In the reaction of the intramolecular cross-linking between Lys-13 (ε-NH3 +) and Leu-129 (α-COO-) in lysozyme using imidazole and l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-l 12 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-l 12 in 6 N HC1 at 110 °C gave a considerable amount of β-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the β-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the β-aspartyl sequence at Asp-101 (101-β-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the β-aspartyl sequence at Asp-101 (101-β-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio. Therefore, it is concluded that the Asp-52 ethyl ester lysozyme reported had been a mixture of the derivatives with the α- and β-aspartyl sequences at Asp-101. The mechanism for the formation of 101-β-lysozyme in these reactions is discussed.
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