Spectroscopic Studies on the Reduction and Reformation of the Disulfide Bonds of Papaya Lysozyme

Abstract

The study of the reduction and reformation of the four disulfide bonds cross‐linking the single polypeptide chain of papaya lysozyme (molecular weight 28000) has been undertaken. The four disulfide bonds were quantitatively reduced and carboxymethylated. Various physicochemical measurements, such as optical rotatory dispersion, circular dichroism and fluorescence showed that the polypeptide chain of the reduced inactive papaya lysozyme appears to behave as a random coil. The conformational changes can be characterized as a complete loss of helical structure and an extensive exposure of the aromatic chromophores. Partial reduction of the protein (with splitting of, most probably, a single disulfide bond) does not affect the enzymatic activity. Physico‐chemical studies indicated that the secondary and tertiary structure of this derivative are very similar with those of the native enzyme. Reoxidation of the completely reduced enzyme, on exposure to air, produces a protein which could not be distinguished from the native molecule with respect to optical rotatory dispersion, fluorescence, solvent perturbation and enzymatic activity. The results of this study show that the primary structure of papaya lysozyme is sufficient to direct the refolding of the reduced protein in order to reform the native three‐dimensional conformation.