57CoB12 was added to serum in vitro to study its binding by the three known serum B12-binders in patients with vitamin B12 deficiency and in normal controls. Gel filtration through columns of Sephadex G-200 was used to separate the low (beta) and high (alpha1 and beta) molecular weight B12-binding fractions. Electrophoresis on filter paper was used to separate the alpha1- and beta-globulins.
The alpha1-globulin fraction in the serum of B12-deficient patients bound more of the added 57CoB12 than did this fraction in normal serum, presumably because this binder of the serum endogenous vitamin B12 is much less saturated in B12-deficiency. However, the total B12 binding capacity of the alpha1-globulin (for endogenous plus added vitamin B12) was lower in B12-deficient than in normal serum.
The low molecular weight beta-binder bound more added 57CoB12 in B12-deficient than in normal serum, whereas the high molecular weight beta binder had a much lower B12-binding capacity in deficient than in normal serum. These abnormalities were independent of the cause of the vitamin B12 deficiency and disappeared after successful treatment with vitamin B12.