Nuclear-Magnetic-Resonance-Spectroscopic Studies of the Amino Groups of Insulin

Abstract

The amino groups of insulin were studied by 1H and 13C NMR spectroscopy in insulin, Zn-free insulin and methylated insulin. By difference spectroscopy it is possible to follow the shift with pH of the .epsilon.-CH2 and .delta.-CH2 proton reasonances of lysine-B29 in insulin. In methylated insulin the dimethyl proton reasonances of glycine-A1, phenylalanine-B1 and lysine-B29 can be followed as a function of pH. In native insulin pKapp [apparent] values of 6.7 and 8.0 are obtained for phenylalanine-B1 and glycine-A1 (the assignment is tentative) and 11.2 for lysine-B29. Separate resonances were observed from the lysine-B29 N.epsilon.-(CH3)2 group for the monomeric and dimeric forms of methylated insulin, which indicates a small change in the environment of lysine-B29 on dimerization. The NMR spectral characteristics of these groups are, in general, consistent with the overall structure of the crystal form of the 2-Zn insulin hexamer.