of 280 nm.
 If it is POLAR, is it Charged or Uncharged? 5. Optical property
 If it is CHARGED, is it Positively Charged or - All molecules with a chiral center are optically
Negatively Charged? active → Can rotate the plane – polarized light
4. R-R Interaction either to the right (dextrolevotatory) or to the left
(levotatory)

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Optical property continued….. o By Le Chatelier’s principle, the more acidic the solution =
- Stereoisomerism- D or L amino acids the more positive ions will be formed, the more alkaline
- D isomer if NH2 group is oriented to the right the solution = the more negative ions will be formed
- L isomer if NH2 group is oriented to the left o Substances having this dual nature are amphoteric and are
- Amino acids that occur in protein are all L form often called ampholytes (from “amphoteric electrolytes”)
- D amino acids are found in bacterial cell wall and
some antibiotics  DIFFERENT FORMS OF AN AMINO ACID:
- 2 D-amino acids found in the human brain: D- 1. UNIONIZED FORM
aspartate and D-serine o Carboxyl group → NOT
NEGATIVE (Didn’t donate
6. Ultraviolet Absorption Spectrum of Aromatic Amino Acids any proton)
- UV absorption properties of proteins are o Amino group → NOT
determined solely by Phe, Tyr and Trp. POSITIVE (Didn’t accept any
7. Acid-Base Properties proton)
Amino Group = can become positively charged when it o Unionized form doesn’t exist
accepts a proton in the body → only used in writing the formula of amino
Carboxyl Group = can become negatively charged when it acids
donates a proton
- An amino acids would possess a basic and acidic side, + 2. DIPOLAR or ZWITTERION FORM
and – charge o A form that exists in the body
- If they possess both positive and negative charge, they
o Amino group → POSITIVELY
are termed as amphoteric molecules
- Amino and carboxylic acid groups of amino acids readily
CHARGED
ionize o Carboxyl group → NEGATIVELY
 pK values of the α-carboxylic acid groups CHARGED
(COOH) = usually lie in a small range around 2.2 o Equal amounts of positive
 pK values of the α -amino groups (NH2) = all charge and negative charge =
near 9.4 ELECTRICALLY NEUTRAL (ZERO
CHARGE)
o ACID BASE PROPERTIES OF AMINO ACIDS
o When you place it in an electrical field (electrophoresis),
o The amino and carboxylic acid group readily ionize
 pK (α-COOH) = pH 2.2
it will not move → Stay in the center because it is neutral
 pK (α-NH2 ) = pH 9.4 in charge.
o Your pK value will serve as a way for me to know the charge of the
amino acid, once placed in a solution with known pH. 3. FULLY PROTONATED (pH 1)
In tagalog: o Fully protonated means all hydrogen ions are attached
 Ano ang silbe ni pK? Si pK ang paraan para malaman  Carboxyl group → INTACT
natin ang charge ng amino acid kapag nilagay natin ang  You have another hydrogen attached to the
amino acid sa solution na alam natin ang pH. amino group → POSITIVELY CHARGED
o Physiological pH (7.4) o Fully protonated forms are
 Amino acids are protonated
ALWAYS POSITIVE
 Carboxylic acid groups are in the conjugated base
(carboxylate) form o pH 1 → ACIDIC MEDIUM
 Amino acid can therefore act as both an acid and base
o For each amino acid there is a particular pH, the isoelectric point
(pI), at which the amino acid exists as the neutral zwitterion.
o Below this pH the amino acid exists as a positively charged ion
(cation).
o Above this pH the amino acid exists as a negatively charged ion
(anion).
4. FULLY IONIZED (pH 11)
o Amino acids in solution exists as an equilibrium mixtrium of
neutral molecules and dipolar ions called “ZWITTERIONS” o Fully dissociated means all
o In acid solution (low pH), the zwitterion converts to a positively hydrogen are removed
charged ion.
o In alkaline solution (high pH), the zwitterion converts to a  Carboxyl group →NEGATIVELY
negatively charged ion CHARGED
 Amino group → Not positively
charged

