Stabilization of proteins by guanidination.

Earlier studies have indicated the marked resistance of two pronase endopeptidases to denaturation in high concentrations of urea or guanidine hydrochloride (Siegel, S., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 3233--3240). One component has only a single residue of lysine and the other has none. The consideration arose that lysine-containing peptide segments may be less stable than those containing arginine because of the fluctuations of the side groups of the former residue. The small epsilon amino groups may not be able to sustain solvation of the hydrophobic arm in an aqueous medium. Arginine residues have shorter hydrophobic arms, larger hydrophilic groups, and higher pKa values and, thus may be less motile than lysine. The hypothesis was tested by guanidination of seven globular proteins (bovine carbonic anhydrase, chymotrypsinogen, alpha-lactalbumin, serum albumin, ribonuclease, hen egg lysozyme, and horse heart cytochrome c). Conversion of lysine residues to homoarginine was between 90 and 99%. Tritium-hydrogen isotope exchange revealed that all proteins except lysozyme demonstrated reduced out-exchange after guanidination. The results with lysozyme were not unexpected since only this protein has a high arginine to lysine ratio. These findings suggest that high arginine to lysine ratios contribute to protein stability.