Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography

Alexander Kurosky; Brian T. Miller; Susan L. Knock

doi:10.1016/0021-9673(93)80534-F

Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography

Alexander Kurosky, Brian T. Miller, Susan L. Knock

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	281-287
Number of pages	7
Journal	Journal of Chromatography A
Volume	631
Issue number	1-2
DOIs	https://doi.org/10.1016/0021-9673(93)80534-F
State	Published - Feb 12 1993
Externally published	Yes

ASJC Scopus subject areas

Analytical Chemistry
Biochemistry
Organic Chemistry

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10.1016/0021-9673(93)80534-F

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@article{b42ba2ee318d4a0785079535380d771d,

title = "Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography",

abstract = "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.",

author = "Alexander Kurosky and Miller, {Brian T.} and Knock, {Susan L.}",

note = "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.",

year = "1993",

month = feb,

day = "12",

doi = "10.1016/0021-9673(93)80534-F",

language = "English (US)",

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pages = "281--287",

journal = "Journal of Chromatography A",

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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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JO - Journal of Chromatography A

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Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography

Abstract

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