o Fully dissociated forms are ALWAYS NEGATIVE

o pH 11 and above → ALAKALINIC MEDIUM

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Protein and Amino Acid Chemistry Part 1
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 Does an amino acid carry a fixed charge? (Is it always positive?
Is it always negative? Is it always zwitterion?) NO. Amino acids
does not carry a fixed charge.
 What determines the charge of an amino acid? PH OF THE
MEDIUM
o It becomes FULLY PROTONATED if the medium is
VERY ACIDIC (Amino acids become positively
charged)
o It becomes FULLY DISSOCIATED if the medium is
VERY BASIC/ALKALINIC (Amino acids become
negatively charged.)
o In between the two, they become ZWITTERION
o Basis of PROTONIC EQUILIBRIUM of AMINO ACIDS
 TITRATION OF AMINO ACIDS – Protonic Equilibria or Titration of
Amino Acids
o Acid-base titration involves the gradual addition or removal
of protons
o Step by step dissociation of amino acid – Starting from the
fully protonated form (positive form) up to the fully
dissociated form (negative form)
(Picture Above) When you do Protonic Equilibria, you start from the
most positive, pass through the zwitterion form, and you end up
with the most negative form.
o You start at pH 1 → At pH 1, it will be at its most positive
form
o As you increase the pH, eventually it becomes negatively
charged
o At pH 11 and above, it is negatively charged
o Uses:
1. It can predict the charge of the amino acid in a given
solution with known pH
2. It can devise a procedure of separating amino acids
based on their charges
3. From the titration curve of the amino acid, one can know
the regions of buffering power of the amino acid. These
are the relatively flat portions of the curve, extending for
approximately 1 pH on either side of the pK values
o Amino acids contain ionizable groups Predominant ionic
form of these molecules in solution depends on the pH
o Titration of an amino acid illustrates the effect of pH on amino
acid side chains
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o Isoelectric Point (pHI or pI or IpH)
- That pH at which an amino acid bears no net
charge and hence do not move in an electrical
field
- That pH exactly at the midpoint between the pK
values on either side of the zwitterion species
General Rule regarding Isoelectric Points
IpH of neutral amino acids = in the neighborhood of 6.0
IpH of acidic amino acids = very much below 6.0
IpH of basic amino acids = very much above 6.0
o pKa Values
- All amino acids have their own pKa
- Each amino acid has either two or three pKa values These
values differ among the amino acids
- It is a measure of tendency of a group to give up a proton, with
that tendency decreasing tenfold as the pKa increases by one
unit
- The acid strengths of weak acids are expressed as their pKa. The
charge on an amino acids- the algebraic sum of all the positively
and negatively charged groups present- DEPENDS upon the
values of its functional groups and on the pH surrounding
medium.
- Altering the charge on amino acids and their derivatives by
varying the pH facilitates the physical separation of amino acids,
peptides, and proteins.
If we want to know the pH or charge of a specific amino acid in a
solution with known pH, the following should be remembered:
1. When the pH of the solution is lower or less than the pKa
of the amino acid, the amino acid GETS the HYDROGEN,
therefore the amino acid will be PROTONATED
pH solution < pKa = A.A is PROTONATED
2. When the pH of the solution is more or greater than the
pKa of the amino acid, the amino acid will be
DEPROTONATED and the solution GETS the HYDROGEN.
pH solution > pKa = A.A. is DEPROTONATED
