Oxidized dextrans of increasing molecular weight were bound covalently to Erwinia carotovora asparaginase. The resulting conjugates retained 50% of their enzyme activity and showed marked resistance to proteolysis by trypsin and chymotrypsin and inactivation by asparaginase-specific antibody. When tested in-vivo, the larger molecular weight conjugates showed prolonged circulatory survival in both immune and non-immune animals and failed to elicit full type III hypersensitivity or anaphylactic reactions when injected into sensitized guinea-pigs. Rabbits could tolerate multiple doses of the asparaginase conjugate without developing an immunity to the enzyme. A conjugate showing increased circulatory half-life and lowered antigen reactivity should have therapeutic potential.

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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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