BIOCHEMISTRY

Protein and Amino Acid Chemistry Part 2

Dr. Alcantara
 PEPTIDES  VASOPRESSIN
Definitions  Also known as Antidiuretic Hormone (ADH)
 Peptides  Secreted in response to low pressure or high
- Has a lower molecular weight Na+ concentration
- Typically consists less than 50 amino acids  Act by stimulating the kidneys to retain water
 Protein Concentrated urine
- Molecules with more than 50 amino acids  CYS-TYR-PHE-GLN-ASN-CYS-PRO-ARG-GLY-NH2
- Consists of one or more polypeptide chains
 Oligopeptides 3. Met-Enkephalin and Leu-Enkephalin
- Polymers consisting of 2 to 10 amino acids (Less than 10, Around 2) o Belong to a group of peptides called OPIOID CELLS
o Dipeptide = 2 amino acids o Found predominantly in the nervous tissue cells
o Tripeptide = 3 amino acids o OPIOID PEPTIDES
 Polypeptides  Molecules that relieve pain and produce pleasant
- Have more than 10 amino acid residues (More than 10, Below 50) sensations
 Bind to receptors in certain cells of the brain and induce
 Peptides perform prominent roles in the neuroendocrine system as analgesia (deadening of pain sensations)
hormones, hormone-releasing factors, neuromodulators or  Represent one of the body’s own mechanisms for control
neurotransmitters. of pain
o Enkephalin receptors also bind to morphine, heroin and other
 PEPTIDES WHICH HAVE BIOLOGICAL SIGNIFICANT ACTIVITIES: addicting opiate drugs (although these are not peptides)
1. Glutathione o Pentapeptides that only differ only in their C-terminal amino
 Significant for its antioxidant property. acid residues
 Met-enkephalin = TYR-GLY-GLY-PHE-MET
 Glutathione must be reduced for it to function as an
 Leu-enkephalin = TYR-GLY-GLY-PHE-LEU
antioxidant.
o ϒ-glutamyl-L-cysteinylglycine
4. Atrial Natriuretic Factor
o Contains an unusual ϒ-amide bond (wherein the ϒ-carboxyl
o Peptide with 28 amino acid residues
group of the glutamic acid residue, not the α-carboxyl group,
o Produced by specialized cells in the heart and the nervous
contributes to the peptide bond)
system
o Found in almost all organisms
o Stimulates the production of a dilute urine (an effect opposed
o Involved in many important biological processes like:
 Protein and DNA synthesis to that of vasopressin)
 Drug and environmental toxin metabolism
 Amino acid transport 5. Substance P and Bradykinin
 *REDUCING AGENT reducing component is o Stimulate the perception of pain Opposed by Opioid
the –SH group of the cysteine residue (reduced Peptides
glutathione GSH) o Pain = protective mechanism in animals that warns of tissue
o Protects cells from the destructive effects of oxidation damage
Reacts with substances such as peroxides (R-O-O-R, by
products of O2 metabolism) 6. Glucagon
o In red blood cells, Hydrogen Peroxide oxidizes the Iron of o Pancreatic Hormone
haemoglobin to its ferric form (Fe+3) o Has 29 amino acid residues
 METHEMOGLOBIN = product of this reaction o Opposes the action of Insulin
 Glutathione protects against the formation of
methemoglobin by reducing H2O2 in a reaction 7. Corticotropin (ACTH)
catalysed by the enzyme glutathione peroxidase o Contains 39 amino acid residues
o Hormone of the Anterior Pituitary Gland Stimulates the
2. Oxytocin and Vasopressin Adrenal Cortex
o Differ only by two residues
o Both contain 9 amino acid residues 8. L-Aspartylphenylalanine Methyl Ester
o Produced by the cleavage of polypeptide precursors within o Commercially synthesized dipeptide
different specialized cells in the hypothalamus o Artificial sweetener better known as ASPARTAME or
o After synthesis, they are transported down the nerve tracts NUTRASWEET
into the Posterior Pituitary Gland (where they are stored) o Promotes neurotoxins, causing neural degeneration
o Each peptide is secreted in response to specific signals from
the hypothalamus
 OXYTOCIN
 Stimulates contraction of uterine muscles
during childbirth
 Stimulates ejection of milk by the mammary
glands during lactation
 In males, Oxytocin may have a regulatory role in
the synthesis of Testosterone
 CYS-TYR-ILE-GLN-ASN-CYS-PRO-LEU-GLY-NH2