In general, whatever is higher between the amino acid and
solution, WINS
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CATION(+1) → ZWITTERION → ANION (-1)
(Picture above is Titration Curve of Glycine)
Example: Glycine (NEUTRAL)
o It has an alpha-carbon, hydrogen, carboxyl group, amino group, and a
side group hydrogen
o Carboxyl group is read as pK1 while the Amino group is read as pK2
o Carboxyl group- pK1 = 2.34; Amino group- pK2 = 9.6
o If glycine is placed in a solution with a pH of 1, the pH is lower than pK1
and pK2 therefore the amino acid will be PROTONATED, it will have a
POSITIVE charge, so + 1
o If glycine is placed in a solution with a pH of 7, the pH is higher than pK1,
therefore the carboxyl group will be DEPROTONATED, BUT the pH is
lower than pK2 so the amino group will be PROTONATED. There will be
both a positive and negative charge in the amino acid making it equal
so the charge is NEUTRAL or 0
o If glycine is placed in a solution with a pH of 12, the pH will be higher on
both pK1 and pK2, the amino acid will become DEPROTONATED.
Therefore it will not gain but the charge is NEGATIVE, so -1.
o In Protonic Equilibra, we write it from the most POSITIVE to the most
NEGATIVE:
pH 1 = +1 ; pH 7 = 0 ; pH 12= -1
pK1 = +1 ; Zwitterion = 0 ; pK2 = -1
o Is pH 7 the Isoelectric point? NO, because even if we put it on a pH of 8,
the charge would still be 0.
o For us to get the ISOELECTRIC POINT, we should use the following
formula:
o To get the IpH, we look for the zwitterion (0) and look for the values
before and after it. So in this example, pK1 and pK2
o pK1= 2.34 ; pK2= 9.6
o IpH is neutral because it is neighborhood around pH 6.0
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Carboxyl group
Amino group
R group
OH
(Picture above is Tyrosine in its Zwitterion Form)
Example: Tyrosine (NEUTRAL)
o Tyrosine has a pK1 of 2.2 ; pK2 of 9.11 ; pKr of 10.07
o If tyrosine is placed in a solution with a pH of 1, the pH is lower than the
pK1, pK2, and pKr. Given this situation, everything will be PROTONATED.
The charge is POSITIVE, so +1
o If tyrosine is placed in a solution with a pH of 5, the pH is higher than
pK1 so the carboxylic group will be DEPROTONATED. The pH is lower
than the pK2 so the amino group will be PROTONATED. Same goes with
the pKr, it will be PROTONATED. The charge will be NEUTRAL or 0
o If tyrosine is placed in a solution with a pH of 9.6, the pH is higher than
pK1 so the carboxylic group will be DEPROTONATED. The pH is higher
than pK2 so the amino group will be DEPROTONATED. The pH is lower
than pKr so the R group is PROTONATED. The charge is NEGATIVE, -1
o If tyrosine is placed in a solution with a pH of 12, the pH is higher than
pK1, pK2, and pK3. Everything will be DEPROTONATED. The charge will
be NEGATIVE, -2
o Now we write it from the most POSITIVE to the most NEGATIVE:
pK1 = +1 ; Zwitterion = 0 ; pK2 = -1 ; pKr = -2
o What is the Isoelectric point? First, look for the 0 charge (zwitterion)
then look for the pK value before and after it, for this case what’s before
and after the 0 charge is pK1 and pK2
o So we add pK1 and pK2 and divide it by 2
o IpH is neutral because it is neighborhood around pH 6.0
(Look at the Titration Curve of Glycine at the LEFT)
o Notice that the dots (pK values) are not connected by a straight line →
Somehow flattened around the pK values → Indicate that there is a
buffering capacity of the amino acids at the pK values
 Amino acids are good buffers → They are good buffers at
their pK values
o There is no buffer effect at the isoelectric point → Straight/Steep Line
BESHYWAP 9
 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 1
Dr. Alcantara

