BIO 203 Biochemistry I

by

Seyhun YURDUGÜL,Ph.D.

Lecture 4
The Chemistry of Amino Acids
 Content Outline
• Aminoacids
• Classifications of Amino Acids
• The Isoelectric Point
• Optical Properties of the Amino Acids
• The Peptide Bond
• Separation of Amino Acids
 Aminoacids(aa)

• The building blocks of proteins or polypeptide

chains
• All peptides and polypeptides: polymers of
alpha-amino acids.
• 20 α-amino acids relevant to the make-up of
mammalian proteins
Chemical nature of the amino acids

– Several other amino acids : found in the body free

or in combined states
– (i.e. not associated with peptides or proteins).
– These non-protein associated amino acids
perform specialized functions.
 Structure of amino acids

• The α-amino acids in peptides and proteins

(excluding proline) consist of
• a carboxylic acid (-COOH)
• and an amino (-NH2) functional group
• attached to the same tetrahedral carbon atom.
• This carbon : α-carbon.
 Structure of amino acids
• Distinct R-groups, that distinguish one amino
acid from another,
• also attached to the α-carbon
• except in the case of glycine where the R-
group: hydrogen
• The fourth substitution on the tetrahedral α-
carbon of amino acids is hydrogen.
 Aminoacids in action

– Several of the amino acids found in proteins:

– also serve functions distinct from the formation of
peptides and proteins,
– e.g. tyrosine
– in the formation of thyroid hormones
– or glutamate
– acting as a neurotransmitter.
 Classifications of
Amino Acids(a.a.)

• Each of 20 α-amino acids

• can be distinguished by the R-group substitution on the
α-carbon atom.
• At present two broad classes of amino acids: based
upon whether the R-group:
• is hydrophobic
• or hydrophilic.
 Classifications of
Amino Acids
• hydrophobic aa tend to repel the aqueous
environment
• and, therefore, reside predominantly in the
interior of proteins.
• these amino acids does not ionize nor
participate in the formation of H-bonds.
 Classifications of
Amino Acids
• hydrophilic aa: tend to interact with the
aqueous environment,
• often involved in the formation of H-bonds
• predominantly found on the exterior surfaces
of :
• proteins or in the reactive centers of enzymes.
Acid-Base properties of the amino
acids

• The α-COOH and α-NH2 groups in amino acids:

• capable of ionizing
• as are the acidic and basic R-groups of the amino acids
• the following ionic equilibrium reactions may be
written due to ionizability:
• R-COOH <--------> R-COO- + H+
• R-NH3+ <---------> R-NH2 + H+
Acid-Base properties of the amino acids

• The equilibrium reactions demonstrate that:

• amino acids contain at least two weakly acidic groups.
• However, the carboxyl group:
• a far stronger acid than the amino group.
• At physiological pH (around 7.4) the carboxyl group:
unprotonated
• and the amino group will be protonated.
Acid-Base Properties of the Amino Acids

• An amino acid with no ionizable R-group is electrically

neutral at a pH:
• termed “zwitterion”.
• Like typical organic acids, the acidic strength of the
carboxyl, amino and ionizable R-groups in amino
acids:
• can be defined by the association constant, Ka
• or more commonly the negative logarithm of Ka, the
pKa.
 Acid-Base properties of the amino
acids

• net charge:
• the algebraic sum of all of the charged
groups present) of any amino acid, peptide
or protein,
• depend upon the pH of the surrounding
aqueous environment.
 The isoelectric point
(pI)
• As the pH of an amino acid or protein solution
changes:
• so too does the net charge:
• can be observed during the titration of any amino
acid or protein.
• When the net charge of an amino acid or protein =
zero
• the pH will be equivalent to the isoelectric point: pI.
• Titration curve for alanine
Functional significance of amino acid R-groups
What are R-groups required for?

• In solution
• the nature of the amino acid R-groups dictate
structure-function relationships of peptides and
proteins:
• hydrophobic amino acids generally be encountered in
the interior of proteins:
• shielded from direct contact with water.
Functional significance of amino acid R-groups:
What are R-groups required for?