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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
 STRUCTURE OF PROTEINS
A. Formation of the Peptide Bond
Example, you want to form the dipeptide Alanyl-Serine:
 In the formation of the peptide bond:
- You need to react 1 carboxyl and 1 amino group.
- You remove water → DEHYDRATION REACTION
*-OH comes from your COOH
*H+ come from your NH2
 Of all the biochemical reactions of amino acids, the most important is the
formation of peptide bonds
 Amino acid can be polymerized to form chains
 Can be represented as a CONDENSATION REACTION (elimination of a water
molecule) Results to formation of CO-NH linkage
 CO-NH Linkage = amide linkage known as the PEPTIDE BOND
 After peptide bonds are incorporated into a peptide, an individual amino
acids (monomeric units) is now referred to as AMINO ACID RESIDUES
 Polypeptides are linear polymers Each amino acid participates in two
peptide bonds and is linked to its neighbors in a head-to-tail fashion rather
than forming branched chains
 Residues at the two ends of the polypeptide each participate in just
one peptide bond
 Residue with a free amino group (leftmost residue) = AMINO
TERMINUS/N-TERMINAL
 Residue with a free carboxylate group (right) = CARBOXYL
TERMINUS/C-TERMINAL
 Variations in the length and the amino acid sequence of polypeptides
contribute to the diversity of the shape and biological functions of
proteins
o When you name the chain, you always start with the LEFT SIDE.
o On the left side or at the start of the chain, you always have a free amino
group Amino group that did not participate in peptide bond
formation N-TERMINAL AMINO ACID
o On the right side of the chain or at the end of the chain, you always have
a free carboxyl group Carboxyl group that did not use C-
TERMINAL AMINO ACID
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 EVIDENCES THAT A PEPTIDE BOND EXIST IN PROTEINS
1. Positive reaction to the Biuret test
o Biuret Test = general test for proteins
o For as long as you have at least 2 peptide bonds, it will react
with the Biuret Test.
2. Relatively few titratable amino and carboxyl functions
o There will be relatively few titratable amino and carboxyl
functions as you form the peptide chains
o For every amino acid, there will be 1 amino group and 1
carboxyl group that is titratable For every amino acid,
there is at least 2 titratable groups.
o When you form the bonds, you always use either the amino
group and carboxyl group You expect to the number of
titratable groups to be less.
ALANYL-SERYL-LYSYL-ASPARTYL-METHIONINE
o When the amino acids are taken separately:
 Alanine = 2 titratable groups (Carboxyl Group and
Amino Group) 
 Serine = 2 titratable groups (Carboxyl Group and
Amino Group) 
 Lysine = 3 titratable groups (Carboxyl Group, Amino
Group, and Amino Group at the R-Group) 
 Aspartic Acid = 3 titratable groups (Carboxyl Group,
Amino Group, and Carboxyl Group at the R-Group)
 Methionine = 2 titratable groups (Carboxyl Group and
Amino Group) 
 Total Titratable Groups = 12

o When you form the chain:
 Total Titratable Groups = 4
 Why 4?
- 4 Peptide Bonds X 2 (2 Titratable Groups Used = 1
Carboxyl Group + 1 Amino Group) = 8
- 12 Total Titratable Groups (when amino acids are
taken separately) – 8 = 4
Addition Notes: To know the number of titratable group, look
whether there is a BASIC or ACIDIC amino acid present because
their side chains contain an extra amino or carboxyl group.
Exercise:
1.) Peptide contains 30 amino acids, all 30 are POLAR uncharged.
How many titratable amino and carboxyl groups would you have?
Ans: 2 because polar uncharged do not have any extra carboxyl
and amino. It would only be the N and C-terminal.
2.) Peptide contains 30 amino acids, all 28 are POLAR uncharged
and 2 are POSITIVELY charged. How many titratable amino and
carboxyl groups could you have? Ans: 4 because the positively
charged contain an extra 2 amino groups.
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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
3. Hydrolyzed by enzymes which are specific for the peptide bond Forms of Beta Pleated sheet
o Enzymes are specific when it comes to the type of bond a. Antiparallel beta-sheet
that react on. - Neighboring H-bonded polypeptide chains run in
opposite direction
4. X-ray diffraction studies proved that existence of peptide linkages - More stable
between amino acids in hemoglobin and myoglobin
5. Synthesis of insulin

