Studies on the Cobalamin-Binding Protein of Human Saliva

Abstract

The association constant for binding of cyanocobalamin to the salivary binder is 15 × 10⁹M⁻¹at 26 °C, pH 7.4. The reaction is exothermic, δH° being –11.6 kcal/mol. In the forward reaction there is an entropy change of +8.0 cal/degree per mole. The binding strenght is only slightly influenced by pH changes between 2 and 10, or by exposure to 7.5 mol/1 guanidin. The cyano-cobalamin-binding capacity of mixed saliva from 90 normal subjects is from 8 to 110 nmol/1, mean 47 nmol/1. In saliva from 14 persons with pernicious anaemia, the binding capacity is of a similar size. Stokes radius of the salivary binder, determined by gel filtration, is 4.36 nm. On the basis of this value and the sedimentation constant from the literature, a value of 71,000 daltons is obtained for the molecular mass. By gel filtration of the salivary binder, with proteins of known molecular mass as standards, a value of 88,000 daltons is estimated. The reason for this somewhat higher value may be the rather high frictional ratio of 1.58. The sterical orientation of the cobalamin molecule on binding to the salivary binder has been studied by employing different cobalamin derivatives. From the results it is suggested that the binding occurs from the edge of the corrin moiety, presumably at the A- or the D-pyrrol ring. The number of combining sites on the salivary binder is found to be one.

Key Words:

• Cobalamins
• molecular mass
• saliva
• Stokes radius
• thermodynamic data
• vitamin B₁₂