Biochemistry Practical Sessions & Contact Information

Contact Information

• Biochemistry Department contact is located in Room F3-04-031
• Contact via Email: [email protected]
• Contact via Ext: 7814
• Consultation times are Mondays and Wednesdays 12:00-13:00 pm

Practical Sessions

• Properties of proteins will be taught on 24 February & 27 February
• The Biuret test will be taught on 3 February & February
• Casein isolation from milk will be taught on 10 & 13 March
• TLC/Paper Chromatography will be taught on 17 & 20 March
• Vitamin C Isolation will be taught on 7 & 10 April
• Test for sugars will be taught on 14 & 17 April
• Periodate oxidation will be taught on 5 May & 8 May
• Acetylation of sugars will be taught on 12 & 15 May
• Theory test 1 is on 27 March 2025; Time - 8.30am – 10.00am
• Theory test 2 is on 8 May 2025; Time - 8.30am – 10.00am
• Practical test is on 15 May 2025; Time - 8.30am – 10.00am

Content Outline

Topics to be covered include:

• Introduction to amino acids
• Amino acid categories: General, Standard protein, Modified protein, and Non-protein
• Stereochemistry and physical properties of amino acids
• UV absorption spectra, melting points, and solubility
• Chemical properties: electrolyte behavior, reactions of -NH2 and -COOH groups

Central Dogma

• DNA is transcribed into RNA, which is then translated into proteins, which are crucial for cellular activities

Protein Functions

Dynamic functions include:

• Enzyme catalysis
• Transport and storage of molecules
• Mechanical work like coordinated motion
• Immune protection and defense
• Control of growth and differentiation
• Generation and transmission of nerve impulses

Structural proteins contribute to:

• Mechanical strength and support, like collagen
• Hair and nails, like keratins
• Proteins are key for biochemical cellular activities, requiring insight into their molecular structure and properties
• Amino acids are the monomeric building blocks of proteins

Amino Acid Categories

• There are approximately 300 different amino acids in nature
• Divided into standard protein, modified protein, and non-protein amino acids

Standard Protein Amino Acids

• All organisms use the same 20 amino acids to construct linear, unbranched protein molecules
• Defined as those amino acids for which a specific codon exists in the genetic code
• Amino acids are bi-functional or amphoteric molecules, acting as either an acid or a base

Carbon Atoms

• When the R group contains additional carbons in a chain, they are designated beta, gamma, delta, epsilon, etc., proceeding away from the carbon

Aliphatic R Groups in Amino Acids

• An aliphatic compound has carbon and hydrogen atoms joined in straight, branched, or non-aromatic rings
• Examples include Glycine, Alanine, Valine, Leucine, Isoleucine
• Valine, Leucine, and Isoleucine are essential

Hydroxyl or Sulphur-Containing R Groups in Amino Acids

• Examples include Serine, Threonine, Cysteine, and Methionine
• Threonine and Methionine are essential amino acids

Aromatic R Groups in Amino Acids

• An aromatic compound has conjugated ring systems
• Examples include Phenylalanine, Tyrosine, and Tryptophan
• Phenylalanine and Tryptophan are essential

Cyclic Amino Acids

• Proline contains a secondary nitrogen (R-N-R) and is nonessential

Acidic Amino Acids

• Acidic amino acids and their neutral amides include Aspartic Acid, Glutamic Acid, Asparagine, and Glutamine
• Aspartic Acid, Glutamic Acid, Asparagine, and Glutamine are all nonessential

Basic Amino Acids

• Basic amino acids include Histidine, Lysine and Arginine which are all essential

Polarity Classification of R-Groups

Amino acids with Neutral R-groups:

• The term "neutral" means the R-groups do not have positive or negative charges
• They interact poorly with H₂O and are nonpolar (Hydrophobic)
• Two types of hydrocarbon side chains are found in this group: Aliphatic and Aromatic

Aliphatic (Gly, Ala, Val, Leu, Ile, and Pro):

• Aliphatic R-groups become more hydrophobic as the hydrocarbon chain length increases

Aromatic (Phe and Trp):

• Aromatic refers to unsaturated cyclic or ring hydrocarbon structures
• Aromatic R-groups are more hydrophobic or nonpolar than aliphatic R-groups
• Neutral Nonpolar AA's also include sulfur containing AA namely Met

Amino Acids with Polar Uncharged R Groups

• Alcohol (-OH) functional group examples include Ser, Thr, and Tyr
• Amide functional groups examples include Asn and Gln
• Sulphydryl (-SH) functional group example is Cys
• These R-groups are hydrophilic and interact with H₂O due to hydrogen bonding
• Polarity is from partial charges in permanent dipole bonds in non-ionizable R-groups

Acidic Amino Acids

• Contain carboxylate groups and behave as Brönsted acids (proton donors), yielding negatively charged groups
• At pH 7, the full negative charge in the ionizable carboxyl functional group makes them polar charged

Basic Amino Acids

• Act as Brönsted bases (proton acceptors) to yield positively charged groups
• Are polar charged due to a full positive charge at pH 7 in an ionizable functional group
• Histidine has a positive charge because of the protonated imidazole group (imidazolium ion)
• Lysine has the amino group (alkylammonium ion) positive charge
• Arginine has the guanidine group (guanidinium ion) positive charge

Modified Protein Amino Acids

• 4-Hydroxyproline is a derivative of collagen, that comprises one third of its residues, also contributes to H-bonding increasing its strength

• N-methyllysine:

• Methylated lysine residues play a key role in epigenetics

• The methylation of specific lysine residues of certain histones in a nucleosome alters the binding of DNA to those histones, affecting gene expression

• Desmosine derivative:

• Is found in protein its structure can be visualized as formed from four lysine residues with their R-groups joined to form a substituted pyridine ring.