(Picture Above is Titration of Glutamic Acid)

Example: Glutamic Acid (ACIDIC)
o Glutamic Acid has a pK1 of 2.2 ; pKr of 4.3 ; pK2 of 9.7 (Rounded-off
kaya mas mataas)

o If glutamic acid is placed in a solution with a pH of 1, the pH is lower

than the pK1, pKr, and pK2. Therefore, everything will be PROTONATED.
The charge is POSITIVE, +1
o If glutamic acid is placed in a solution with a pH of 3.5, the pH is higher
than pK1 so the carboxyl group will be DEPROTONATED. On the other
hand, pKr and pK2 will become PROTONATED. The charge is 0
o If glutamic acid is placed in a solution with a pH of 7, the pH is higher
than pK1 and pKr so the carboxyl group and R group respectively will
become DEPROTONATED, while the pK2 will become PROTONATED.
The charge will be NEGATIVE, -1
o If glutamic acid is placed in a solution with a pH of 12, the pH is higher
in all pK values. So everything will be DEPROTONATED. The charge will
become NEGATIVE, -2
o Write it from the most POSITIVE to the most NEGATIVE:
pK1 = +1 ; Zwitterion = 0 ; pKr = -1 ; pK2 = -2
o To compute for the Isoelectric point, you add the pK values before and
after the zwitterion (the 0). So in this case we add pK1 and pKr
(Picture Above) HOW TO USE THE pK VALUE TABLE:
 If you look at the pK value:
 pK1 = ALPHA-CARBOXYL GROUP
 pK2 = ALPHA-AMINO GROUP
 pKr = R-GROUP
 ↓ pK value – it will dissociate first
 In the case of BASIC AMINO ACIDS, pK of Alpha-Amino
Group is ↓ than pK of the R-Group = Alpha-Amino Group
dissociates first
 In the case of your ACIDIC AMINO ACIDS, pK of R-Group is
↓ than pK of Alpha-Amino Acids = R-Group dissociates first
 For all the amino acids, the ALPHA-CARBOXYL GROUP has
the lowest pK value → It is always the Alpha-Carboxyl
Group that dissociates first.
ADDITIONAL NOTES ABOUT TITRATION FROM PACIS’ TRANS
I added information from Pacis’ trans on the next pages for a more in depth
discussion on titration 

