ISSN:
1573-4927
Keywords:
human transcobalamin II
;
genetic variants
;
cobalamin binding capacity
;
cobalamin binding affinity
;
exchange reaction
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract Electrophoresis and subsequent autoradiography of 57Co-cobalamin (57Co-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° 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00502982
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