Arginine and Its Possible Relation to the Physiological Activity of Insulin.

Abstract

Recently 1 we stated that insulin (21 units per mg.) gives positive color reactions with picric acid, with m-dinitrobenzene, and with m-dinitrobenzoic acid (1.3.5). It seemed that the presence of labile sulfur in insulin sufficed to account for the color reactions obtained. But among the aminoacids that yield positive reactions with the above mentioned reagents, arginine was conspicuous for the strength of its picric acid reaction.

Color reactions specific for arginine have been devised by Harden and Norris, 2 and more recently by Sakaguchi. 3 On testing our insulin we found both reactions strongly positive. Insulin, similar in purity to our preparation, yielded with the van Slyke method 35 per cent of diamino nitrogen in one instance 4 and 10 per cent of arginine nitrogen in another. 5 Since only small amounts of insulin were at our disposal, we could not attempt to isolate arginine by acid hydrolysis. 6 We, therefore, tried an enzymatic hydrolysis, which may be carried out with relatively small amounts of insulin (21 mg.). In addition, we expected this enzymatic method would throw some light upon the structure of the insulin molecule, as well as yield information concerning certain enzymatic problems. 7 The method has the advantage of being highly specific, thereby eliminating many sources of error. 8

We determined the arginine from the amount of NH₃ liberated from 440 units of insulin (21 mg. containing a total of 3.4 mg. of nitrogen) by thie combined action of trypsin + trypsin-kinase, 9 erepsin, arginase and urease (18 hours, 37° C., pH = 7.4, 0.2 M phosphate buffer). As a contiol we used the amount of NH₃ which was liberated under identical conditions from the same amount of insulin by the combined action of trypsin + trypsin-kinase, erepsin and urease, but omitting the arginase.