Porck H J, Frants R R, Lindemans J, Hooghwinkel G J, Planta R J
Biochem Genet. 1986 Feb;24(1-2):103-14. doi: 10.1007/BF00502982.
Electrophoresis and subsequent autoradiography of 57Co-cobalamin (57 Co-Cbl)-labeled serum show intensity differences between the genetic variants of human transcobalamin II (TC2), suggesting differences in the unsaturated (apo-) TC2 concentration. In order to distinguish between variant-specific differences in the Cbl binding affinity and those in the total-TC2 concentration, techniques were developed to determine total, apo-, and holo-TC2. Prolonged incubation at 37 degrees C with a 20-fold excess of 57Co-Cbl resulted in an almost complete exchange of endogenously bound Cbl, which allowed determination of the total TC2. The holo-TC2 concentration of both gene products in TC2 heterozygotes could be estimated by comparison of the labeling levels of apo- and total TC2, using densitometric quantification of the autoradiographs. By means of ion-exchange chromatography, TC2 could be separated from other Cbl-binding proteins, permitting a simple quantitative assay of apo- and total TC2, the results of which correlate fairly well with those measured by an immunoadsorption assay. The results obtained in the present investigation indicate that the variant-specific variation in the apo-TC2 concentration is caused by differences in the total-TC2 concentration rather than in the Cbl binding affinity.
对用⁵⁷Co-钴胺素（⁵⁷Co-Cbl）标记的血清进行电泳及随后的放射自显影，结果显示人转钴胺素II（TC2）基因变体之间存在强度差异，提示不饱和（脱辅基）TC2浓度存在差异。为了区分钴胺素结合亲和力的变体特异性差异与总TC2浓度的差异，开发了测定总TC2、脱辅基TC2和结合钴胺素的TC2（全酶形式的TC2）的技术。在37℃下与20倍过量的⁵⁷Co-Cbl长时间孵育，导致内源性结合的钴胺素几乎完全交换，从而能够测定总TC2。通过比较脱辅基TC2和总TC2的标记水平，利用放射自显影片的光密度定量分析，可以估算TC2杂合子中两种基因产物的全酶形式的TC2浓度。借助离子交换色谱法，可将TC2与其他钴胺素结合蛋白分离，从而对脱辅基TC2和总TC2进行简单的定量测定，其结果与免疫吸附测定法测得的结果相当吻合。本研究获得的结果表明，脱辅基TC2浓度的变体特异性差异是由总TC2浓度的差异而非钴胺素结合亲和力的差异引起的。
