Stevens J, Dixon H B
Department of Biochemistry, University of Cambridge, UK.
Biochim Biophys Acta. 1995 Oct 25;1252(2):195-202. doi: 10.1016/0167-4838(95)00118-e.
The N-terminal residue of a protein or peptide may be converted into a 2-oxoacyl group by non-enzymic transamination. This group may then be removed, to obtain the peptide chain shortened by one residue, by treatment with phenylene-1,2-diamine. Hitherto this scission has required a pH of 4-5, but we find that the reaction will proceed well at pH 7 in the presence of concentrated phosphate buffer. We describe a method using reverse-phase HPLC for determining the extent of scission in model peptides; this method also allows products to be isolated and identified. The new scission conditions have been tested by removing the N-terminal residue from cystatin, an inhibitor of cysteine peptidases; electrospray mass spectrometry was used to assess how this protein reacted.
蛋白质或肽的N端残基可通过非酶转氨作用转化为2-氧代酰基。然后可以通过用1,2-苯二胺处理除去该基团，从而获得肽链缩短一个残基的产物。迄今为止，这种断裂需要pH值为4-5，但我们发现，在浓磷酸盐缓冲液存在下，该反应在pH值为7时也能顺利进行。我们描述了一种使用反相高效液相色谱法测定模型肽中裂解程度的方法；该方法还可以分离和鉴定产物。通过从半胱氨酸蛋白酶抑制剂胱抑素中去除N端残基，对新的裂解条件进行了测试；采用电喷雾质谱法评估该蛋白质的反应情况。
