Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange ( P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem. 255, 10828–10833 ). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.