Hopkins-Cole reaction

The intense blue color which is obtained when tryptophan, in the presence of an aldehyde, is treated with concentrated sulfuric acid containing an oxidizing agent (Adamkiewicz-Hopkins-Cole reaction) was beheved to involve formation of a tetrahydro-j8-carboline intermediate, since most l,2,3,4-tetrahydro-j8-carbohne derivatives yield a similar color with concentrated sulfuric acid containing an oxidizing agent. The two colors have now been shown to have different absorption spectra. The nature of the carboline-blue color is still obscure. 

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 Reaction - Indole ring of tryptophan reacts with glacial acetic acid in the presence of concentrated sulphuric acid and forms a purple coloured product. Glacial acetic acid reacts with concentrated sulphuric acid and forms glyoxalic acid, which in turn reacts with indole ring of tryptophan in the presence of sulphuric acid forming a purple coloured product. 

Hopkins-Cole reaction.—Add to 2 cc. of the egg-white solution 5 drops of a solution of glyoxylic acid (see Appendix). Pour down the side of the tube concentrated sulphuric acid so that two layers are formed. If no color develops examine the tube in a few minutes. 

Albumin by difference. Determination of total protein and measurement of the globulin fraction by a method specific for globulins, e.g. the Hopkins-Cole reaction for tryptophan, enables the albumin to be estimated by difference. 

Immunochemical techniques are available for the determination of many of the individual globulins. The globulin fraction as a whole can be determined by procedures based on the Hopkins-Cole reaction for tryptophan since globulins, unlike albumin contain tryptophan residues in their structure. 

Properties. The n-amanitin crystallizes from methanol as fine needles, melting with decomposition at 254-255° C. It is dextrorotatory [ ] = + 191° (in methanol). It is strongly reducing, particularly for ammoniacal silver nitrate and porphyrindine, but does not reduce porphyrexide. It gives a series of color reactions, i.e., Hopkins-Cole, Millon, and Pauly. With concentrated sulfuric acid containing Fe(III), it gives a golden yellow color. 

Shortly before Hopkins and Cole isolated tryptophane, they studied the Adamkiewicz reaction—the production of a violet colour when concentrated sulphuric acid is added to a protein dissolved in glacial acetic acid—and found that it was caused by the presence of glyoxylic acid in the glacial acetic acid, from which it arose by the action of sunlight. On applying the glyoxylic reaction to tryptophane a very intense colour was produced, and hence the presence of tryptophane in the protein molecule is the cause of this reaction. 

F. G. Hopkins and S. W. Cole. On the Proteid Reaction of Adamkiewicz, with Contribu-... 

Tryptophan was first isolated only at the beginning of this century (411). A number of color reactions of proteins were extensively studied in the latter half of last century and numerous attempts were made to isolate the chromogen responsible. The name tryptophan was given to this chromogen in 1890 by Neumeister (645). The chromogen was soon associated with the substance giving rise to indole on bacterial putrefaction of proteins. The failure of many early attempts to isolate tryptophan was probably due to the fact that it is destroyed on acid hydrolysis. The successful isolation by Hopkins and Cole (411) used enzymic hydrolysis of casein, but the chief reasons for their success were their discovery of mercury salts as... 

Initially amino acids were hard to identify because reaction conditions used by chemists to break up the protein usually destroyed amino acids in the process. But as techniques improved, these units were identified by such chemists as Emil Fischer, Sidney W. Cole, and Frederick Gowland Hopkins. The difficulty was then to decide how amino acids were joined to make up different proteins. In the mid-1940s the first complete analysis of the sequence of amino acids that makes up a protein was achieved. More quickly followed, such as the work by Frederick Sanger in which he unraveled the amino acid sequence for insulin. 

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