Purification of nitrate reductase from spinach spinacia oleracea l. by immunoaffinity chromatography using a monoclonal antibody

Fido, R.J.

Plant Science (Shannon) 50(2): 111-116


ISSN/ISBN: 0168-9452
Accession: 006233215

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NADH-Nitrate reductase (EC from spinach (Spinacia oleracea L. v. Noorman) has been purified to apparent homogeneity by immunoaffinity chromatography using a monoclonal antibody linked covalently to Sepharose 4B followed by affinity chromatography. A pre-column of covalently linked non-immune rat .gamma. globulin prevented non-specific binding. The enzyme, released with 1 M KNO3, was purified 1550-fold to a specific activity of 24.8 .mu.mol NO2- produced min-1, mg protein-1 with a recovery of 60% of applied NADH-NR activity. Proteolytically 'nicked' subunits, detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were removed by 5'-AMP Sepharose chromatography (Fido and Notton, Plant Sci. Lett., 37 (1984) 87).