o pK1 = 2.2 ; pKr = 4.3

o IpH is acidic because it is very much below 6.0

#GrindNation
“Strength In Knowledge” BESHYWAP 10
 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 1
Dr. Alcantara
A. TITRATION OF NEUTRAL AMINO ACIDS (Example: Alanine)
 During titration with a strong base (such as NaOH) Alanine loses
two protons in a stepwise fashion
 In a strongly acidic solution (pH 1) Alanine is present mainly in
the form in which the carboxyl group is uncharged or undissociated
Alanine has a net charge of +1 (Ammonium group is protonated)
 Lowering H+ concentrations Results in carboxyl group losing its
proton to become a negatively charged carboxylate group (TAKE
NOTE: Protons are first lost from the group with the lowest pKa)
 At this point, Alanine has no net charge and is electrically neutral
pH at which this occurs is called ISOELECTRIC POINT (Because
there is no net charge at the isoelectric point) → Amino acids are
least soluble at this pH.
 Isoelectric point for alanine may be calculated as follows:
 The IpH of Alanine is therefore 6.0
 As the titration continues, the ammonium group loses its proton
Leaving an uncharged amino group Alanine then has a net
negative charge because of the carboxylate group
(Picture Above) TITRATION OF ALANINE (NEUTRAL AMINO ACID)
 When you titrate, you always start with the most positive form. At
pH 1 = Alanine is +1 (Refer to picture above -- Structure on the LEFT:
MOST POSITIVE FORM OF ALANINE)
 When you titrate, you add Sodium Hydroxide (NaOH). When you add
NaOH to a medium that started from pH 1, you will have a higher pH.
 pH 7 = Neutral
 ↓ pH 7 = Acidic
 ↑ pH 7 = Basic
 NaOH has an –OH that is negatively charged → It will attract a
hydrogen ion.
 It has 2 hydrogen ions that it can attract. Which one will it
attract first?
- It depends on the strength of the acids → DISSOCIATION
CONSTANT → The stronger the acid, the higher the
dissociation constant. The weaker the acid, the lower is the
dissociation constant. → This means that if the acid is
stronger, it will dissociate faster.
- In this case, the CARBOXYL GROUP (-COOH) will donate the
proton first. The dissociation of this one is designated as
pK1.
- The higher the dissociation constant, the lower the pK value.
(If ↓pH = more acidic → If more acidic = ↑ dissociation
constant → And if ↑ dissociation constant = ↓pK value)
- pK1 = always represents the dissociation of the ALPHA-
CARBOXYL GROUP (pH 2.3 for Alanine)
#GrindNation
“Strength In Knowledge”
- Once the alpha-carboxyl group has dissociated (COOH
becomes COO-), this now becomes your ZWITTERION (Refer
to picture on the left -- Structure in the MIDDLE:
ZWITTERION)
- As you add more base, the hydrogen in the AMINO GROUP
will now dissociate, and you designated that as pK2.
- pK2 = always represents the dissociation of the ALPHA-
AMINO GROUP (pH 9.7 for Alanine)
- Once the alpha-amino group has dissociated (NH3+
becomes NH2), Alanine now becomes -1. (Refer to picture
above -- Structure on the RIGHT: MOST NEGATIVE FORM OF
ALANINE)
pK VALUES OF ALANINE:
 What does these pK values represent?
pH of pK1 = 2.3 This means that if the pH of the medium is at
exactly 2.3, the form of Alanine is 50% +1 and 50% Zwitterion.
- At exactly the pK value, half of it will be fully protonated, half
of it will be dissociated.
- When you go above 2.3, there will be more of Zwitterion
The predominant form of Alanine is Zwitterion.
- When you go below 2.3, there will be more of +1 The
predominant form of Alanine is +1.
pH of pK2 = 9.7 This means that if the pH of the medium is at
exactly 9.7, the form of Alanine is 50% Zwitterion and 50% -1.
- When you go above 9.7, there will be more of -1 The
predominant form of Alanine is -1.
- When you go below 9.7 and above 2.3, there will be more of
Zwitterion The predominant form of Alanine is
Zwitterion.
ISOELECTRIC POINT (IpH):
 pH wherein an amino acid is 100% Zwitterion
 This means that at pH 6, Alanine is 100% Zwitterion.
 Rule regarding Isoelectric Points:
 Neutral Amino Acids = Have an IpH around 6 (6± = 5-7)
 Acidic Amino Acids = Have an IpH ↓ 5
 Basic Amino Acids = Have an IpH ↑ 7
RECALL (ALANINE):
 pK1 = 2.3
 pK2 = 9.7
 IpH = 6.0
o What is the predominant form of Alanine if it is placed in a medium
with pH 4?
- ZWITTERION → Because 4 is above 2.3 (pK1) and below 9.7 (pK2)
o What are the exact values wherein Alanine is a good buffer?
