Hawke D H, Lahm H W, Shively J E, Todd C W
Beckman Research Institute of the City of Hope, Division of Immunology, Duarte, California 91010.
Anal Biochem. 1987 Nov 1;166(2):298-307. doi: 10.1016/0003-2697(87)90578-1.
A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 N HCl (30 min at room temperature) was used for cleavage as originally described by G.R. Stark (Biochemistry 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90 degrees C were 5.6 X 10(-5), 5.1 X 10(-4), and 8.6 X 10(-4) s-1, respectively. In order to obtain complete couplings within 30-40 min at 50 degrees C, the effect of pyridine catalysis was studied. The addition of 0.225 M pyridine resulted in roughly doubling the rates at 50 and 70 degrees C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50 degrees C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.
对基于异硫氰酸酯的肽和蛋白质环化降解化学方法的重新研究表明，试剂三甲基甲硅烷基异硫氰酸酯（TMS - ITC）在偶联效率和缺乏复杂副反应方面给出了更好的结果。按照G.R.斯塔克最初的描述（《生物化学》8, 4735, 1968），使用乙酸酐（在不同温度下10分钟）活化羧基末端，并用6 N盐酸（室温下30分钟）进行裂解。利用缓激肽（一种九肽）的减法化学方法探索了有效偶联的反应条件，并通过反相高效液相色谱法分离反应产物。产物通过快原子轰击质谱分析，结果表明是N - 乙酰化的起始原料和缓激肽的N - 乙酰化去 - Arg9衍生物。在50、70和90℃下测得的准一级速率常数分别为5.6×10⁻⁵、5.1×10⁻⁴和8.6×10⁻⁴ s⁻¹。为了在50℃下30 - 40分钟内实现完全偶联，研究了吡啶催化的效果。加入0.225 M吡啶使50和70℃下的速率大致翻倍。对于缓激肽，在50℃下于吡啶催化剂存在下与TMS - ITC的反应在15分钟内完成。为了将此方法应用于蛋白质分析，合成了氨基酸的硫代乙内酰脲衍生物，并通过反相高效液相色谱法进行分离。这些衍生物也通过质谱进行了表征。在3 nmol抹香鲸肌红蛋白上对上述反应条件进行了三个降解循环的测试。样品首先以90%的产率与对苯二异硫氰酸酯衍生的氨丙基玻璃偶联。第一个循环中甘氨酸的近似初始产率为30%。前三个循环与肌红蛋白预测的羧基末端序列完全一致。这些结果证明了一种新型斯塔克试剂用于自动化羧基末端化学方法的可行性。
