Evaluation of Biotin-Dye Conjugates for Use in an HPLC Assay To Assess Relative Binding of Biotin Derivatives with Avidin and Streptavidin

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• 作者：D. Scott Wilbur ; Pradip M. Pathare ; Donald K. Hamlin ; Milah B. Frownfelter ; Brian B. Kegley ; Wai-Yee Leung ; and Kyle R. Gee
• 刊名：Bioconjugate Chemistry
• 出版年：2000
• 出版时间：July 2000
• 年：2000
• 卷：11
• 期：4
• 页码：584 - 598
• 全文大小：204K
• 年卷期：v.11,no.4(July 2000)
• ISSN：1520-4812

文摘

In this investigation, studies were conducted to determine if size exclusion HPLC could be used toassess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives fromavidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, moleculesthat can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of thechromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with theHPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin includeddansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue,Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugateswere initially evaluated to determine their peak characteristics on two different size exclusion HPLCcolumns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of anequal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes testedhad association rates within a factor of 3× (slower) that of biotin. The relative dissociation rate ofbiotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with alarge excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resultingin very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, wasselected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cblconjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that theiminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLCassay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation ratethan the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b boundtightly with Av in the absence of added biotin, it was completely released within 1 h when challengedby an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicatethat CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-ratesof biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cblconjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.