 LEVELS OF PROTEIN STRUCTURES

A. Primary Structure
- Refers to:
 quantitative amino acid composition
 sequence of amino acids
 number of peptide chains
- Most abundant amino acids in proteins:
 Leu, Ala, Gly, Ser, Val and Glu
- Rarest in proteins are:
 Trp, Cys, Met and His
- The backbone of a protein refers to the atoms that participate in
the formation of peptide bonds
- The 3-dimensional shape of a folded polypeptide is result of the
interactions among the R groups
b. Parallel beta-sheets
- H-bonded chains extend in the same direction
B. Secondary Structure
- Due to the formation of hydrogen bonds between peptide bonds
- Two types:
o Coils or Helices
 Intrachain hydrogen bonding
o Sheets or Pleats
 Interchain hydrogen bonding

1. Alpha-helix
- Stabilized by inter-residue hydrogen bonds formed bet.
the H atom attached to a peptide N and the carbonyl O
atom.
- Each peptide bond participates in hydrogen bonding
- An alpha-helix forms spontaneously as it is the lowest
energy, most stable conformation for a polypeptide
chain.
- There are 3.6 amino acid residues per turn with a pitch
(distance bet. corresponding pts. per turn) of .54 nm (5.4 A); Factors that destabilize the beta-pleated sheets:
spacing per residue is .15 nm (1.5 A) 1. Presence of adjacent similarly charged amino acids
- Amino acid R groups extend outward from the helix 2. Presence of adjacent bulky R groups
These are the branched amino acids:
Val, Ile, Leu, Pro, Met
Factors that destabilize the alpha-helix:
C. Tertiary Structure
1. Presence of adjacent similarly charged amino acids
- 3-dimensional structure protein conformation
2. Presence of adjacent bulky R groups
- Results from folding of a polypeptide
- These are the branched amino acids:
- Indicates how 2o structures- helices, sheets, bends, turns and loops
Val, Ile, Leu, Pro, Met
assemble to form domains
3. Presence of Proline
- The 3-dimensional shape of folded polypeptide is result of the
- contains rigid ring that prevents the N-C bond from
interactions among the R-groups
rotating
- no N-H group available to form intrachain hydrogen
Bonds Recall:
How do you call the individual amino acid in primary structures?
Ans: Residue. How about in the secondary structures? Ans:
2. Beta-pleated Sheet
Residue. How do you call the individual amino acid in tertiary
- Second most commonly occurring protein 2o structure
structure? Ans: Domains
- Formed when 2 or more almost fully extended polypeptide
chains lie side by side
- H-bonds are interchain
- R groups 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