• Conversely, the hydrophilic amino acids: found on the

exterior of proteins as well as in the active centers of
enzymatically active proteins.
• Indeed, naturally certain amino acid R-groups:
• allow enzyme reactions to occur.
Examples from different R-groups
– The imidazole ring of histidine:
– allows it to act as either a proton donor or acceptor at
physiological pH:
– frequently found in the reactive center of enzymes.
– Equally important:
– ability of histidines in hemoglobin to buffer the H+ ions
from carbonic acid ionization in red blood cells.
– It is this property of hemoglobin that allows it to exchange
O2 and CO2 at the tissues or lungs, respectively.
Examples from different R-groups
– The primary alcohol of serine and threonine as well as the
thiol (-SH) of cysteine:
– allow these amino acids to act as nucleophiles during
enzymatic catalysis.
– Additionally, thiol of cysteine:
– able to form a disulfide bond with other cysteines:

– Cysteine-SH + HS-Cysteine <--------> Cysteine-S-S-Cysteine

 Properties of Cystine
• This simple disulfide :
• identified as cystine.
• The formation of disulfide bonds between cysteines present
within proteins:
• important to the formation of active structural domains in a
large number of proteins.
• Disulfide bonding between cysteines in different polypeptide
chains of oligomeric proteins:
• tend to order the structure of complex proteins, e.g. the
insulin receptor.
Optical properties of the amino
acids
– A tetrahedral carbon atom with four(4) distinct
constituents is said to be chiral.
– The only one amino acid not exhibiting chirality:
– glycine
– since its '"R-group" is a hydrogen atom.
Optical properties of the amino
acids
– Chirality describes:
– handedness of a molecule that is observable by
the ability of a molecule to rotate the plane of
polarized light
– either to the right (dextrorotatory)
– or to the left (levorotatory).
– All of the amino acids in proteins exhibit the
same absolute steric configuration as L-
glyceraldehyde.
 Optical properties of the amino
acids
• Therefore, they are all L-alpha-amino acids.
• D-amino acids :
• never found in proteins, although they exist
in nature.
• D-amino acids are often found in polypeptide
antibiotics.
 Optical properties of the amino
acids
• The aromatic R-groups in amino acids:
• absorb ultraviolet light(U.V.) with an absorbance
maximum in the range of 280 nm.
• The ability of proteins to absorb U.V. light:
• predominantly due to the presence of the
tryptophan
• which strongly absorbs U.V. light.
 The peptide bond:

• Peptide bond formation:

• a condensation reaction
• leading to the polymerization of amino acids into
peptides and proteins.
 The peptide bond:
• Peptides are small consisting of few amino acids.
• e.g. A number of hormones and neurotransmitters,
several antibiotics and antitumor agents.
• Proteins:
• polypeptides of greatly divergent length.
 The peptide bond:

• The simplest peptide, a dipeptide:

• contains a single peptide bond
• formed by the condensation of the carboxyl group of
one amino acid
• with the amino group of the second with the
concomitant elimination of water.
 The peptide bond
• The presence of the carbonyl group in a peptide bond:
• allows electron resonance stabilization to occur:
• such that the peptide bond exhibits rigidity not unlike
the typical -C=C- double bond.
• The peptide bond is, therefore, said to have partial
double-bond character.
 Structure of peptide bond:

• Resonance stabilization forms of peptide bond

 Solubility of proteins
• Proteins show a variation in solubility:
• depending on their solution ionic
environments.
• This property:
• characterized in:
• salting-in, salting-out, and dialysis of proteins.
 Salting-in:

• The effects of salts such as sodium chloride;

• on increasing the solubility of proteins:
• often referred to as salting-in.
 Salting-in
• When low concentrations of salt:
• added to a protein solution,
• the solubility increases.
• This could be explained by the following:
 Salting-in
• Salt molecules stabilize protein molecules by:
• decreasing the electrostatic energy:
• between the protein molecules;
• which increase the solubility of proteins.
 Ionic strength
• measures the concentration of charges in
solution.
• As the ionic strength of a solution increases,
• the activity coefficient of an ion decreases.
 Ionic strength
• Formula:
• Γ/2 = ½ Σ Mi Zi2
• Mi= the molarity of the ion
• Zi= the net charge of the ion(regardless of its
sign)
• Σ= the sum
• Γ= Gamma letter in Greek
 Ionic strength
• The relationship between the ionic strength
and the molarity of a solution of ionizable salt:
• depends on the number of ions produced and
their net charge.
Salt type Salt example Ionic strength

