Mislovicová D, Chudinová M, Vikartovská A, Gemeiner P
Institute of Chemistry, Slovak Academy of Sciences, Slovak Republic.
J Chromatogr A. 1996 Jan 26;722(1-2):143-9. doi: 10.1016/0021-9673(95)00530-7.
A method based on the flow microcalorimetric determination of catalytic activity of immobilized enzyme in a so-called enzyme thermistor was used to monitor the process of lectin affinity chromatography of invertase on Concanavalin A-bead cellulose. The strong biospecific interaction between Concanavalin A and invertase was employed to determine the bound enzyme and this principle was used for the investigation of an alternative direct method for monitoring the lectin affinity chromatography of glycoenzymes. The results obtained by flow microcalorimetry showed that the catalytic activity of invertase immobilized on Concanavalin A-bead cellulose can be compared directly with the thermometric value delta Tmax. The validity of the method was also confirmed by the enzyme thermistor post-column method, which is based on the determination of the product from the immobilized invertase enzymatic reaction. The adsorption and desorption in the chromatography column were examined by flow microcalorimetry in small samples withdrawn from the column. Attention has been given to the operating parameters and the storage stability of the affinity sorbent. The binding ability of the affinity matrix decreased with the number of consecutive chromatographic runs, although its storage stability was satisfactory.
一种基于流动微量热法测定所谓酶热敏电阻中固定化酶催化活性的方法，被用于监测转化酶在伴刀豆球蛋白A - 珠状纤维素上进行凝集素亲和色谱的过程。利用伴刀豆球蛋白A与转化酶之间强烈的生物特异性相互作用来测定结合的酶，并且该原理被用于研究一种监测糖酶凝集素亲和色谱的替代直接方法。流动微量热法得到的结果表明，固定在伴刀豆球蛋白A - 珠状纤维素上的转化酶的催化活性可直接与温度测量值ΔTmax进行比较。酶热敏电阻柱后法也证实了该方法的有效性，该方法基于对固定化转化酶酶促反应产物的测定。通过从柱中取出的小样品，利用流动微量热法对色谱柱中的吸附和解吸进行了检测。已关注亲和吸附剂的操作参数和储存稳定性。尽管其储存稳定性令人满意，但亲和基质的结合能力随着连续色谱运行次数的增加而降低。
