Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes

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Abstract
A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydrate (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285000) is made up of eight apparently identical subunits of Mr 35000. The enzyme is sensitivie to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (.apprxeq. 12% of control) when assayed in buffers devoid of Zn2+. An of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 .mu.M Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constantfor E.cntdot.S.cntdot.Pb2+ complex) = 25.3 .+-. 3.0 .mu.M; KiS (dissociation constant for E.cntdot.Pb2+ complex) = 9.0 .+-. 2.0 .mu.M]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.