TY - JOUR
T1 - Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography
AU - Kurosky, Alexander
AU - Miller, Brian T.
AU - Knock, Susan L.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants NS 29261 (A.K.) and NS 07185 (W.D. Wllis, Jr.), by Grant H-l 190 from the Robert A. Welch Foundation (A.K.), and a grant from the Pearl and Aaron Forman Research Foundation (B.T.M.). We thank Ms. Angelina Mouton for preparation of the manuscript and Mr. J. Steve Smith and Ms. Anna Garcia for expert technical assistance.
PY - 1993/2/12
Y1 - 1993/2/12
N2 - A procedure employing C18 reversed-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 · 10-2 s-1 and 1.08 · 10-2 s-1, respectively in 0.05 M sodium 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.
AB - A procedure employing C18 reversed-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 · 10-2 s-1 and 1.08 · 10-2 s-1, respectively in 0.05 M sodium 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.
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U2 - 10.1016/0021-9673(93)80534-F
DO - 10.1016/0021-9673(93)80534-F
M3 - Article
C2 - 8450022
AN - SCOPUS:0027439644
SN - 0021-9673
VL - 631
SP - 281
EP - 287
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1-2
ER -