Henrik Olesen

Nature of vitamin B12 binding: III. Thermodynamics of binding to human intrinsic factor and transcobalamins

Abstract Thermodynamic parameters for binding of cyanocobalamin to intrinsic factor, transcobalin I, and transcobalamin II have been determined by Sephadex gel filtration. The equilibrium constant at pH 7.4, 26° was 6·10 9 M −1 for intrinsic factor, and about 300·10 9 M −1 for the two transcobalamins. The index of heterogeneity was near unity for all three binders. The reaction between cyanocobalamin and intrinsic factor was exothermic, ΔH ° being —22.7 kcal/mole and ΔS ° —31 cal/degree per mole. There was no significant change in heat content on vitamin B 12 binding to transcobalamins. These latter reactions were entropy driven, ΔS ° being +66.8 cal/degree per mole for transcobalamin I and +49.3 cal/degree per mole for transcobalamin II. Exposure to guanidin resulted in a decrease in free energy of reaction of 2.5 to 5.0 kcal/mole for the three binders. However, despite the use of 7 M guanidin there still remained a considerable binding strength, and especially so for transcobalamin I. The free energy of reaction for cyanocobalamin and intrinsic factor was only moderately affected at pH between 2 and 10. For transcobalamin I and transcobalamin II the binding strength was unchanged between pH 4 and 11.

Plasma Clearance of 57Cobalt-Labelled Vitamin B12 Bound in Vitro and in Vivo to Transcobalamin I and II

Radioactive-labelled B12 bound to transcobalamin I has an initial distribution volume twice the plasma volume, a fractional turnover rate of 45 to 60 per cent per day, a biologic half-life in plasma of 6 days and an extra-vascular distribution of 74 to 81 per cent. Transcobalamin II had a fractional turnover rate of 1536 and 1944 per cent per day, a biologic half-life of 12 hours and an extravascular distribution of 90 per cent.

Folic Acid Binding by Human Plasma Albumin

Binding of [3H]folate to pure human plasma albumin was studied by Sephadex G-200 gel filtration in a steady-state system. The experiments showed the presence of two binding sites per molecule of albumin and an equilibrium constant of 0.9x10(3) 1/mol. With normal human plasma, only albumin bound exogenous [3H]folate with nearly the same equilibrium constant. In human blood plasma at concentrations of less than 10(-5) mol/1, 50% of folate was free, and 50% was bound to albumin. Binding was maximal at about pH 6 and negligible above pH 8 and below pH 4.5. Neither human transferrin nor Cohn fraction II was able to bind [3H]folate.

Solid-Phase Cobalamin Assay Using Cobalamin-Binding Protein from Dog Stomach

Ninety-five samples from 42 animals were studied for the presence of a cobalamin-binding protein fulfilling the criteria of occurring in major quantities and being homogeneous regarding size and binding constant. The binder from dog stomach thus found was made insoluble by coupling to bromoacetyl-cellulose and used for solid-phase assay of serum cobalamins. Equilibrium between free and bound radioactive cobalamin was unaffected by centrifugation of the samples, thus allowing the straightforward use of the Scatchard equation for calculation of results. The precision of the method was 0.05 to 0.10 at concentrations above 100 pmolX1(-1), the sensitivity was about 30 pmolX1(-1), the accuracy could be based on aqueous solutions of cyanocobalamin, and the reference interval was as for the Lactobacillus leichmannii method, i.e. 160-480 pmolX1(-1).

Changes in the ultraviolet and circular dichroism spectra of aquo-, hydroxy-, azido-, and cyanocobalamin when bound to human intrinsic factor or human transcobalamin I.

