Precipitation reactions of insulin

Abstract

We here report the results obtained by the addition of various reagents to the aqueous solution of insulin as it is prepared for injection in human cases. Potency tests were made on normal rabbits, the dose given being equivalent to 20 gms. of pancreas except in a few of the earlier experiments where the equivalent of 40 gms. was taken. Only those instances where some positive test was obtained with the same material treated at the same time are reported. Up to date 29 reagents embracing a wide range in chemical nature, have been tried of which two (formaldehyde and ethyl acetate) gave no precipitate of any kind. Next comes a list of 6 which gave a definitely negative result, i. e., contemporary experiments with the same material gave some positive tests with other reagents. It should here be explained that on the addition of the reagents the precipitate is thrown down by centrifuge taken up in sterile water and injected, the supernatant liquid is dried down, with or without dialysis, depending on the nature of the precipitant, and similarly dissolved and injected. The following 6 gave definite negatives: ether, petroleum ether, toluene, xylene, chloroform and cadmium chloride. Five more are probably destructive to the principle, phosphotungstic, phosphomolybdic and tannic acids, MgSO4 and NaSO4 although the evidence is not yet conclusive. Mechanical difficulties were encountered with phenyl hydrazine, pyrogallic and picric acids, which have eliminated them from our list. Too little has been done with UO2Ac2, Zn SO4 and NaCl to justify any inferences. Turning now to the positive results we find that absolute ethyl alcohol is unique in being the only reagent encountered so far that throws down a precipitate from aqueous solution but leaves the insulin in solution.