A Comparison of the Properties of Chicken Serum with Other Vitamin B₁₂ Binding Proteins Used in Radioisotope Dilution Methods for Measuring Serum Vitamin B₁₂ Concentrations

Abstract

The effects of pH, ionic strength, and vitamin BIZ concentration on the vitamin B,,-binding capacities of chicken serum, normal human serum, chronic myelogenous leukaemia serum, and hog intrinsic factor concentrates are compared. The influence of pH on Biz-binding capacity was shown to be greatest at low pH and that of ionic strength was most evident below 0.1 ionic strength, particularly at acidic pH. The effect of Bi2 concentration on Biz-binding capacity was shown to vary with different binders, the pH of binding, and the time of incubation.It was possible to select conditions for carrying out the binding of BIZ to chicken serum so that the amount of BI2 bound was virtually unaffected by the kind of variations in pH, ionic strength, or concentration of Biz that could occur in a radioisotope dilution assay of serum vitamin Biz. Under these conditions it was further demonstrated that the binding was unaffected by the presence of denatured human serum. In addition chicken serum had a very great binding capacity and affinity for BIZ, was very stable on storage and was compatible with the use of protein-coated charcoal.Some conclusions on the optimal use of BI2-binding protein in the radioisotope dilution assay of serum vitamin BIZ are made and the high potentiality of chicken serum in such a system is suggested.In recent years many attempts have been made to set up satisfactory ahnatives to microbiological assays of serum vitamin B,,$ by use of the technique of radioisotope dilution.There are three main steps in such techniques. First the serum vitamin B,, is released from its protein complexes. Secondly the released BIZ is allowed to compete with a known amount of isotopically labelled B12 in binding to a limited amount of B,,-binding protein. And lastly the protein-bound portion of the B, , is separated from that remaining free.

Activation Parameters for the Carbon−Cobalt Bond Homolysis of Coenzyme B12Induced by the B12-Dependent Ribonucleotide Reductase fromLactobacillus leichmannii

“…Finally, it must be pointed out that such steric compression of the axial Co−N bond of AdoCbl would come at a substantial energy cost. However, it is very clear from studies of vitamin B 12 binding proteins that at least some proteins are capable of binding cobalamins very tightly, generating large amounts of binding energy. − In the extreme case, the haptocorrin from chicken serum binds CNCbl with a binding constant, K b , of 9.3 × 10 15 M -1 at 37 °C, representing the release of 22.7 kcal mol -1 of binding free energy. Should the intrinsic affinity of AdoCbl for RTPR be this high, the observed rather weak binding ( K b = 2.4 × 10 4 , Δ G b = −6.2 kcal mol -1 ) suggests that a large amount of binding free energy (as much as 16.5 kcal mol -1 ) is potentially available for distortion of the coenzyme by compression of its axial Co−N bond.…”

Structural and Enzymatic Studies of a New Analogue of Coenzyme B12 with an α-Adenosyl Upper Axial Ligand

“…It is clear that proteins are capable of binding cobalamins extremely tightly, as seen in the affinity of B 12 binding proteins for CNCbl (66)(67)(68) which, in the case of haptocorrin, can have binding constants as high as 5 × 10 16 M -1 , representing nearly 23 kcal mol -1 of binding free energy (68). AdoCbl-requiring enzymes bind AdoCbl much less tightly, so significant binding free energy may well be available for distorting the coenzyme, a process which may be an integral part of normal catalysis (vide infra).…”