Fuglsang Anders, Nilsson Dan, Nyborg Niels C B
Royal Danish School of Pharmacy, Department of Pharmacology, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark.
J Biochem Biophys Methods. 2004 Feb 27;58(2):139-51. doi: 10.1016/j.jbbm.2003.10.004.
A new method for determination of first-order elimination constants for dipeptides is presented. The peptides are hydrolysed by plasma enzymes into amino acids, and ortho-phthaldialdehyde (OPA) is used to react with free primary amino groups. The concentration of free amino groups can, thus, be followed using simple spectrophotometry. A mathematical model for the concentration of free primary amino groups with time is presented through which the elimination constant, and thus the half life, can be determined by curve fitting. The method is applied to inhibitors of angiotensin-converting enzyme derived from the primary structure of milk proteins. The results show that these dipeptides have in vitro half lives ranging from 4.3-64 min, when incubated with 50% rat plasma. This explains why these casokinins in vivo only cause a very moderate and short-lasting inhibition. The model for calculation of elimination constant is limited to dipeptides that do not contain a C-terminal proline. The derivatization method can be applied to longer peptides as a crude indicator of peptide hydrolysis, but does not allow calculation of their elimination constants per se.
本文提出了一种测定二肽一级消除常数的新方法。肽被血浆酶水解成氨基酸，邻苯二甲醛（OPA）用于与游离伯氨基反应。因此，游离氨基的浓度可以通过简单的分光光度法进行跟踪。提出了一个游离伯氨基浓度随时间变化的数学模型，通过曲线拟合可以确定消除常数，进而确定半衰期。该方法应用于源自乳蛋白一级结构的血管紧张素转换酶抑制剂。结果表明，当与50%大鼠血浆孵育时，这些二肽的体外半衰期为4.3 - 64分钟。这解释了为什么这些酪蛋白激肽在体内仅引起非常适度且持续时间短的抑制作用。消除常数的计算模型仅限于不含C末端脯氨酸的二肽。衍生化方法可应用于较长的肽，作为肽水解的粗略指标，但本身不允许计算其消除常数。
