Welsch D J, Nelsestuen G L
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Biochemistry. 1988 Sep 20;27(19):7513-9. doi: 10.1021/bi00419a050.
Acetylation of prothrombin fragment 1 in acetate-borate buffer at pH 8.5 resulted in the appearance of increased light absorbance at about 250 nm. Protease digestions resulted in isolation of a single peptide (residues 94-99) with intense absorbance at about 250 nm (estimated extinction coefficient of 5000 M-1 cm-1). Amino acid analysis showed the expected composition except for the absence of His-96. Instead, an unidentified amino acid which had a ninhydrin product with absorption properties similar to those of proline eluted near aspartate. When sequenced, this peptide (YP?KPE containing epsilon-amino-acetyllysine) lacked histidine at the third position but gave a high yield of a PTH derivative that eluted near PTH-Gly from the HPLC column. Fast atom bombardment mass spectrometry of the derivatized 94-99 peptide showed a mass that was 74 units higher than expected. The histidine degradation product was identified as a di-N-acetylated side chain with an opened imidazole ring and loss of C2 of the ring. While a similar degradation pattern has previously been reported during acylation of histidine, the high chemical reactivity exhibited by His-96 was unusual. For example, under conditions sufficient for quantitative derivatization of His-96, His-105 of fragment 1 was not derivatized to a detectable level. Furthermore, His-96 in fragment 1 was at least an order of magnitude more susceptible to degradation than His-96 in the isolated 94-99 peptide. His-96 is therefore one of several neighboring amino acids of the kringle portion of fragment 1 that displays highly unusual chemistry (see also Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946-4952] and Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325-334, Elsevier Science, New York]).(ABSTRACT TRUNCATED AT 250 WORDS)
凝血酶原片段1在pH 8.5的乙酸 - 硼酸盐缓冲液中乙酰化后，在约250 nm处出现吸光度增加。蛋白酶消化后分离出一个单一肽段（第94 - 99位氨基酸残基），在约250 nm处有强烈吸光度（估计消光系数为5000 M-1 cm-1）。氨基酸分析显示，除了缺少His-96外，组成符合预期。相反，在天冬氨酸附近洗脱了一种未鉴定的氨基酸，其茚三酮产物的吸收特性与脯氨酸相似。测序时，该肽段（YP?KPE，含ε-氨基乙酰赖氨酸）在第三位缺少组氨酸，但从HPLC柱上洗脱的PTH衍生物产量很高，其洗脱位置靠近PTH - 甘氨酸。对94 - 99肽段进行衍生化后的快原子轰击质谱显示，其质量比预期高74个单位。组氨酸降解产物被鉴定为具有开环咪唑环且环上C2缺失的二 - N - 乙酰化侧链。虽然之前报道过组氨酸酰化过程中存在类似的降解模式，但His-96表现出的高化学反应活性并不寻常。例如，在足以使His-96定量衍生化的条件下，片段1的His-105未衍生化至可检测水平。此外，片段1中的His-96比分离出的94 - 99肽段中的His-96对降解的敏感性至少高一个数量级。因此，His-96是片段1kringle部分几个相邻氨基酸之一，表现出非常特殊的化学性质（另见Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946 - 4952]和Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325 - 334, Elsevier Science, New York]）。（摘要截短至250字）
