Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions.
The effects of additives on the nonenzymatic deamidation of an Asn residue in a peptide and racemization of Asp and/or Asn in lysozyme were investigated at pH 6 and 100 degrees C. These chemical reactions were accelerated by the addition of phosphate ions. Several salts suppressed the deamidation in the presence of phosphate ions, while the salts did not affect the deamidation in the absence of phosphate ion at pH 6 and 100 degrees C. The results indicated that the effect of the salts was due to the suppression of phosphate catalysis. On the other hand, trifluoroethanol (TFE), which induces the conversion of random coiled polypeptides to secondary structured ones, dramatically suppressed the deamidation of an Asn residue in a peptide. The rate of deamidation in the presence of TFE was comparable to that of asparagine (free amino acid), which was very slowly deamidated. Because TFE could not suppress the deamidation of free asparagine, the suppression of the deamidation of an Asn residue in a peptide was attributed to suppression of the catalysis by the peptide bond in the carboxyl terminus. Since the inactivation of lysozyme was caused by multiple chemical reactions such as the deamidation and racemization, it was expected that the inactivation of lysozyme could be prevented by the addition of salts or TFE. Thus, it was confirmed that salts and TFE suppressed the lysozyme inactivation at pH 6 and 100 degrees C.
Tomizawa
H
H
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Yamada
H
H
Wada
K
K
Imoto
T
T
D000577:Amides; D004151:Dipeptides / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008970:Molecular Weight; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011489:Protein Denaturation; D012492:Salts / Q000494:pharmacology; D013237:Stereoisomerism; D014270:Trifluoroethanol
D000577:Amides; D004151:Dipeptides; D007202:Indicators and Reagents; D012492:Salts; D014270:Trifluoroethanol; D009113:Muramidase