Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides.
Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides.
复制标题
肽基脯氨酸二肽顺反异构化动力学研究。
DOI：
10.1016/0021-9673(96)00241-5
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Horváth,C
中科院分区：
文献类型：
--
作者：
Thunecke,F;Kálmán,A;Kálmán,F;Ma,S;Rathore,AS;Horváth,C
采用CZE和HPLC柱内孵育法和特设溶出法研究了含肽基脯氨酸基团的二肽的顺反相互转化平衡和动力学参数。后者的使用是可能的，因为固体肽的构象组成，并因此其特设的解决方案，是充分不同的解决方案在平衡。该方法结合1H-NMR和CZE分析，对Phe-Pro在10°C、pH 8.4的硼酸钠水溶液中的顺反异构化反应得到了非常相似的结果，平衡常数和速率常数的平均值分别为0.34和6.6·10−5s− 1。通过CZE或HPLC进行柱内孵育方法，使构象异构体在色谱柱的前半部分分离，然后在色谱柱中孵育，使其相互转化并达到平衡。随后在柱的第二半中分离得到四个峰。因此，通过测量作为反应时间的函数的构象异构体组成，可以评估速率常数。在10°C下，通过CZE柱内孵育法测定的Phe-Pro异构化的正向速率常数为1.42·10−4s− 1，是通过特设溶出法获得的值的两倍。据信熔融石英毛细管的内壁对异构化具有催化作用。计算机模拟也被用来获得进一步的洞察毛细管内壁的催化活性，这种异构化。而Phe-Pro在含水硼酸盐缓冲液，pH 8.4，与37厘米长的毛细管的实验和模拟配置文件是在极好的协议，四倍更快的相互转化率必须使用匹配的实验配置文件获得的57厘米长的毛细管在其他相同的条件下。采用高效液相色谱柱内孵育法证实了十八烷基硅胶固定相对异构化反应的催化作用。Phe-Pro的顺-反异构化的总速率（其需要在固定相和流动相中的反应）在0°C下比通过NMR在游离溶液中使用含有65%（v/v）磷酸钠（pH 6.5）和35%（v/v）甲醇的移动的相测量的速率快约6倍。本文的结果表明，CZE中的壁或HPLC中的固定相对反应的影响不可忽略。
The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with1H-NMR and CZE analyzes yielded very similar results for the cis-trans isomerization of Phe-Pro in aqueous sodium borate, pH 8.4 at 10°C with an average value of 0.34 and 6.6·10−5s−1for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42·10−4s−1determined by the CZE in-column incubation method for Phe-Pro isomerization at 10°C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0°C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.