Further observations on the chemical and physical properties of insulin

Abstract

Probably all active workers with insulin have observed considerable variation in the potency of the final product by whatever method obtained. Experience in this laboratory had led us to reinvestigate especially the influence of the exact H ion concentration upon potency at the several stages of the aqueous method. 1

It has been found that after thorough extraction in N/5 HCl neutralizing with N/1 NaOH to P_H of 5.0 to 5.7 gives the largest yield of potency in the first filtrate.

The reaction is controlled both titrometrically to phenolphthalein and electrometrically (error ±0.2P_H). Stopping at a P_H of 4.0 to 4.4 gives more rapid filtration but not so much available potency at once. However if the reaction after filtration is at once readjusted to 5.3 or thereabouts the potency is rendered available or, if it be allowed to stand for 24 hours at P_H 4.0 to 4.4 the potency increases. In fact the filtrate may be kept at room temperature until there is an abundant growth of bacteria and yeasts without destroying the potency.

In the final concentration it has been found that potency is best preserved in a fairly acid medium .06 to .08 N HCl; though a reaction as high as P_H 5.7 preserves for several weeks if an antiseptic like tricresol is added at once.

Pasteurization temperature maintained for half an hour does not destroy the potency. In fact the concentrated extract purified of proteins can be heated to 80° for half an hour, the exact effect depending upon the reaction. At a P_H of 6.2 to 7.0 or higher heating to this point seems regularly to increase the potency on rabbits. Heating at a P_H of 4.4 to 5.7 the potency is usually diminished or carried down with the coagulum if proteins are present.