• Cystine consists of two cysteine residues joined enzymatically

• Disulphide isomerase form a disulphide covalent bond.

• These bonds stabilize the tertiary or folded structure of proteins

• O-Phosphoserine: derived enzymatically from serine.

• The addition and removal of the phosphate group regulates enzyme activity

Non-Protein Amino Acids

• B-Alanine and y-aminobutyric acid are examples
• Homoserine and Homocysteine are free or combined but never in proteins

Physical Properties of Amino Acids

UV absorption spectra:

• Proteins absorb UV light in the range of 190-300 nm
• Proteins are most sensitively detected in the Far UV range from 190-240nm due to absorption from the amide bond (max - 214 nm)
• Proteins also absorb light in the Near UV, corresponding to 250-300nm, mainly due to aromatic amino acids and disulfide bonds
• Measurement of light absorbance at 280nm is an easy way to estimate protein concentration

Solubility of Amino Acids:

• Generally soluble in H₂O; insoluble or slightly soluble in alcohol and insoluble in ether
• Exceptions include: Tyrosine is more soluble in hot water
• Cystine is difficult to solubilize in both hot and cold H₂O
• Proline and hydroxyproline are soluble in alcohol and ether
• AAs are generally soluble in dilute acids and bases, forming corresponding AA salts

Chemical Properties of Amino Acids

Electrolyte Behavior determines understanding acids and bases:

• The technology of separating, identifying and quantifying different AAs based on their electrolyte behavior

Predominant Ionic Forms

• In aqueous solution, equilibrium exists between zwitterionic, and anionic species of an AA
• Predominant ionic form depends on the pH of the solution and the nature of the R-group

pKa and its Measurement

• pKa measures the tendency of an ionisable group to donate a proton (H+)
• Tendency decreases 10-fold as the pKa increases by one unit
• e.g. molecule A has a pKa value = 1 while molecule B has a pKa value = 2, Molecule A is 10 times more acidic than B

Acid Dissociation

pH = pKa + log([A- ]/[HA] )

• This equation relates the pH of a solution and the dissociation constant (Ka) of an ionizable group
• When pH = pKa, the acid is half neutralized i.e. 50% free acid (HA) + 50% conjugate base (A) or [HA] = [A]

Amino Acid Ionizable Groups

pKa values vary for different amino acids' carboxyl (-COOH) and amino (-NH3+) groups, as well as ionizable R-groups AA's with Charged R-groups:

• The ionization pattern of amino acids includes a third ionizable function in their R group

Group 1: Aspartic and glutamic acid, cysteine and tyrosine

• In this group four different net charge species are possible +1, 0, -1, and -2.
• At pH 7 the side chain -COO¯ group contributes a full negative charge
• The -SH group of cysteine and phenolic hydroxyl group of tyrosine can exist also as the negative charge species.
• Only 5% of the -SH group of cysteine is ionized at pH 7.
• Only 0.1% of phenolic hydroxyl group of tyrosine is ionized at pH 7. Group 2: Lysine, Arginine and Histidine
• In this category four different net charge species are possible +2, +1, 0, and -1.
• Lys and Arg at pH 7 will contribute a net positive charge to the AA.
• The R group of His contributes approximately 10% of positively charged species at pH 7.

Isoelectric Point

• At this point An amino acid has no net charge (0)
• Calculated with the following formula: pI = (pKa1 + pKa2)/2
• For AA's with Charged R-groups use the formula pI = (pKa(n) + pKa(n+1)) / 2, where n is the number of positive charges on the fully protonated AA

Chemical Reactions:

• O-Phthalaldehyde Reaction. Quantitative technique to detect picomolar quantities, exposure to radiation induces fluorescence, readily reacts with primary amino acids
• Ninhydrin Reaction. Quantitative; detects nanomolar quantities. The intensity of blue/purple correlates, with primary amino acids, secondary ones give a slightly different product

Tests that react with amino acids:

• Sanger (1-Fluoro-2,4-Dinitrobenzene/FDNB) Reaction is utilized to detect amino terminal residue from peptides
• Dansyl Chloride Reaction utilized in detecting the N-terminal residue, also employed in detection

Reactions of the Carboxyl Group

• The AA undergoes reduction in an anhydrous medium such as Lithium or Sodium Borohydride to generate the corresponding 1º alcohol
• This reaction is used to identify the carboxyl terminal AA residue of a peptide

Questions

With the aid of appropriate ionic structures illustrate the complete ionic transition of Glu when titrated with NaOH. With the aid of appropriate ionic structures illustrate the complete ionic transition of Arg when titrated with NaOH. Assume that pKa1 = 2.63; pKa2 = 9.04 and pKa3 = 12.48 for Arg. Calculate the isoelectric point of this AA?