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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
Features: o Protein Purification
- Amino acid residues that are distant from each other - Proteins are separated, isolated from other substances
in the 1o structure come into close proximity when the - Precedes protein analysis
polypeptide folds - Fractional procedures- to eliminate selectively the other
- When polypeptide folds, proteins become more components of the mixture so that only the required protein
compact; most water molecules are excluded from the remains
proteins’ interior making interactions bet. polar and Characteristics of proteins and other biomolecules that are
nonpolar AA possible used in the various separation techniques:
- Large globular proteins (> 200 AA) often contain
several compact units called domains Characteristics Procedure
- Domains are structurally independent segments that Paper chromatography, Thin layer
have specific functions (e.g. binding an ion) Solubility
chromatography
Ion Exchange chromatography,
Types of Interaction that stabilize tertiary structures: Charge
electrophoresis
- Hydrophobic interactions Polarity Hydrophobic interaction chromatography
- Electrostatic interactions (salt bridges) Gel filtration chromatography, SDS PAGE,
- Hydrogen bonds Size
Ultracentrifugation
- Covalent bonds (disulfide bonds) Binding
Affinity chromatography
Specificity
D. Quaternary Structure
- Quaternary structure describes the characteristic manner in which o Protein Solubility
the individual , folded polypeptide chains fit each other or interact - Utilize differences in protein solubility, which is a complex function
with one another so that they can act as one single molecule of pH, temperature, salt concentration, and other factors
- Exhibited only by proteins containing more than one polypeptide - The solubility of proteins is generally lowered at high salt
chain concentrations, an effect called “salting out”
- Most proteins with molecular masses above 100 kD, consist of more - The addition of a salt in the right amount can selectively precipitate
than one polypeptide chain some proteins, while others remain in solution
 Each polypeptide component is called a subunit - Ammonium Sulfate ((NH4)2SO4) is often used for this purpose
- Oligomeric proteins are those with more than one subunit because of its high solubility in water
 Subunits maybe identical or different
 Protomers are identical subunits o Chromatography
- Mobile phase: dissolved substances
Interactions that hold subunits together: - Liquid phase: percolated through a column containing porous solid
- Hydrophobic interactions- principal force that hold matrix
sub-units together - The retarding force depends on the properties of each solute
- Electrostatic interactions- contribute to the proper
alignment of sub-units Types of Chromatography
- Hydrogen bonds o Uses strips of filter paper as the
- Interpolypeptide disulfide binds 1. Paper chromatography
stationary phase
o Powerful chromatographic technique
Recall: o For high resolution separation of
What stabilizes primary structure? Ans: Peptide. What stabilizes proteins, peptides and amino acids
secondary alpha structures? Ans: Hydrogen intrachain. How 2. High Performance
o Principle of separation may be based on
about secondary beta pleated sheets? Ans: Hydrogen interchain. Liquid chromatography
the CHARGE, SIZE, or HYDROPHOBICITY
How about tertiary structures? Ans: All bonds. How about (HPLC)
of proteins
quaternary structures? Ans: All bonds but mainly by hydrophobic o Most commonly the peptides are bound
bonds. to a reversed phase column
o Together with electrophoresis-
separates substances in terms of the
basis of CHARGES
o Charged particles
- Bind to oppositely charged groups
- Been immobilized on the matrix
o Most frequently used matrix materials
- Cellulose and agarose
3. Ion Exchange
o Anion Exchangers
chromatography
- Anions bind to cationic groups
 PROTEIN ANALYSIS - Most frequently used matrix with
To study a protein in detail, proteins must be separated from other attached diethylaminoethyl
proteins. The protein must first be purified. The amino acid sequence can (DEAE) groups
be determined by the partial breakdown into fragments followed by
stepwise analysis proceeding from one end chain to the other

