ROLE OF DIVALENT-CATIONS IN THE NOVEL BACTERICIDAL ACTIVITY OF THE PARTIALLY UNFOLDED LYSOZYME

It was recently found that partial denaturation of hen egg white lysoz yme at 80 degrees C for 20 min at pH 6.0 produces a strong bactericida l lysozyme (HLz) not only against Gram-positive bacteria but also agai nst the Gram-negative ones. The novel antimicrobial action of the mild ly denatured lysozyme was found to operate through a membrane-disrupti ng mechanism independent of its muramidase activity. To evaluate the r ole of Ca2+ and Mg2+, which are known to stabilize the structure of ba cterial membranes, in the antimicrobial activity of HLz, the antimicro bial activity in the presence of divalent cations and chelator EDTA wa s tested. The antimicrobial activity of HLz against test microorganism s Staphylococcus aureus and Escherichia coli K12 progressively decreas ed with an increase in Ca2+ or Mg2+ concentration, whereas 1 mM cation produced nonbactericidal lysozyme, either the native (NLz) or the hea ted enzyme (HLz). However, the inhibitory effect of divalent cations o n the antimicrobial activity was more pronounced on HLz than on NLz. T he antimicrobial activity of HLz against Gram-negative E. coli, which has been abolished in the presence of free Ca2+ (1 mM), was recovered by the addition of 1 mM EDTA, indicating the competition between free and membrane-bound Ca2+ on the part of HLz molecule. Addition of incre asing concentration of NaCl up to 10 mM had no inhibitory effect on th e strong antimicrobial activity of HLz. Thus, the inhibitory effect of divalent cations on the activity of HLz is unlikely to be simply due to charge suppression. Intrinsic fluorescence analysis revealed that C a2+ induces conformational change of the HLz molecules, thus providing evidence that HLz exerts its antimicrobial action against Gram-negati ve bacteria by disrupting the normal electrostatic interactions betwee n divalent cations and components of the outer membrane. Structural ch anges relevant to divalent cations sensitization of HLz are discussed.