RESUMO
Binding of lysozyme with the antibiotics such as penicillin-G, penicillin-V and methicillin at different concentrations and pH was studied by equilibrium dialysis. Co-operative binding isotherms were observed at pH 5.0,7.0 and 9.0 with all the penicillins and the binding ratios decreased slightly with the increase of pH. The Gibbs free energy change calculated on the basis of Wyman's binding potential concept decreased slightly with the increase of pH indicating slight decrease in the binding strength at higher pH in the case of all penicillins. The ultra-violet difference spectra of lysozyme-penicillin complexes showed a less intense peak in the region of 284-300 nm at pH 5.0. Only penicillin-G complex had a peak at pH 7.0 at these wavelengths with less intensity compared to that at pH 5.0. However, none of the penicillins showed discrete peaks in this region at pH 9.0. The appearance of peaks in the difference spectra of all these complexes at pH 5.0 and with only penicllin-G complex at pH 7.0 in the aromatic region indicated hydrophobic interactions with tryptophan residues as the binding sites. In addition, the ionic interactions with lysine residues in lysozyme were also occurring. The conformational changes induced by the binding of penicillins to lysozyme monitored by circular dichroism showed a slight decrease in the aromatic bands in the 320- 250 nm region. However, in the 250-200 nm region, [θ]222nm values obtained at various concentrations of penicillins in the complex indicated an increased α- helical content generating a more ordered structure. These results led to the conclusion that both the hydrophobic and electrostatic interactions prevail in the binding of penicillins to lysozyme.