Distribution of Sulfur in Crystalline Insulin

Abstract

The distribution of the sulfur in insulin has long been a subject of interest. Crystalline insulin contains 3.06 to 3.38% of sulfur. 1 Only part of the total sulfur could be accounted for by cystine, the Folin-Marenzi method yielding 9-10% and the Sullivan method 8-9% of cystine. 2 Methionine has been claimed to be absent. 2 3

A reliable technique for the estimation of methionine in proteins has been established by Baernstein 4 and further developed to include the iodometric determination 4 of cysteine and homocysteine. The Folin method for the determination of cystine has been adapted to the Pulfrich Photometer, 5 resulting in greater specificity. It has been found that the Sullivan method gives more constant and reliable results if the time interval between additions of color reagent and sulfite is regulated 6 (10 seconds measured with a stopwatch).

It was, therefore, thought desirable to determine the distribution of the sulfur in crystalline insulin with the aid of these improved techniques. A sample of such material (crystalline insulin, Lilly T-800) was kindly put at our disposal by Eli Lilly and Company. The insulin was repeatedly extracted with petroleum ether and dried in vacuo at 100°C. Its sulfur content (Pregl) was found to be 3.11 and 3.14%. The ash content was 1.5%. Certain observations 7 may indicate that 1-3% of the total sulfur of this preparation is in the form of sulfate. The presence or absence of such traces of sulfate could not be ascertained directly on account of the large amounts of crystalline insulin necessary.

The experimental data are presented in Table I. The determinations by Baernstein's methods were carried out as described, 4 except for some minor modifications and corrections. 7 .