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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
Ion Exchange chromatography continued…
o Cationic Exchangers
- Cations bind to anionic groups
- Most frequently used matrix
bearing carboxymethyl (CM)
groups
o Separate in the basis of POLARITY
o Separate proteins based on tendency to
associate with stationary matrix
- Coated with hydrophobic groups
- E.g. phenyl sepharose, octyl
sepharose
4. Hydrophobic
o Matrix material is lightly substituted with
interaction
octyl or phenyl groups
chromatography
o Non-polar group surface proteins
- Interact with the solvent
o Both types of groups
- Excluded by polar solvent
o Eluant aqueous buffer
- Decreasing salt concentration
o Separates on the basis of SIZE
o Also called size exclusion or molecular
sieve chromatography
o Molecules are separated according to
their size and shape
o Large molecules CANNOT penetrate into
the pores of the Dextran beads (has
small openings) while small molecules
5. Gel filtration
CAN penetrate.
chromatography
o Expected result:
- The first ones to come out of the
column are the big molecules
- The last ones that will come out
are the smaller molecules
o Proteins are considered as large
molecules so it is expected that they will
come out first of the column
o A molecule (ligand) that specifically
binds to the protein of interest is
6. Affinity covalently attached to an inert matrix
chromatography o Desired protein binds to the immobilized
ligand, whereas other substances are
washed through the column with buffer
o Typically carried out in agarose or
polyacryl-amide gels.
o The molecular separations are based on
sieving effects as well as electrophoretic
mobility
o SDS-PAGE Electrophoresis – a form of
polyacrylamide gel electrophoresis
7. Gel Electrophoresis (PAGE) wherein the detergent sodium
dodecyl sulfate (SDS) is used to denature
proteins.
- used to separate proteins on the
basis of their molecular weights.
Larger molecules are retarded in
the gel matrix, whereas the
smaller ones move more rapidly
 PROTEIN SEQUENCING
The protein must be broken down into fragments small enough to be
individually sequenced. The primary structure of the intact protein is then
reconstructed from the sequences of overlapping fragments
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Steps involved in Protein Analysis:
A. Determination of the amino acid composition of the protein
B. Determination of N and C terminals
C. Determine the amino acid sequence
D. Check numbers of polypeptide chains
E. Identification of component amino acids by chromatography or
electrophoresis
A. Determination of the amino acid composition of the protein
o To determine an amino acid composition, it is necessary to breakdown
the polypeptide chain into its constituent amino acids, separate the
resulting amino acids according to the type and measure the quantities
of each amino acids
o Remember that amino acids are joined by the peptide bonds,
therefore, the bonds will be cut → AA will be separated from one
another (HYDROLYSIS)
a. Acid Hydrolysis- uses hydrochloric acid
b. Basic Hydrolysis- use of NaOH
c. Enzymatic Hydrolysis- use of enzymes
Acid Hydrolysis of Proteins:
o Heat proteins at 100-120oC for 10 to 24 hours in 6N HCl
o Disadvantages:
- All tryptophan and variable amounts of Ser and Thr are destroyed
- Glutamine and Asparagine are deamidated to Glu and Asp
 DEAMIDATION (Note that it is deamiDation)
 Removes AMIDE group at R position
 Converts the amide group to a COOH → Asparagine to
Aspartic acid OR Glutamine to Glutamic acid
- Glutamic acid undergoes intramolecular dehydration (removal of
water within glutamic acid) to form pyrolidone-5-carboxylic acid
- Other amino acids may undergo intermolecular dehydration
forming cyclic anhydrides
Basic Hydrolysis of Proteins:
o Incubate proteins with 2 to 4 N NaOH at 100oC for 4 to 8 hrs
o Disadvantages:
- Cysteine, cystine, Ser, Thr and Arg are decomposed in the process
- Other amino acids may be partially destroyed by DEAMINATION
(Note that it is deamiNation)
 Removal of amino group
 What is left after deamination is called KETO-ACID
 Each amino acid has its corresponding keto-acid
Amino acid Its Keto-Acid
Alanine Pyruvic acid
Aspartic acid Oxaloacetate
Glutamic acid Α-keto glutarate
- Racemization of amino acids occur
o Principal use of basic hydrolysis: To estimate Trp
Why not ONLY Enzymatic Hydrolysis?
It is quite extensive. It has specific bonds to cut. Unlike acid/basic hydrolysis
by which all the peptide bonds are already being cut, in ENZYMATIC
hydrolysis, they can only act on a particular bond. A lot of different enzymes
are required to cut the bonds in the protein.
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Protein and Amino Acid Chemistry Part 2
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Enzymatic Hydrolysis of Proteins Take note whether you are cleaving at the carbonyl or amino side
Digestive enzymes are usually in their inactive form called ZYMOGENS and
are only activated when there is a need for them.

Produced by the stomach HCl

PEPSIN (Pepsinogen if inactive)
activates pepsinogen to pepsin.
EXAMPLE:
Cleaves off peptide bonds whose
carbonyl function is contributed by Met-Leu-Phe-Arg-Ser-Ala-Pro-Glu-Asp-Asn-His-Trp-Met-Val-Lys-Gly-Ile-
Phe, Tyr, Trp, Leu, Glu, Gln Tyr-Glu-Ala-Thr

