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Biochemistry

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 LIPIDS

Lipids are classified as follows:

1. Simple lipids: Esters of fatty acids with various alcohols.

(a) Fats: Esters of fatty acids with glycerol. Oils are fats in the liquid state. A long-chain carboxylic acid; those in animal fats and vegetable oils often have 12–22 carbon atoms.

(b) Waxes: Esters of fatty acids with higher molecular weight monohydric alcohols. Waxes are carboxylic acid esters, RCOOR’ ,with long, straight hydrocarbon chains in both R groups

2. Complex lipids: Esters of fatty acids containing groups in addition to an alcohol and a fatty acid.

(a) Phospholipids: Lipids containing, in addition to fatty acids and an alcohol, a phosphoric acid residue. They frequently have nitrogen containing bases and other substituents,

Eg  glycerophospholipids the alcohol is glycerol

     sphingophospholipids the alcohol is sphingosine.

(b) Glycolipids (glycosphingolipids): Lipids containing a fatty acid, sphingosine, and carbohydrate. These lipids contain a fatty acid, carbohydrate and nitrogenous base. The alcohol  is sphingosine, hence they are also called as glycosphingolipids. Clycerol  and phosphate  are absent  

 

e.g., cerebrosides, gangliosides.

(c) Other complex lipids: Lipids such as sulfolipids and aminolipids. Lipoproteins may also be placed in this category.

3. Precursor and derived lipids: These include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and ketone bodies, hydrocarbons, lipid soluble vitamins, and hormones. Because they are uncharged, acylglycerols (glycerides), cholesterol, and cholesteryl esters are termed neutral lipids

4. Miscellaneous lipids: These include a large number of compounds possessing the characteristics of lipids e.g., carotenoids, squalene, hydrocarbons such as pentacosane (in bees wax), terpenes etc.

NEUTRAL LIPIDS: The lipids which are uncharged are referred to as neutral lipids. These are mono-, di-, and triacylglycerols, cholesterol and cholesteryl esters.

CALCIUM

Total calcium in the human body is 1 to 1.5kg, out of which 99% is seen in bone and 1% in extracellular fluid. The main source of calcium is milk.

The daily requirement of calcium for child is 1200mg/day and for adult it is 500mg/day. During pregnancy /lactation the calcium requirement is 1500mg/day.

The absorption of calcium takes place in 1st and 2nd part of deuodenum. Calcium absorption requires carrier protein, helped by Ca2+ - dependent ATpase.

Factors responsible for increase in calcium absorption include Vitamin D, Parathyroid hormone, acidity and amino acids. Factors such as phytic acid,oxalates, malabsorption  syndromes and Phosphates decreases calcium absorption. The normal calcium level in blood is 9-11mg/dl.

These amino acids have aromatic side chains, which contribute to protein structure, UV absorbance, and biochemical signaling.

1. Phenylalanine (Phe, F)

  • Structure: Contains a benzyl side chain (a phenyl group attached to a CH₂).

  • Properties: Non-polar, hydrophobic.

  • Function: Precursor to tyrosine, dopamine, norepinephrine, and epinephrine.

2. Tyrosine (Tyr, Y)

  • Structure: Similar to phenylalanine but with a hydroxyl group (-OH) on the aromatic ring.

  • Properties: Polar, can participate in hydrogen bonding.

  • Function: Precursor to catecholamines and thyroid hormones; involved in signal transduction via phosphorylation.

3. Tryptophan (Trp, W)

  • Structure: Contains an indole ring (a fused double ring with nitrogen).

  • Properties: Slightly polar, bulky.

  • Function: Precursor to serotonin, melatonin, and niacin; absorbs UV light strongly at 280 nm.

4. Histidine (His, H) (sometimes included due to its aromatic-like imidazole ring)

  • Structure: Has an imidazole ring (five-membered ring with two nitrogen atoms).

  • Properties: Polar, positively charged at physiological pH.

  • Function: Key role in enzyme active sites; acts as a proton donor/acceptor.

Bonus Insight: UV Absorbance

  • Tryptophan and Tyrosine absorb UV light at ~280 nm, which is useful for measuring protein concentration.

  • Phenylalanine absorbs weakly at ~260 nm.

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