Assignment of the histidine proton magnetic resonance peaks of soybean trypsin inhibitor (Kunitz) by a differertial deuterium exchange technique.

Deuterium exchange at the C(2)-H position of the two histidine residues of native soybean trypsin inhibitor (Kunitz) in 2-H2O was followed by 1-H nuclear magnetic resonance (NMR) spectroscopy. The two histidine residues of soybean trypsin inhibitor exchange at significantly different rates at pH* 5.00, 40 degrees. Half-times observed were: peak H1, t1/2=61 plus or minus 2 days; peak H2, T1/2=24 plus or minus 2 days. Differentially deuterated soybean trypsin inhibitor was cleaved by cyanogen bromide into two fragments each containing one histidine residue. The deuterium content of the histidine residue of each separated fragment was analyzed by 1H NMR spectroscopy. Hisidine-71 in fragment 1-114 showed approximately twice the deuterium content of His-157 in fragment 115-181. These results lead to the assignment of 1H NMR peak H1 to His-157 and peak H2 to His-71. These assignments were extended to the histidine peaks of trypsin-modified soybean trypsin inhibitor by converting the differentially deuterated virgin soybean trypsin inhibitor to the modified form. The correlation of histidine peaks in virgin amd modified soybean trypsin inhibitors was the same as proposed earlier on the basis of pK arguments. The results demonstrate that His-71 is the residue whose pK value is raised from 5.27 to 5.91 on trypsin modification of soybean trypsin inhibitor [Markley, J. L., (1973), Biochemistry 12, 2245].