Most important because it activates a. If acted by Pepsin, these will be the following fragments:
the other enzymes like 1. Met-Leu
chymotrypsin, carboxypeptidase, 2. Phe
elastase etc. 3. Arg-Ser-Ala-Pro-Glu
4. Asp-Asn-His-Trp
TRYPSIN Produced by pancreas Enterokinase 5. Met-Val-Lys-Gly-Ile-Tyr
activates Trypsinogen to Trypsin 6. Glu
7. Ala-Thr
Cleaves off peptide bonds whose
carbonyl function is contributed by b. If acted by Statphylococcus aureus V8 protease, these will be the
Lys and Arg following fragments:
Trypsin converts Chymotrypsinogen 1. Met-Leu-Phe-Arg-Ser-Ala-Pro-Glu-Asp-Asn-His-Trp-Met-Val-
to Chymotrypsin Lys-Gly-Ile-Tyr-Glu
2. Ala-Thr
CHYMOTRYPSIN
Cleaves off peptide bonds whose
carbonyl function is contributed by Note that it did not cut between Glu-Asp because Asp is a polar
Phe, Trp and Tyr amino acid. The one on the amino side should be hydrophobic just
Cuts the last bond like Ala
CARBOXYPEPTIDASE
Cleaves off C-terminal amino acids
Cuts the first bond c. If acted by Thermolysin, these will be the following fragments:
AMINOPEPTIDASE 1. Met
Cleaves off N-terminal amino acids
Heat stable bacterial protease 2. Leu-Phe-Arg-Ser-Ala-Pro-Glu-Asp-Asn-His-Trp-Met
3. Val-Lys-Gly
THERMOLYSIN Cleaves off peptide bonds whose 4. Ile-Tyr-Glu-Ala-Thr
amino function is contributed by
Leu, Val and Ile B. Determination of N and C terminals
Found in certain plants like papaya
 Sanger’s reaction
PAPAIN Cleaves off peptide bonds whose  Dansyl chloride reaction
N-terminal reaction
carbonyl function is contributed by  Edman’s reaction
Lys, Leu, Arg, Gly  Reaction with aminopeptidase
Derived from pineapple  Reduction with lithum borohydride
C-terminal reaction  Hydrazinolysis
BROMELAIN Cleaves off peptide bonds whose  Reaction with carboxypeptidase
carbonyl function is contributed by
Lys, Ala, Tyr, Gly

Chemical Reagents that Promote Peptide Bond Cleavage at Specific Residue:

Cleaves peptide bonds whose
carbonyl part is contributed by
CYANOGEN BROMIDE
Methionine

Cleaves peptide bonds whose

carbonyl part is contributed by Glu,
STAPHYLOCOCCUS AUREUS V8
particularly where the amino part is
PROTEASE
contributed by a non-
polar/hydrophobic amino acid

In tagalog:
Pag ang peptide chain ay may Glutamic acid at pagkatapos ng
isang Glutamic acid ay isang hydrophobic amino acid, mag cl-
cleave ka sa carbonyl part ng glutamic acid.

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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
C. Determine the amino acid sequence 4. Reaction with aminopeptidase
Methods in Determining the N-terminal Amino Acid in a Polypeptide Chain:
1. Sanger’s Reaction

Cuts the first bond only, separating the N-terminal from the rest of
the chain

Methods of Determining C-Terminal Amino Acid in Peptide Chain

1. Reduction by lithium borohydride
All bonds are cut with the last amino acid (C-terminal) converted
into a primary alcohol.
Upon subjecting to hydrolysis, all the peptide bonds will be cut while
the N-terminal amino acid is still attached to DNP, thus, the N-
terminal is readily identified.

2. Dansyl Chloride Reaction

2. Hydrazinolysis
All the bonds is cut and converted to hydrazides except for the last
amino acid

All the other amino acids can still be recovered but you will not know
which one is 2nd, 3rd, or 4th. Only the amino acid on the N-terminal
side will be identified. 3. Reaction with carboxypeptidase
Only cuts the last peptide bond
3. Edman’s Reaction
Used to SEQUENCE amino acids

D. Check numbers of polypeptide chains

 If there are two or more peptide chains separate them by oxidation
with performic acids or reduction with mercaptans (destroy disulfide
bonds) and use of denaturing agents (breaks off non-covalent bonds)
 OXIDATION- Using performic acid. Leads to the formation of cysteic
Only the 1st bond is cut. The rest of the chain is intact. The N-
acids
terminal amino acid is going to be separated now as
 REDUCTION- Using mercaptoethanol, thioglycollate and sodium
Phenylthiohydantoin (PTH) plus you get the rest of the chain intact
borohydride. Disulfides are reduced to sulfhydrylform
minus the N-terminal

In Edman’s reaction, you are separating the amino acids one by one
starting from the N-terminal side. This is a way of determining the
sequence of the amino acids in the chain. Sequenator is a machine
that does a series of Edman’s reaction in a shorter period of time.

