Effect of Charge Neutralization at Lysine Residues on the Free Energy of Stabilization of Hen Egg White Lysozyme

Positive charge at lysine residues of hen egg white lysozyme (HEWL) was neutralized through carbamylation reaction and its effect on the conformational stability of the enzyme was studied. Additionally, guanidination of HEWL was also performed, in which the modified derivative retained the positive charge at lysine residues, but with the addition of guanidino group. About 88% and 85% of lysine residues of HEWL were modified in carbamylated (C88) and guanidinated (G85) preparations, respectively. Polyacrylamide gel electrophoresis and Sephadex G-75 gel chromatographic results confirmed the charge and size homogeneity, respectively, of the modified derivatives. A comparison of Stokes radius and frictional ratio values of native (1.31 nm; 0.83), C88 (1.39 nm; 0.87) and G85 (1.20 nm; 0.75) HEWLs suggested an increase in the hydrodynamic volume of C88 HEWL and a more compact conformation for G85 HEWL. Decrease in free energy of stabilization (∆GD2) from 8258 and 8918 cal/mol (for native HEWL) to 6362 and 6137 cal/mol (for C88 HEWL) as obtained from GdnHCl denaturation studies using fluorescence intensity and emission maximum probes, respectively, clearly indicated destabilization of the enzyme upon carbamylation. This destabilization can be attributed to the increase in electrostatic free energy in C88 HEWL. These findings were further supported by ∆GD2 values obtained for G85 HEWL (8092 and 8386 cal/mol), in which retention of positive charge on HEWL was responsible in maintaining the stability of the enzyme. All these results revealed significant contribution of positively charged lysine residues in the conformational stability of HEWL through salt bridges.