| Abstract: | 1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step. |