- 2.3 and 9.7 → Amino acids are good buffers at their pK values.
BESHYWAP 11
 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 1
Dr. Alcantara
B. TITRATION OF ACIDIC AMINO ACID (Example: Glutamic Acid)
(Picture Above) TITRATION OF GLUTAMIC ACID (ACIDIC AMINO ACID)
 Difference is that, you have another carboxyl group at the R-group →
There will now be 3 groups that could dissociate.
 At pH 1 = Glutamic Acid is +1 → Fully Protonated Form (Refer to
picture above – 1st Structure on the Left: MOST POSITIVE FORM OF
GLUTAMIC ACID)
 When you add NaOH, the ALPHA-CARBOXYL GROUP ALWAYS
DISSOCIATE FIRST.
 It is designated as pK1. In the case of Glutamic Acid, pK1 =
2.2
 When the alpha-carboxyl group gets dissociated (-COOH
becomes COO-), Glutamic Acid now becomes ZWITTERION
(Refer to picture above – 2nd Structure from the Left:
ZWITTERION)
 As you add more base (NaOH), it can either be the R-Group or the
amino group that will get dissociated.
 Between those 2, the CARBOXYL GROUP from the R-
GROUP will now get dissociated (Remember: Acids
dissociate first.)
 The dissociation of your R-Group is designated as your pKr.
In the case of Glutamic Acid, pKr = 4.3.
 When the carboxyl group of the R-group gets dissociated
(-COOH becomes COO-), Glutamic Acid now becomes -1
(Refer to picture above – 3rd Structure from the Left: -1)
 The last one that will get dissociated is the ALPHA AMINO GROUP.
 It is designated as pK2. In the case of Glutamic Acid, pK2 =
9.6
 When the amino group gets dissociated (NH3+ becomes
NH2), Glutamic Acid now becomes -2 (Refer to picture
above – 4th Structure from the Left: -2)
 For all the amino acids, ONLY THE ACIDIC AMINO ACID CAN BECOME
-2 → Because it is only them who have 2 carboxyl groups.
 When you compute for the IpH of Glutamic Acid, you only add the pK
values before and after the zwitterion ion. In this case, you only add
pK1 and pKr.
 IpH of Glutamic Acid is 3.2
 RECALL: ACIDIC Amino Acids should have an IpH ↓ 5.
 If you think you got a wrong pK value, you will know that
you are really wrong if it contradicts the rule.  (For
example, you got an IpH value of 7 even if it is an acid
amino acid.)
#GrindNation
“Strength In Knowledge”
RECALL (GLUTAMIC ACID):
 pK1 = 2.2
 pKr= 4.3
 pK2 = 9.6
 IpH = 3.2
o What is the predominant form of Glutamic Acid if it is placed in a
medium with pH 5?
 -1 → Because 5 is above 4.3 (pKr) and below 9.6 (pK2)
o What are the exact values wherein Alanine is a good buffer?
 2.2, 4.3 and 9.6 → Amino acids are good buffers at their pK
values. (No matter how many pK values there are, they are
all buffering zones.)
C. TITRATION OF BASIC AMINO ACIDS (Example: Lysine)
(Picture Above) TITRATION OF LYSINE (BASIC AMINO ACID)
 You have another amino group at the R-group → There will be 3 groups
that could dissociate.
 At pH 1 = Lysine is +2 → Fully Protonated Form (Refer to picture above
– 1st Structure on the Left: MOST POSITIVE FORM OF LYSINE)
 When you add NaOH, the ALPHA-CARBOXYL GROUP ALWAYS
DISSOCIATE FIRST.
 It is designated as pK1. In the case of Lysine, pK1 = 2.18
 When the alpha-carboxyl group gets dissociated (-COOH
becomes COO-), Lysine now becomes +1 (Refer to picture
above – 2nd Structure from the Left: +1)
 As you add more base (NaOH), it can either be the R-Group or the amino
group that will get dissociated.
 Between those 2, the ALPHA-AMINO GROUP from the R-
GROUP will now get dissociated
 It is designated as pK2. In the case of Lysine, pK2 = 8.95
 When the amino group gets dissociated (NH3+ becomes
NH2), Lysine now becomes ZWITTERION (Refer to picture
above – 3rd Structure from the Left: ZWITTERION)
 The last one that will get dissociated is the AMINO GROUP OF THE R-
GROUP.
 The dissociation of your R-Group is designated as your pKr.
In the case of Lysine, pKr = 10.93.
 When the Amino Group of the R-Group gets dissociated
NH3+ becomes NH2) Lysine now becomes -1 (Refer to
picture above – 4th Structure from the Left: -1)
 When you compute for the IpH of Lysine, you only add the pK values
before and after the zwitterion ion. In this case, you only add pK2 and
pKr.
BESHYWAP 12
 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 1
Dr. Alcantara

 IpH of Lysine is 9.94

 RECALL: BASIC amino acids should have an IpH ↑7

REFERENCES

 Biochemistry Lecture Guide (2018)

 PPT
 Lea Pacis Trans
 Gradelifting Fairies Trans
 Dr. Alcantara Recordings

#GrindNation
“Strength In Knowledge” BESHYWAP 13