Rao S V, Anderson K W, Bachas L G
Department of Chemical and Materials Engineering, University of Kentucky, Lexington 40506, USA.
Bioconjug Chem. 1997 Jan-Feb;8(1):94-8. doi: 10.1021/bc960080p.
A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.
开发了一种方法来测定生物素化蛋白质缀合物中生物素的总量。在本案例研究中使用了牛血清白蛋白、碱性磷酸酶和辣根过氧化物酶的缀合物。通过完全酸水解或使用蛋白酶K进行酶消化以从生物素化蛋白质中释放生物素，然后使用耦合的HPLC结合测定系统对生物素进行灵敏检测，来确定生物素化程度。该检测系统基于生物素增强链霉亲和素-FITC的荧光。将通过该方法测定的生物素化程度与通过基于从抗生物素蛋白结合位点置换染料4-羟基偶氮苯-2-羧酸（HABA）的传统比色法获得的值进行比较。结果发现，由于所述方法测定水解后释放的生物素量，因此不会受到与蛋白质表面上的生物素从抗生物素蛋白置换HABA的能力相关的空间位阻问题的影响。因此，该方法可以更准确地测定生物素化程度。还确定，与蛋白酶K的酶消化相比，生物素化蛋白质的酸水解在释放共轭生物素方面更有效。
