Isolation and Characterization of 101-β-Lysozyme That Possesses the β-Aspartyl Sequence at Aspartic Acid-101

Hidenori Yamada, Tadashi Ueda, Ryota Kuroki, Toshimitsu Fukumura, Takanori Yasukochi, Tatori Hirabayashi, Kahee Fujita, Taiji Imoto

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)7953-7959
Number of pages7
JournalBiochemistry
Volume24
Issue number27
DOIs
Publication statusPublished - Dec 1 1985

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Muramidase
Aspartic Acid
Biochemistry
Diatomaceous Earth
Derivatives
Chitin
aspartylglycine
Affinity chromatography
Carbodiimides
Peptides
Proteins
Esterification
Cross Reactions
High performance liquid chromatography
Reverse-Phase Chromatography
Affinity Chromatography
Adsorbents
Hydrolysis
Esters
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Yamada, H., Ueda, T., Kuroki, R., Fukumura, T., Yasukochi, T., Hirabayashi, T., ... Imoto, T. (1985). Isolation and Characterization of 101-β-Lysozyme That Possesses the β-Aspartyl Sequence at Aspartic Acid-101. Biochemistry, 24(27), 7953-7959. https://doi.org/10.1021/bi00348a017

Isolation and Characterization of 101-β-Lysozyme That Possesses the β-Aspartyl Sequence at Aspartic Acid-101. / Yamada, Hidenori; Ueda, Tadashi; Kuroki, Ryota; Fukumura, Toshimitsu; Yasukochi, Takanori; Hirabayashi, Tatori; Fujita, Kahee; Imoto, Taiji.

In: Biochemistry, Vol. 24, No. 27, 01.12.1985, p. 7953-7959.

Research output: Contribution to journalArticle

Yamada, H, Ueda, T, Kuroki, R, Fukumura, T, Yasukochi, T, Hirabayashi, T, Fujita, K & Imoto, T 1985, 'Isolation and Characterization of 101-β-Lysozyme That Possesses the β-Aspartyl Sequence at Aspartic Acid-101', Biochemistry, vol. 24, no. 27, pp. 7953-7959. https://doi.org/10.1021/bi00348a017
Yamada, Hidenori ; Ueda, Tadashi ; Kuroki, Ryota ; Fukumura, Toshimitsu ; Yasukochi, Takanori ; Hirabayashi, Tatori ; Fujita, Kahee ; Imoto, Taiji. / Isolation and Characterization of 101-β-Lysozyme That Possesses the β-Aspartyl Sequence at Aspartic Acid-101. In: Biochemistry. 1985 ; Vol. 24, No. 27. pp. 7953-7959.
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T1 - Isolation and Characterization of 101-β-Lysozyme That Possesses the β-Aspartyl Sequence at Aspartic Acid-101

AU - Yamada, Hidenori

AU - Ueda, Tadashi

AU - Kuroki, Ryota

AU - Fukumura, Toshimitsu

AU - Yasukochi, Takanori

AU - Hirabayashi, Tatori

AU - Fujita, Kahee

AU - Imoto, Taiji

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N2 - 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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