Insulin 



413 



201 (2161) 



Further observations on the chemical and physical properties 



of insulin. 



By H. A. PIPER, H. A. MATTILL and JOHN R. MURLJN. 



[From the Physiological Laboratory of the University of 

 Rochester , Rochester, N. Y.] 



Probably all active workers with insulin have observed con- 

 siderable variation in the potency of the final product by what- 

 ever 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 HC1 

 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 phenolphtha- 

 lein 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 avail- 

 able potency at once. However if the reaction after filtration 

 is at once readjusted to 5.3 or thereabouts the potency is ren- 

 dered 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 bac- 

 teria 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 HQ ; 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 



i Murlijo, Clough, Gibbs and Stokes: Jour. Biol. Chem., 1923. 



