Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography.
Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography.
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通过高效液相色谱法对肽的特定残基的生物素化进行动力学分析。
DOI：
10.1016/0021-9673(93)80534-f
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Knock,SL
中科院分区：
文献类型：
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作者：
Kurosky,A;Miller,BT;Knock,SL
采用C18反相高效液相色谱法（HPLC），研究了生物素与N-羟基琥珀酰亚胺酯反应后，多肽中特定残基的生物素化动力学。利用该HPLC方法，我们测定了两种模型肽[生物素-Ser 108] ProA-产卵激素（108-121）和pGlu-Lys-Trp-Ala-Pro中赖氨酰残基生物素化的伪一级反应速率常数（k′1），在25°C下，0.05M磷酸钠缓冲液（pH 8.2）中分别为1.22 · 10−2s− 1和1.08 · 10−2s−1。两种肽的各自反应半衰期为57 s和64 s。此外，建立了HPLC分析方法，用于测定生物素化试剂在酸性和碱性pH值下的水解时间过程，并用于评价生物素试剂的均匀性。
A procedure employing C18reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method we determined the observed pseudo-first-order reaction rate constant (k′1) of biotinylation of lysyl residues in two model peptides [biotinyl-Ser108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22 · 10−2s−1and 1.08 · 10−2s−1, respectively in 0.05Msodium phosphate buffer, pH 8.2, at 25°C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic alkaline pH and for evaluating biotin reagent homogeneity.