Hopkins-Cole test

Proteins respond to the following color tests (a) biuret, pink to purple with an excess of alkali and a small amount of copper sulfate (b) ninhydrin. a blue color when boiled with ninhydrin (triketohydrindene hydrate), which is intensified by the presence of pyridine (c) Millon s test for tyrosine, a brick-F color or precipitate when boiled with mercuric nitrate in an excess of nitric acid (d) Hopkins-Cole test for tryptophan, a violet zone with a salt of glyoxylic acid and stratified over sulfuric acid and (e) xanthoproteic test, a brilliant orange zone when a solution in concentrated nitric acid is stratified under ammonia. Almost all so-called alka-loidal reagents will precipitate proteins in slightly acid solution. 

Approximately this quantity of mercuric sulfate is necessary to precipitate the tryptophane completely, as judged by the Hopkins-Cole glyoxylic acid test. 

Brustier and coworkers utilized the Adamkiewicz-Hopkins-Cole reaction as an identity test for gramicidin121. The test detects the indole ring structure of the tryptophane residue. 

Hopkins-Cole Reaction. Glyoxylic Acid Reaction.—This reaction is produced with proteins by the action of glyoxylic acid, CH(OH)2— COOH (p. 52). To a little protein solution add an equal volume of a solution of glyoxylic acid made by reducing oxalic acid with sodium amalgam. Mix thoroughly and then introduce an equal volume of concentrated sulphuric acid by means of a pipette reaching to the bottom of the test tube so as not to mix the acid and the solution. A reddish-violet color at the zone between the two liquids shows the presence of protein. 

Hopkins-Cole Reagent (test for presence of the indole ring in the tryptophan moiety of a protein or peptide). Add 5 mL cold water to 10 g of magnesium in a conical flask and add 250 mL of a cold saturated aqueous oxalic acid solution with vigorous stirring. Filter, add 25 mL of glacial acetic acid and dilute with distilled water to the 1.0 L mark. Place 10 drops of the saturated protein or peptide solution in a test tube, add 10 drops of concentrated nitric acid and heat in a water bath for three minutes. Allow the mixture to cool and add 4 mL 6N NaOH. A positive test is confirmed by the formation of a yellow color. 

Tryptophane is not obtained in any large amount by the hydrolysis of proteins by acids and is best prepared by the action of trypsin. According to Hopkins and Cole, the protein is digested in alkaline solution by trypsin, until the solution gives a maximal coloration when tested with bromine water the solution is then acidified, boiled and... 

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