Farries T C, Harris A, Auffret A D, Aitken A
Pharmacia LKB Biochrom Ltd, Cambridge, England.
Eur J Biochem. 1991 Mar 28;196(3):679-85. doi: 10.1111/j.1432-1033.1991.tb15865.x.
Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.
酰基肽水解酶是一种从N端乙酰化肽中去除修饰残基的酶，已从羊肝脏中纯化出来，并发展成为一种用于对封闭肽和蛋白质进行测序的工具。其不稳定性对该哺乳动物酶在蛋白质化学中的应用造成了重大限制。与琼脂糖凝胶偶联，然后用二甲基辛二亚胺进行分子内交联，提高了其热稳定性，并使其更能抵抗SDS或N，N-二甲基甲酰胺的失活作用。所得的酶制剂可重复使用，并且在切割较长的乙酰化肽时更有效。因此，它在蛋白质序列分析之前用于解封乙酰化蛋白质很有用。完整的蛋白质和许多分离的肽仍然太大而无法直接切割，但在本文中我们描述了一种克服这一困难的方法。将蛋白质片段化，然后用异硫氰酸玻璃吸收非乙酰化肽。N端肽保留在溶液中，并用稳定化的酰基肽水解酶解封。无需色谱分离。然后可以通过自动Edman降解获得N端序列。该方法已在一种大型合成肽上成功得到验证。