• 1:1 KCl, NaBr M

• 2:1 CaCl2, Na2HPO4 3XM
• 2:2 MgSO4 4XM
• 3:1 FeCl3 6XM
• 2:3 Fe2(SO4)3 15XM
 Ionic strength
• Type:
• refers to the net charge on the ions.
• Only the net charge on an ion:
• used in calculating the ionic strength.
 Salting-out:

• When the ionic strength of a protein solution:

• increased by adding salt,
• the solubility decreases,
• and protein precipitates.
• This could be explained by the following:
 Salting-out:

• The salt molecules compete with the protein

molecules;
• in binding with water.
• Some salts, such as high concentrations of
ammonium sulfate,
• have general effects on solvent structure that
lead to:
• decreased protein solubility and salting-out.
 Salting-out:

• In this case,
• the protein molecules tend to associate with each
other;
• because protein-protein interactions become
energetically more favorable than protein-solvent
interaction.
• Proteins have characteristic salting-out points,
• and these:
• used in protein separations in crude extracts.
 Salting-out:

• The most effective region of salting-out:

• at the isoelectric point of the protein:
• because all proteins exhibit minimum
solubility in solutions of constant ionic
strength;
• at their isoelectric points.
 Salting-out:

• The salt commonly used:

• ammonium sulfate because of:
• its large solubility in water.
• its relative freedom from temperature effects.
• it has no harmful effects on most of the
proteins.
Various techniques in separation of
amino acids
• A) Ion-exchange chromatography.
• Depends on electrical charge
• two main mechanisms for effecting separation
in ion-exchange chromatography.
• One is titration,
• The other: increasing the pH.
Ion exchange chromatography apparatus
 Separation of amino acids

• As pH increases,
• the amino acids go from a positive to a neutral
charge state,
• and so released from the fixed anionic sites in
the resin (stationary phase)
 Separation of amino acids
• One other mechanism:
• competitive ion exchange.
• The positive amino acids show a higher affinity for
the ion-exchange sites than do the eluting cations
(H+, Li+ or Na+)
• and so are bound strongly to the anionic sites.
• eluted by the continuous flow of cation;
• or by increasing cation concentration developed by
gradient formation.
 Separation of Amino Acids
• Amino acid analyzers, such as the Beckman
System 6300TM
• based on step gradients-- isocratic procedures
that step through a set of 3-6 solutions of
varying pH and cation concentration.
• Both titration and elution adjustments are
made with each eluant step.
 Separation of amino acids
• The modern liquid chromatographic-high
performance liquid chromatography (HPLC)
techniques
• Modern enhancement of ion exchange
chromatography:
• use three solutions and continuous gradients.
• HPLC is; to a lesser extent; also called as: ‘high
pressure liquid chromatography’.
HPLC (High Performance Liquid Chromatography)
 Separation of amino acids
• An amino acid analyzer: a special instrument
for the separation process.
• Gradient capability allows for easier methods
development and results in flatter baselines--
especially important at high sensitivity.
• As with dedicated analyzers, the eluants are
either sodium- or lithium-based.
Amino acid analyzer
 LITERATURE CITED
• Devlin,T.M. Textbook of Biochemistry with Clinical
Correlations,Fifth Edition,Wiley-Liss Publications,New
York, USA, 2002.
• Lehninger, A. Principles of Biochemistry, Second edition,
Worth Publishers Co., New York, USA, 1993.
• Matthews, C.K. and van Holde, K.E., Biochemistry,
Second edition, Benjamin / Cummings Publishing
Company Inc., San Francisco, 1996.
• Segel, I.H., Biochemical Calculations, Wiley Publications,
New York, USA, 1976.