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Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
E. Identification of component amino acids by chromatography or  Ion Exchange Chromatography
electrophoresis - Separation on the basis of differences in net charges of amino acids
 Paper Chromatography at different pH conditions with the use of ion-exchange resins which
- Separation on the basis of differences in solubility of amino acids are either poly-anions or poly-cations
between two immiscible solvents - Applicable to low molecular weight polypeptides as well as amino
- Principle: acids
There are 2 phases.
The solvents usually used are: 1. Cation Exchange Chromatography
o A hydrophobic organic solvent (MOBILE PHASE) o Cation exchanger
 Non-polar phase o Polysulfonic
 Which means non polar amino acids are more soluble in o Carry fixed negative charge
the mobile phase o pH gradient: low pH to high pH
 Expected result is that non-polars will travel farther from
the starting point. In Cation Exchange, we fill the glass column with polysulfonic
 And, the LONGER the R-group, the FARTHER it goes. exchanger which is negatively charged. Place the amino acid mixture
 The non-polars with SHORTER R-groups, more or less they using a medium with pH 1. The amino acid mixture that you would like
are in the middle. to separate is first dissolved at pH 1 mixture
o Water molecules bound to the cellulose (STATIONARY PHASE)
 Polar phase Recall Protonic Equilibria:
 Polars are more soluble in this phase
 Expected result is that the polars are located close to the
point of origin.

1. In starting point, that is where amino acid mixture that you want to o Start form most positive at pH 1
separate is being placed. o Adding more base, pH of the medium will increase; from +1
2. Immerse filter paper in the developing solvent to zwitterion form
3. Let it stand o As you add more base, medium becomes negatively
4. Developing solvent is made up of N-glutanol glacioacetic acid mixture charged; medium will also become negative
5. Observe that the wet part will go up o α carboxyl group dissociates first
6. Pull out filter paper when reached certain part of the filter paper
7. Dry At pH 1, what is the charge of the amino acids?
8. Spray with ninhydrin o All amino acids are positively charged
- Acts with the amino groups of the amino acids Basics are +2
9. Observe bluish purple dots on the filter paper with varying distances Neutrals are +1
(different locations) depending upon the solubility of the amino acids. Acidics are +1
o Why are basics +2? It’s because there is an amino group at
Identifying: R position which is also positively charged. In terms of
1. Measure the distance from the starting point to the center of the dot attachment to the column, basics are most attached
→ Rf Value followed by neutrals then acidics

Wash down the column with a buffer pH that’s going up. From pH 1,
2. In the Rf value table, there are corresponding amino acids per value. next, use pH 3, then pH 5, pH 7, then pH 9. As you increase the pH of
3. Doing that for all the dots and you will come up with what amino the medium, what happens to the amino acids?
acids that you have separated. o Amino acids become negatively charged. Acidics will
become negative first because they have a low isoelectric
point

Recall Isoelectric Points:

o General Rule:
 Neutral AA: IpH = 5-7
 Acidic AA: IpH = <5
 Basic AA: IpH = >7

Example:
Lysine, Proline, Methionine Below IpH is positive, that’s pH 1. Above IpH, they are negative. This
means that the first to reach zwitterion is the first to become negative.
Among the 3, Lysine is the only polar, thus it must be the one closest to the Acidics have zwitterion at 4, thus above 4, it will become negative
point of origin. Between Proline and Methionine, Met has a longer R group, already. Whereas the neutrals, at 4, it’s still positive, they only become
thus it must be the one farthest from the point of origin. zwitterion in between 5 to 7; expect them to become negative above
7. Basics are the last to become negative because they have an
 Thin Layer Chromatography (TLC) isoelectric of 8 above.
- Same principle as paper chromatography
- Makes use of glass plates for support

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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
Cation Exchange continued…..
Going back to Cation exchange, once the acidics become negative, they
will be repelled by the column because it is also negatively charged.
Given that in Cation exchange we start with the pH 1, expect that the
acidic amino acids will come out in the first test tube, followed by the
neutrals, then basics.
 Electrophoresis
- Separation on the basis of acid-base property of amino acids
facilitated with the use of electrical field
- In electrophoresis, an ampholyte (protein, peptide or amino acid) in
a solution buffered at a particular pH is placed in an electrical field.
Depending on the relationship of the buffer pH to the IpH of the
molecule, the molecule will either move toward the cathode (-) or
the anode (+), or remain stationary (pH = IpH)

EXAMPLE:
If you got Glutamic acid in your sample using a buffer of 7:
Picture on the Left:
This just shows how Ion
exchange chromatography
is done At pH 7, Glutamic acid is on the anode side at -1 because it is above 4.2 and
below 9.6.

