13.3 Protein Purification and Biochemical Analysis of Proteins, Carbohydrates, and Lipids

13.3 Protein Purification and Biochemical Analysis of Proteins, Carbohydrates, and Lipids

Quick Answer: The TOS lists separation and purification under proteins (area B), analysis under carbohydrates (area C), and oxidations and reductions under lipids (area D). Expect purification-table arithmetic, choosing a chromatographic step from a protein's pI and size, reading SDS-PAGE results, and the classic colour tests for amino acids, sugars, and fats. Section 13.2 covers sequencing and denaturation.

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Purifying a Protein

1. Homogenize the tissue and remove debris by differential centrifugation: nuclei pellet at about 1,000 g, mitochondria at about 10,000-20,000 g, and microsomes at about 100,000 g.
2. Salting out: protein solubility first rises at low ionic strength (salting in), then falls at high ionic strength as salt competes for water. Ammonium sulfate fractionation precipitates different proteins at different saturations. Solubility is lowest at the protein's pI.
3. Dialysis through a semipermeable membrane with a defined molecular-weight cut-off removes salts and small molecules.
4. Column chromatography:

The Purification Table

Specific activity should rise at each step even as total activity (yield) falls. Purity is reached when specific activity stops increasing and a single band appears on a gel.

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Electrophoresis

• SDS-PAGE: sodium dodecyl sulfate binds at about 1.4 g per g of protein, giving similar charge per unit mass. A reducing agent (2-mercaptoethanol or DTT) breaks disulfide bonds. Polypeptides then separate by size, with migration distance roughly linear in log molar mass. Comparing reducing and non-reducing gels reveals disulfide-linked subunits.
• Native PAGE separates folded proteins by charge, size, and shape and keeps activity.
• Isoelectric focusing (IEF): in a pH gradient, each protein migrates until it reaches the pH equal to its pI, where its net charge is zero.
• Two-dimensional electrophoresis: IEF in one direction, then SDS-PAGE at right angles, can resolve thousands of proteins.
• Western blotting: proteins are transferred from the gel to a membrane and detected with specific antibodies.

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Protein Assays and Amino Acid Tests

The Kjeldahl method estimates crude protein as nitrogen x 6.25, because proteins average about 16% nitrogen.

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Carbohydrate Tests

Sucrose is non-reducing: it is negative in Benedict's test until acid hydrolysis frees glucose and fructose. Section 13.6 explains reducing ends.

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Lipid Analysis: Hydrolysis, Oxidation, and Reduction

Worked values: Tripalmitin (807.3 g/mol) has a saponification value of $\frac{3 \times 56{,}106}{807.3} = 208$ mg KOH/g. Triolein (885.4 g/mol, three C=C) has a saponification value of 190 and an iodine value of $\frac{3 \times 253.8}{885.4} \times 100 = 86$.

Typical iodine values: coconut oil about 7-10 (mostly saturated), olive oil about 75-94, and soybean oil about 120-143.

• Reduction (hydrogenation): $H_2$ over Ni converts liquid oils to solid fats (margarine). Partial hydrogenation forms trans fatty acids, which raise cardiovascular risk.
• Oxidative rancidity: radical autoxidation starts at the bis-allylic $CH_2$ groups of polyunsaturated chains, forming hydroperoxides that break down to odorous aldehydes. Antioxidants (vitamin E, BHT) trap the chain-carrying radicals.
• Hydrolytic rancidity: lipases release free fatty acids, such as butyric acid in spoiled butter.
• Saponification with NaOH or KOH gives glycerol and soap. The acrolein test (heating with $KHSO_4$) detects glycerol by its sharp odour.