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Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.

The influence of Tris, phosphate ions, imidazole, carbonate ions, bicarbonate ions and hydroxyl ions on the rate of activation of calf intestinal alkaline phosphatase after its dissolution in aqueous solution was investigated. The kinetic data are consistent with a general base-catalysed reaction in which the base reacts with an unprotonated enzyme species formed by ionization of a group with pK = 8.9.Mammalian alkaline phosphatases are zinc metallo enzymes catalysing the hydrolysis of phosphomonoesters, and the transfer ofphosphoryl groups to suitable aminoalcohol acceptors. It has long been recognised that dilution of serum, with buffer, has resulted in an activation of alkaline phosphatase [l]. Similarly, reconstitution of lyophilised serum or thawing of frozen serum results in a slow activation of alkaline phosphatase [2,3]. However, this phenomenon has not been satisfactorily characterised. It has been attributed variously to dissociation of partially active lipoprotein complexes [4], reversible association of the enzyme with Tris to form a more active addition complex [5], or from calculations of entropy changes to exposure of additional active sites [6].This paper demonstrates that rapid dissolution of partially purified calf intestinal alkaline phosphatase into buffered solution is followed by an increase in activity, the rate of which depends on pH and buffer concentration. The attendant kinetics of activation are consistent with general base catalysis in which the conjugate base of the buffer activates the enzyme by reaction with an unprotonated species formed by ionisation of a group with pK = 8.9.

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