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Red cell adenosine deaminase from normal subjects and from a patient with hereditary hemolytic anemia with a 40-fold increase in activity were purified using antibody affinity chromatography. The purified enzymes were completely homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were no differences in the molecular weight, specific activity, polyacrylamide gel electrophoresis, Michaelis constant for adenosine, inhibition constant for guanylurea, utilization of 2-deoxyadenosine, thermal stability, optimum pH, immunological reactivity, amino acid composition and tryptic peptide mapping. These results strongly suggest that increased red cell adenosine deaminase activity is caused by an overproduction of a structurally normal enzyme.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified in high yield from human erythrocytes using a simple procedure based on two chromatographic steps. In the first step enzyme is bound to a diaminohexane-Sepharose column and eluted with salt. The second strap is column chromatography on Separose-bound purine riboside prepared by cross-linking the riboside to the matrix with 1,4-butanediol-diglycidyl ether. The overall purification factor was approximately 750,000 and the purity was at least 96%. The procedure is suitable for small sample sizes and for purification of the variant forms of the enzyme obtained from a single individual.

Adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) has been purified 468,000-fold from pooled human erythrocytes. The procedure developed was used to isolate the enzyme from up to 23 liters of packed erythrocytes at one time. An easily prepared affinity column bed material employing adenosine as the ligand was used as the final step in the purification. During elution from the affinity column there was approximately a 3:1 partition of adenosine deaminase between gel bed and column buffer. There was no apparent difference in the partitioning of unresolved or partially resolved preparations of the electrophoretically different forms of the enzyme on the affinity column. Gel filtration and electrophoresis on polyacrylamide gels of increasing concentration revealed no differences in the Mr of these electrophoretically different forms. The four bands resolved by electrophoresis of the different forms on polyacrylamide gels under nondenaturing conditions yielded a single band when electrophoresis was carried out in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. Partially resolved preparations of the different electrophoretic forms of adenosine deaminase also gave rise to a single band of the same mobility when electrophoresed on polyacrylamide gels under these conditions. The band had the mobility of a protein of Mr of 36,000. This Mr is approximately the same as estimated for the nondenatured enzyme.

Human malaria infected erythrocytes show a dramatic increase in adenosine deaminase activity in vitro. Using recently developed culture techniques, adenosine deaminase-deficient human erythrocytes were infected in vitro with the major human pathogen Plasmodium falciparum. Adenosine deaminase activity was undetectable in the uninfected host red cells, but increased by 2-fold over normal levels in these cells with an 8% parasitemia. The enzyme in these cells appeared unique in that its activity was markedly elevated over that of other parasite purine enzymes, was not cross-reactive with antibody against human erythrocyte adenosine deaminase, and though inhibited competitively by deoxycoformycin was relatively insensitive to erythro-9-(2-hydroxy-3-nonyl) adenine. The use of adenosine deaminase-deficient erythrocytes for the in vitro cultivation of Plasmodium provides a unique system for the study of parasite enzyme and allows further insight into the purine metabolism of the intraerythrocytic malaria parasite.

In summary we have shown the following:1) Human erythrocyte ADA has been purified approximately 800,000 fold (sp. act. 538 micronmol/min/mg at 37 degrees) to apparent homogeneity using antibody affinity chromatography. 2) The enzyme was shown to be a single polypeptide chain with estimated molecular weight of approximately 38,000. 3) The 3 electrophoretic forms of erythrocyte ADA purified simultaneously by this technique were indistinguishable by SDS gel electrophoresis under reducing conditions. 4) Several properties of the highly purified ADA were identical to properties observed with an impure preparation of the enzyme.