Figure 1. Characterization of ADA from IgG preparations.

(A) Typical elution profile showing separation of the substrates and products of three reactions of purine metabolism on a column with Toyopearl TSK HW-40 S gel. PNP, purine nucleotide phosphorylase; HX, hypoxanthine. A 400 μl mixture containing 5′-AMP, adenosine, inosine and hypoxanthine was injected into an XK 16/20 column packed with 10 ml of Toyopearl TSK HW-40 S gel and equilibrated with 0.01 M sodium phosphate buffer. The nucleotides and nucleosides, which are specifically retained by the gel matrix, were subsequently eluted in the same buffer. The ratio between the peak areas was used to calculate the rates of the corresponding enzymatic reactions. (B) ADA2 activity in ten commercial IgG preparations in comparison with the ADA2 activity in human plasma. ADA2 is co-purified and concentrated together with the immunoglobulin fraction during industrial processing of human plasma using cold ethanol precipitation. (C) pH dependence of ADA activity in IgG preparations. The acid optimum pH of the reaction is a characteristic feature of the ADA2 isoenzyme. (D) Determination of K_m value for ADA in IgG preparations using a Lineweaver–Burk plot.