Lysine:

At pH 7, Lysine is on the cathode side at +1 because its charge it is above 2.4

and below 9
2. Anion Exchange Chromatography
o Anion exchanger
Alanine:
o Polyamino
At pH7, Alanine is 0, stationary at the center
o Carry fixed positive charges
Changing the buffer pH to 3, where will you find Glutamic acid?
o pH gradient: high pH to low pH
Stationary, 0, because 3 is above 2.2 and below 4.2
In Anion Exchange, we fill the glass column with polyamino exchanger
Lysine: Still +1 because it is above 2.4 and below 9
which is positively charged. Place the amino acid mixture using a
Alanine: Still stationary because it is above 2.3 and below 9.7
medium with pH 11. The amino acid mixture that you would like to
separate is first dissolved at pH 11 mixture

At pH 11, what is the charge of the amino acids?

o All amino acids are negative
Acidics are -2 Picture on the Left:
Neutrals are -1 This just shows how
Basics are -1 Electrophoresis is done
o Why are acidics -2? It’s because they have an extra carboxyl
group at R position. So the ones that are attached to the
column are the acidics because this time the column is
positive

As you lower the pH of the solution, slowly, the amino acids that started
as negative will now become positive. Which one will become positive
first?
o Basics will become positive because they have a high
isoelectric point, followed by the neutrals and then acidics

 High Performance Lipid Chromatography (HPLC)

- Used pressures of 5000-10,000 psi are used to force the solutions
rapidly through the resin
- Separations that formerly required hours can now be done in minutes

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 BIOCHEMISTRY
Protein and Amino Acid Chemistry Part 2
Dr. Alcantara
 DENATURATION REFERENCES
o Cutting NON-COVALENT bonds  Biochemistry Lecture Guide (2018)
o Occurs when a protein loses its native:  PPT
- Secondary → hydrogen bonds between peptide bonds destroyed →  Lea Pacis Trans
it uncoils  Maria Bernabe Trans
- Tertiary → R-R interactions destroyed → shape of protein is  jpa Trans
destroyed  Gradelifting Fairies Trans
- Quaternary → peptide chain interactions are destroyed
 Dr. Alcantara Recordings
o There is cleavage of non-covalent bonds. The primary structure is
not altered by denaturation (Peptide bonds are intact)
o Can be reversible SOMETIMES.

So when you subject proteins to denaturation, you are going to destroy

the secondary, tertiary, and quaternary structures. Primary structure
is NOT altered by denaturation

Primary structure is not altered by denaturation; this means that

peptide bonds are intact.

Secondary structures: hydrogen bonds between the peptide bonds are

altered; means proteins will uncoil or will become linear

Tertiary structures: bonds between R-R interactions are cut between

the side chains, destroying the shape/conformation of the protein and
its function.

Quaternary structure: bonds between the chains are destroyed

Denaturing Agents:
1. Strong acids or bases
2. Organic solvents
3. Detergents
4. Reducing agents
5. Salt concentration
6. Heavy metal ions
7. Temperature changes
8. Mechanical stress
Chemical Alterations
o Decrease in solubility
- Most visible effect in globular proteins
o Many chemical groups which were inactive become exposed
and more readily detectable

Physical Alterations
o Increased viscosity
o Decreased rate of diffusion
o Increased levorotation
o Cannot be crystallized

Biological Alterations
o Increased digestibility
o Enzymatic or hormonal activity is destroyed
o Antibody functions are altered

 RENATURATION
o A process wherein certain denatured globular proteins will
regain their native structure and biological activity if returned
to conditions in which the native conformation is stable
e.g. Bovine pancreatic ribonuclease: a digestive enzyme
from cattle that degrades RNA

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