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The Physical Chemistry of Insulin. 11. Hydrogen Ion Titration Curve of Crystalline Zinc Insulin. The Nature of its Combination with Zinc1-:' BY CHARLES TAXFORD AND
JACK
EPSTEIN
RECEIVED ALXUST 21, 103.3
A complete hydrogen ion titration curve has been obtained for crystalline zinc insulin (one atom of Z n per 11,500 g.) in aqueous solution of ionic strength 0.075, a t 25 '. Comparison with the correspouding curve for zinc-free insulin indicates that the zinc is combined with imidazole groups, a i has previously been found for the combination of zinc with serum albumin. However, two imidazole groups are involved per zinc ion. Polarographic rneasureinerits confirm this conclusion. I n contrast t o the titration curve of zinc-free insulin, the present curve is riot reversible. I t was observed, however, that identical "reversed" curves are obtained after exposure to acid and to base. The difference between the direct and reversed curves lies largely in the region of incomplete solubility: the direct curve represciits cquilibrium between hydrogen ion and zinc insulin crystals; the reversed curve, on the othcr haiid, rcpresrnts equilibrium between hydrogen ion and an amorphous zinc insulin precipitate, which is formed when zinc itisuliti is freshly precipitated after solution in either acid or h:tsc. Thc p H of reversed mixtures was found to drift \villi time so ;tb to approach the direct titratioii curve, and, a t thc satnc tiinc, zinc insulin crystals were formed.
The original purpose of the present study was t u elucidate the nature of the interaction between insulin and zinc ions, both from a comparison of the titration curve of zinc-free insulin (cf. preceding paper4) with t h a t of zinc insulin, and from polarographic measurements. The solution to this problem proved t o be straightforward. As in the case of serum albumin,5,6 zinc ions appear t o combine preferentially with imidazole groups. In contrast to serum albumin, however, each zinc ion combines with two rather than one imidazole group. X second problem of great interest presented itself, however, as this work got under way. For, not only was the expected differencefound between the behavior of zinc insulin and that o f zinc-free insulin, but, in addition, entirely unexpected tiifferences were found between the behavior of suspensions of zinc insulin between pH 3.3 and S, and of similar mixtures which had first been dissolved by the addition of acid or alkali, and then returned t o this pH region. LIuch of the paper is therefore concerned with this phenomenon. Experimental The sanie procedures and reagents \ i c r c u i e t l :IS i n t h c preceding paperJ except for the folloiiiiig. Insulin.-A sample of fivc tirnci reci-~~t,tlIizctlh i \ iiic zinc insulin, Lot No. T-2842. was supplied 1)). Eli L i l l ~ .L t I ( i Co. The sample writs statcrl to have :in activit) of 2 i . 5 u.,/nig., arid a zinc content of 0.429yo, i . c z . , 0 . i l i zinc ions licr insulin monomer.' Since the ana1ytic;tl proceduw l>y ivhicli this zinc content was measured, t - , S . P.XI\., 1,. Z)!). i i subject to considerable error,R the zinc contciit \ \ , I \ rctlctcrmined by us, using a polarographic mrthod.': A wrne\rh:tt higher value was found, equiialcilt to (1.9.5 (1.05 ZII in-ulin monomer. IYithin the experinicntal crror, therefore, the zinc content is 1.O Zn; insulin inoiionii'r. . ~ n dthis figure
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(1) Presented a t the 1 2 4 t h meetinp of t!ir American Clieniir.il 5 8 ciety, Chicago, Ill., Sept 6 - 1 I . 195.3 (2) This investigation has received support from a resedrcli
