8 min readConcept sheetDone with the colour tests in the exam; salting out and isoelectric point are viva favourites

Precipitation reactions of proteins

Proteins stay dissolved by their charge and water shell; heat, salt, heavy metals, acid reagents, alcohol or the isoelectric pH upset this balance and the protein precipitates.

Salting outHalf-saturated ammonium sulphate → globulins; full saturation → albumin

Isoelectric pointNet charge zero, least soluble; casein pI about 4.6

Metals vs acid reagentsMetal cations bind protein anions (pH above pI); picric, tannic, TCA anions bind protein cations (pH below pI)

Reversible?Salting out is reversible; heat coagulation and heavy metals denature

Aim and principle

Aim: To study the precipitation of proteins by heat, at the isoelectric pH, by salting out, and by heavy metal salts, alkaloidal reagents and alcohol. Principle: A protein stays in solution because its charged groups hold a shell of water and like charges keep the molecules apart. Heat unfolds (denatures) globular proteins such as albumin; the exposed water-hating groups stick together as a coagulum, best near the isoelectric pH. At the isoelectric pH (pI) the net charge is zero, molecules stop repelling each other and solubility is lowest; casein precipitates at about pH 4.6. Salting out: a high concentration of ammonium sulphate takes away the water shell; globulins come out at half saturation and albumins at full saturation, without denaturation. Heavy metal ions (Pb2+, Hg2+, Ag+, Cu2+) combine with negatively charged carboxyl groups on the alkaline side of the pI to form insoluble metal proteinates. Alkaloidal (acid) reagents such as picric, tannic, trichloroacetic and sulphosalicylic acids combine through their anions with positively charged amino groups on the acid side of the pI. Alcohol lowers the dielectric constant of water and dehydrates the protein, so it precipitates.

Requirements

Reagent or item	Made of	What it does
Protein solutions	Egg white diluted with 0.9% NaCl (keeps globulins dissolved); casein in dilute alkali, or skimmed milk	The samples
1% acetic acid and pH paper	Dilute acetic acid	Brings the protein near its pI for the heat and isoelectric tests
Ammonium sulphate	Saturated solution and the solid salt	Half and full saturation for salting out
Heavy metal salts	5% lead acetate, 5% mercuric chloride, 5% copper sulphate, 1% silver nitrate	Form insoluble metal proteinates (poisonous: gloves)
Alkaloidal reagents	Saturated picric acid, 5% tannic acid, 10% trichloroacetic acid (TCA), 20% sulphosalicylic acid	Form insoluble protein salts
Ethanol	95% ethanol	Dehydrates the protein
Apparatus and safety	Test tubes, boiling water bath, funnel and filter paper, pipettes with a bulb; gloves, goggles	Mercury and lead salts are poisons; TCA burns skin

Procedure

Typical volumes are given. Follow the exact volumes in your lab manual.

1
Heat coagulation: 5 mL egg albumin solution in a tube; heat only the upper part, keeping the lower part as a control. If it is only cloudy, add 1-2 drops 1% acetic acid and heat again.
White coagulum in the heated part
2
Isoelectric precipitation: to 5 mL casein solution (or diluted milk) add 1% acetic acid drop by drop, checking the pH with paper.
Curd at about pH 4.6; it redissolves in excess acid
3
Salting out: to 5 mL egg white solution add 5 mL saturated ammonium sulphate (half saturation); mix, stand 5 minutes and filter.
Precipitate = globulins
4
Add solid ammonium sulphate to the filtrate, shaking, until a little stays undissolved (full saturation).
Precipitate = albumin; add water to show it redissolves
5
Heavy metals: to 2 mL neutral protein solution in separate tubes add 5% lead acetate, 5% mercuric chloride or 5% copper sulphate, drop by drop.
White (bluish with copper) precipitate
6
Alkaloidal reagents: to 2 mL protein made slightly acidic with 2 drops acetic acid, add a few drops of picric acid, tannic acid, TCA or sulphosalicylic acid in separate tubes.
Yellow with picric acid; white or buff with the others
7
Alcohol: to 2 mL protein add 5 mL 95% ethanol and mix.
White precipitate

Test | Observation | Inference

Test	Observation	Inference
Heat coagulation	Heated upper part turns white and opaque, more so with a drop of acetic acid; lower part stays clear	Heat-coagulable protein (albumin) denatured
Isoelectric precipitation	White curdy precipitate at about pH 4.6; redissolves in excess acid	Casein precipitates at its pI, where its net charge is zero
Half saturation with ammonium sulphate	White precipitate	Globulins present
Full saturation with ammonium sulphate	White precipitate in the filtrate; redissolves when diluted with water	Albumin present; salting out is reversible
Heavy metal salts	White precipitate (bluish-white with copper sulphate)	Metal proteinates formed; protein acts as an anion
Alkaloidal reagents	Yellow (picric acid) or white to buff (tannic acid, TCA, sulphosalicylic acid) precipitate	Protein salts formed; protein acts as a cation
Alcohol	White precipitate	Protein dehydrated and precipitated

Which side of the isoelectric point?

Heavy metals: pH above pIThe protein carries negative -COO⁻ groups, which bind Pb2+, Hg2+, Ag+, Cu2+. Keep the solution neutral or slightly alkaline.

Alkaloidal reagents: pH below pIThe protein carries positive -NH3+ groups, which bind the anions of picric, tannic, trichloroacetic and sulphosalicylic acids. Acidify slightly first.

At the pI itself the protein has no net charge and precipitates on its own; that is isoelectric precipitation.

Precautions and result

Wear gloves and goggles: mercuric chloride and lead acetate are poisons, and TCA and picric acid burn skin (dry picric acid can explode, so keep it wet).
Add heavy metal salts drop by drop; the precipitate may redissolve in excess of some reagents.
In heat coagulation add only 1-2 drops of acetic acid; too much gives the protein a charge and stops coagulation.
Saturate fully with solid ammonium sulphate (a little should stay undissolved) before judging albumin.
Never pipette by mouth; collect heavy-metal waste in the marked container.
Result: The given protein was precipitated by heat, at its isoelectric pH, by ammonium sulphate (globulin at half, albumin at full saturation), by heavy metal salts, by alkaloidal reagents and by alcohol.

Nutrition link

Precipitation explains many kitchen and clinic facts. Curd and paneer form when casein reaches its isoelectric pH (about 4.6) through lactic acid or lemon juice. Cooking egg is heat coagulation, which makes the protein easier to digest. Tannins in tea bind proteins and also reduce iron absorption, so tea with meals is discouraged in anaemia. Raw egg white or milk is given in heavy-metal poisoning to bind the metal. TCA and sulphosalicylic acid remove protein before blood tests and detect albumin in urine.

Exam angle

Short answer: “What is the isoelectric point? Give the pI of casein.” The pH at which a protein has no net charge and is least soluble. Casein precipitates at about pH 4.6.

Short answer: “Why is egg white given in mercury or lead poisoning?” Its proteins combine with the heavy metal ions to form insoluble metal proteinates, which can then be removed by vomiting or stomach wash before the metal is absorbed.

Structured question: “Explain salting out of proteins with ammonium sulphate.” Principle: at high concentration the salt takes away the water shell, so protein molecules come together and precipitate. Globulins come out at half saturation, albumins at full saturation. The protein is not denatured, so it redissolves on dilution; this is used to separate and purify proteins and enzymes. At low salt, solubility rises instead (salting in).

Remember it

Half for globe, full for album: half-saturated ammonium sulphate brings down globulins; full saturation brings down albumin.