UBC Theses and Dissertations
Protection of the proteolytic activity of crude papain and chemical modification of papain by tetrathionate Arteaga Mac Kinney, Guillermo Eleazar
In the first chapter, sodium tetrathionate (TT), a sulfhydryl blocking agent, is assessed for its ability to protect the proteolytic activity (PA) of papaya latex during air, sun or vacuum drying, and of crude papain during storage. By means of Taguchi's L₂₇ (3¹³) fractional factorial design, it was found that the addition of 1% TT significantly increased the retention of PA of papaya latex when it was air dried at a temperature of 55°C. This protection of PA was found to be 23% higher than the one given by the addition of 1% sodium metabisulfite, the compound commonly used in the commercial processing of papaya latex. When drying was carried out either under 27 inches vacuum at 50°C or in the sun, the protective effect of TT on the PA was not significantly different from that of metabisulfite. The PA of crude papain during storage at room temperature was also protected by TT. A loss of 20% of the original PA occurred over a period of 13 wk when crude papain contained 1% TT, compared to a loss of 45% when the crude enzyme preparation contained 1% metabisulfite. In the same chapter five different oxidants for synthesis of TT from thiosulfate are compared, namely: iodine, hydrogen peroxide, ferric chloride, cupric sulfate and sodium vanadate. The results indicated that hydrogen peroxide or sodium vanadate were not only effective in the oxidation but also much less expensive than iodine, which is the most popular oxidant for the synthesis of TT. The results obtained in this chapter warrant the use of TT in the commercial production of commercial papain to prevent the destruction of the enzymes during harvesting, storage, transportation and processing. In the second chapter, chemical modification of pure papain by TT is discussed. Optimization techniques were applied for improving the precision of two methods used in this study: circular dichroism (CD) and proteolytic activity determination. Simplex optimization significantly improved repeatability and signal to noise ratio of the CD scan of papain. A new optimization approach, which was a combination of a central composite rotatable design and simplex optimization, was successfully applied to achieve maximum precision for the proteolytic activity assay of papain using casein as a substrate. This approach may also be applied to other analytical methods to improve the reliability of the experimental data. Influential factors in the inactivation of PA of papain by using TT and reactivation of the inactivated papain by cysteine were carried out using two Taguchi's L₁₆ (2¹⁵) fractional factorial designs. The results indicated that when inactivation was carried out at pH 6.8, with a reaction time of 5 min at 22°C, and a molar ratio of TT to papain of 10, the inactivation reaction was highly reversible upon addition of 20 mM cysteine. Although some interactions of the factors were significant, 70% reactivation was achieved in most cases. Analysis of UV absorbance, near-UV and far-UV CD spectra indicated that there were no major changes in the spectra in papain upon the chemical modification of the enzyme with TT. Secondary structure computed from far-UV CD spectra also demonstrated no significant changes upon this modification. Sulfhydryl data and pH-fluorescence profiles of the modified papain support the hypothesis that reversible blocking by TT results from binding with the single reactive cysteine residue present in papain. Quenching of the intrinsic fluorescence of papain when the modification was carried out using high molar ratios of TT to papain was suggestive of modification of tryptophan residues in the enzyme during the oxidation reaction with TT. Precipitation or insolubilization of pure papain, and of the proteins of papaya latex and commercial papain was observed upon the chemical modification with TT under certain conditions. Addition of β-mercaptoethanol and TT at levels of 100 mM and 50 mM, respectively, precipitated 90% of pure papain. Solubility studies together with electrophoretic analysis of the precipitated papain suggested formation of insoluble aggregates due to the insoluble aggregation as a result of inter-molecular disulfide bonds formation. TT was found to be a competitive inhibitor of both reversible and irreversible inhibition of the enzyme action, when carbobenzoxyglycine p-nitrophenyl ester was used as a substrate. The second order inactivation constant in the absence of substrate was computed to be 16,919 M⁻¹sec⁻¹, indicating that the reaction had a high rate.
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