Biochem Lab M3

Amino Acids

Building blocks of proteins; contain an amino group (NH₂), carboxyl group (COOH), hydrogen atom, and R-side chain bonded to an alpha carbon

Essential Amino Acids

Amino acids that cannot be synthesized by the body and must be obtained from diet

Chirality of Amino Acids

All amino acids except glycine are chiral, having four different groups attached to the alpha carbon

Amphoteric Property of Amino Acids

Amino acids can act as both an acid (–COOH group) and a base (–NH₂ group)

Zwitterion

Dipolar ion form of amino acids at their isoelectric point (pI), making them electrically neutral

UV Absorptive Amino Acids

Aromatic amino acids (tryptophan, tyrosine, phenylalanine to lesser extent) absorb in UV

Chromatography

Separation process invented by Mikhail Tsvet; separates mixture components based on affinities for stationary and mobile phases

Stationary Phase

Solid or liquid fixed in place

Mobile Phase

Solvent or mixture of solvents that moves through stationary phase

Retention Factor (Rf)

Measurement of separation in chromatography; depends on solubility and adsorptive properties of sample

Paper Chromatography

Technique where sample is spotted on filter paper, solvent carries sample components up the paper

Ninhydrin Reagent

Reacts with amino acids to give colored products (Ruhemann’s purple) for visualization in chromatography

Biuret Test

Detects peptide bonds; in alkaline solution, cupric ions form purple

Ninhydrin Test

Detects amino groups (α-amino acids); produces purple or brown colored compounds.

Xanthoproteic Test

Detects aromatic amino acids (tyrosine, tryptophan, phenylalanine); nitration by HNO₃ gives yellow, turns orange with alkali

Millon’s Test

Detects phenolic amino acid (tyrosine); produces red-colored complex with Millon’s reagent

Sakaguchi Test

Detects guanidino group of arginine; reacts with α-naphthol and oxidizing agent to produce red color

Hopkins-Cole Test

Detects indole group of tryptophan; reaction with glyoxylic acid and H₂SO₄ gives purple color.

Lead Acetate Test

Detects sulfur-containing amino acid (cysteine); NaOH liberates Na₂S which reacts with Pb²⁺ to form black precipitate.

Protein

Polymers of amino acids; albumin is a simple globular protein

Primary Protein Structure

Linear sequence of amino acids

Secondary Protein Structure

Local folding into α-helices or β-sheets

Tertiary Protein Structure

3D folding pattern stabilized by interactions between R-groups

Quaternary Protein Structure

Assembly of multiple polypeptide chains

Protein Denaturation

Process that alters protein conformation, disrupting secondary, tertiary, and quaternary structures, leading to loss of function

Heat as Denaturing Agent

Increases atomic vibrations, disrupting hydrogen bonds and hydrophobic interactions

pH as Denaturing Agent

Alters charges on amino acid R-groups, disrupting ionic bonds.

Organic Solvents as Denaturing Agent

Ethanol and similar solvents disrupt hydrogen bonding, leading to denaturation

Heavy Metals as Denaturing Agent

Mercury, lead, silver bind disulfide groups, disrupting disulfide bridges and salt linkages, causing protein precipitation

Antidotes for Heavy Metal Poisoning

Protein-rich substances (e.g., egg whites, milk) bind heavy metals to form insoluble complexes removed by emesis

Enzymes

Special proteins that catalyze biochemical reactions by speeding up reaction rates

Substrate

Reactant acted upon by an enzyme

Product

Substance formed from enzyme-substrate reaction

Active Site

Region of enzyme where substrate binds and reaction occurs

Factors Affecting Enzyme Activity

Temperature, substrate concentration, inhibitors, and pH

Competitive Inhibition

Inhibitor binds to active site, preventing substrate binding

Uncompetitive Inhibition

Inhibitor binds reversibly to enzyme-substrate complex.

Noncompetitive Inhibition

Inhibitor binds to site other than active site, reducing enzyme activity

Catalase

Enzyme that protects cells by catalyzing hydrogen peroxide into water and oxygen

Effect of Temperature on Enzymes

Higher temperature may denature enzyme; lower temperature reduces activity

Effect of pH on Enzymes

pH changes enzyme conformation; enzymes have optimal pH for activity

Amylase

Enzyme in saliva and pancreas that hydrolyzes polysaccharides (starch) into simpler sugars (glucose)

Starch-Iodine Complex

Iodine forms a blue complex with starch; as amylase hydrolyzes starch, color disappears.

Fehling’s Test

Positive (brick-red precipitate) indicates presence of reducing sugars like glucose; confirms starch hydrolysis by amylase