Purification of histaminase (diamine oxidase) from human pregnancy plasma by affinity chromatography.
The present study describes the use of affinity chromatography to achieve a high degree of purification of histaminase (diamine oxidase, EC 1.4.3.6) from plasma of women in the third trimester of pregnancy. The procedure is based upon the binding of histaminase to cadaverine, a diamine substrate for the enzyme, which is coupled to Sepharose. Contaminant proteins were removed by high concentrations of NaCl (up to 1.0M), and the histaminase was then eluted from the column with a buffer containing 300--400 units/ml of sodium heparin. The purification technique has the following characteristics: (1) in optimal experiments, 3000-fold purification of enzyme was obtained; (2) the yield of enzyme was as great as 25%; (3) the binding of histaminase to the amine groups of the cadaverine appears to represent a true "affinity" phenomenon since enzyme bound to DEAE-cellulose under neutral pH conditions was eluted at much lower concentrations of NaCl (less than 0.4 M). The enzyme purified by the present procedure has the following properties: (1) disc gel polyacrylamide electrophoresis showed two protein bands for 1000--3000-fold pure histaminase; the major band may represent a contaminant protein, while the minor band corresponded to the position of histaminase activity; (2) a 90 000 molecular weight subunit for the plasma histaminase was identified on calibrated sodium dodecyl sulfate gels; this value agrees well with previous estimations for the subunit size of human placental histaminase; (3) the purified enzyme behaved as classical histaminase (diamine oxidase) in that it was totally inhibited by low concentrations of aminoguanidine, but was less inhibited by semicarbazide and by inhibitors of monoamine oxidase, and the enzyme was active against histamine and putrescine, but not against the monoamines benzylamine and tryptamine. Also, the enzyme was strongly inhibited by NaCl.
D006631:Amine Oxidase (Copper-Containing) / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D002103:Cadaverine; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004589:Electrophoresis, Disc; D005260:Female; D006801:Humans; D007700:Kinetics; D010949:Plasma / Q000201:enzymology; D011247:Pregnancy; D011263:Pregnancy Trimester, Third; D011485:Protein Binding
D006631:Amine Oxidase (Copper-Containing); D002103:Cadaverine