The ultraviolet and the circular dichroic spectra of aquo-, hydroxy-, azido-, and cyanocobalamin free or bound to purified human intrinsic factor or purified human transcobalamin I were recorded. Except for azidocobalamin-transcobalamin I, the molar absorption for the gamma1-band in the ultraviolet spectra increased with a factor between 1.2 and 1.4 when the cobalamins were bound to protein. The ultraviolet spectrum of azidocobalamin-transcobalamin I changed so that the gamma2-band became the most intense. Minor changes were observed in the other ultraviolet spectra. The most prominent change in the CD-spectra of protein bound cobalamins was the increase in negative elipticity above 400 nm. This increase was most intense for cyano- and azidocobalamin-transcobalamin I. The study emphasizes that the mechanism by which human intrinsic factor and human transcobalamin I bind cobalamins differ.

Transferrin Determination by Laurell Electrophoresis in Antibody Containing Agarose Gel

Serum transferrin has been analysed by Laurell electrophoresis in antibody containing agarose gel. The coefficient of correlation for this method and an autoanalyzer method was 0.90.The normal range for iron treated healthy females was 247 to 457 μg per cent.

Stokes radius of 57Co-labelled vitamin B12- transcobalamin I and II and 125I-labelled insulin estimated by sephadex G-2000 gel filtration in human plasma AT 37°

Stokes radius for [57Co]B12-transcobalamin II determined in Tris buffer by Sephadex G-200 gel filtration is 26 A, while it increases to 77 A at 37°, when the mobile phase is normal human plasma depleted of lipoprotein. This is thought to be caused by the presence of antibodies to transcobalamin II in normal plasma. The elution of [57Co]B12-transcobalamin I and [125I]insuli is not affected by the use of plasma as eluant.

Characterization of a Cobalamin-binding Plasma Protein from a Patient with Hepatoma

A cobalamin-binding protein has been purified by affinity chromatography from plasma of a patient with hepatoma and a 10,000-fold increase in the concentration of the plasma cobalamin-binding capacity. The protein behaved as normal transcobalamin I in gel filtration, agar gel electrophoresis, immunoelectrophoresis, precipitation by ammonium sulphate, and cobalamin-binding studies. The protein contained 38 per cent carbohydrate, and the relative molecular mass based on amino acid and carbohydrate analyses was 69,000. The molar absorption coefficient of cyanocobalamin bound to the protein was determined to be 36,000 at 362 nm. On amino acid sequencing, one amino terminal was found, and the first 13 residues were determined as Glu-Ile-Ser/Cys-Glu-Val-Ser/Cys-Glu-Glu-Asx-Tyr-Ile-Arg-Leu/Ile.

HEPATITIS-ASSOCIATED (AU/SH) ANTIGEN DETERMINATION IN 10,000 PATIENTS IN A COPENHAGEN GENERAL HOSPITAL

Abstract 10,000 consecutively admitted patients were investigated for hepatitis-associated (AU/SH) antigen (H.A.A.). 35 patients had a positive result. 6 of these had acute hepatitis, 7 chronic hepatitis or cirrhosis of the liver, and 22 no apparent liver disease. Of the 35 H.A.A.-positive patients, 5 were under treatment with corticosteroids. On quantitation of H.A.A., 3 of the steroid-treated patients had the highest figures. 4 out of 16 patients became H.A.A.-negative in the follow-up period. Hospital personnel, if informed of patients who are H.A.A.-positive, can take steps to avoid transmitting infection.

Purification and characterization of rabbit haptocorrin.

Rabbit haptocorrin (R-binder) has been purified from serum by affinity chromatography on cobalamin-Sepharose and Blue Sepharose. It proved to be a protein with a relative molecular mass of 60 000 and an amino acid content very similar to that of other haptocorrins (Nexø, E. and Olesen, H. (1981) in B12 (Dolphin, D., ed.), Wiley Interscience, New York/London, in the press). The pH optimum (pH 6-9) for binding of cyanocobalamin and the affinity to dicyanocobinamide were like those of human and hog haptocorrins. In spectral studies, the extinction coefficient of cyanocobalamin at 363 nm (gamma 1-band) increased by about 16% on binding to rabbit haptocorrin. Binding of azidocobalamin gave spectra changes similar to those for binding to rabbit transcobalamin.