Enzymes are protein catalyst produced by a cell and responsible ‘for the high rate’ and specificity of one or more intracellular or extracellular biochemical reactions.

Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Enzyme reactions are always reversible.

The substance, upon which an enzyme acts, is called as substrate. Enzymes are involved in conversion of substrate into product.

Almost all enzymes are globular proteins consisting either of a single polypeptide or of two or more polypeptides held together (in quaternary structure) by non-covalent bonds. Enzymes do nothing but speed up the rates at which the equilibrium positions of reversible reactions are attained.

 In terms of thermodynamics, enzymes reduce the activation energies of reactions, enabling them to occur much more readily at low temperatures - essential for biological systems.

Protein electrophoresis is a laboratory technique used to separate proteins based on their size, charge, or other physical properties. It plays a vital role in diagnosing and monitoring various diseases, especially those involving abnormal protein production or structure.

Types of Protein Electrophoresis

1. SPE (Serum Protein Electrophoresis)

• Principle: Separation of serum proteins based on their charge.

• Major Fractions:

  • Albumin

  • Alpha-1 globulin

  • Alpha-2 globulin

  • Beta globulin

  • Gamma globulin

• Clinical Applications:

  • Multiple Myeloma: Detects monoclonal spike (M-protein) in gamma region.

  • Chronic Infections: Polyclonal increase in gamma globulins.

  • Nephrotic Syndrome: Decreased albumin, increased alpha-2 globulin.

  • Liver Disease: Altered albumin and beta-gamma bridging.

2. Hemoglobin Electrophoresis

• Principle: Separation of hemoglobin variants based on charge differences.

• Common Hemoglobins:

  • HbA, HbA₂, HbF, HbS, HbC, HbE

• Clinical Applications:

  • Thalassemia: Elevated HbA₂ or HbF levels.

  • Sickle Cell Disease: Presence of HbS.

  • Hemoglobinopathies: Differentiates variants like HbC, HbE, etc.

3. SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis)

• Principle: Proteins are denatured and separated by molecular weight.

• SDS: Imparts uniform negative charge, eliminating charge-based separation.

• Clinical Applications:

  • Protein Purification: Identifies specific proteins in research and diagnostics.

  • Genetic Disorders: Detects abnormal or truncated proteins.

  • Muscular Dystrophies: Analyzes dystrophin protein expression.

  • Cancer Research: Studies tumor markers and oncogenic proteins.

CLASSIFICATION OF ENZYMES

1. Oxidoreductases : Act on many chemical groupings to add or remove hydrogen atoms. e.g. Lactate dehydrogenase

2. Transferases Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules. e.g. Aminotransferase.

3. Hydrolases Add water across a bond, hydrolyzing it. E.g. Acetyl choline esterase

4. Lyases Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds. e.g. Aldolase.

5. Isomerases Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others. e.g. Triose phosphate isomerase

6. Ligases Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP. e.g. Acetyl CoA carboxylase

IONIZATION OF WATER, WEAK ACIDS AND WEAK BASES

The ionization of water can be described by an equilibrium constant. When weak acids or weak bases are dissolved in water, they can contribute H+ by ionizing (if acids) or consume H+ by being protonated (if bases). These processes are also governed by equilibrium constants

Water molecules have a slight tendency to undergo reversible ionization to yield a hydrogen ion and a hydroxide ion :

H₂O = H⁺ + OH⁻

The position of equilibrium of any chemical reaction is given by its equilibrium constant. For the general reaction,

A+B = C + D

The amino acids buffer system

Amino acids contain in their molecule both an acidic (− COOH) and a basic (− NH₂) group. They can be visualized as existing in the form of a neutral zwitterion in which a hydrogen atom can pass between the carboxyl and amino groups. 

By the addition or subtraction of a hydrogen ion to or from the zwitterion, either the cation or anion form will be produced 

Thus, when OH⁻ ions are added to the solution of amino acid, they take up H⁺ from it to form water, and the anion is produced. If H⁺ ions are added, they are taken up by the zwitterion to produce the cation form. In practice, if NaOH is added, the salt H₂N - CH₂ - COONa would be formed. and the addition of HCl would result in the formation of amino acid hydrochloride.