Biomimetic-dye affinity chromatography for the purification of mitochondrial L-malate dehydrogenase from bovine heart.
Seven biomimetic anthraquinone triazinyl dye-ligands, bearing as triazine-linked terminal moiety (keto)carboxylated structures mimicking substrates and inhibitors of malate dehydrogenase (MDH), were immobilised on cross-linked agarose Ultrogel A6R. These biomimetic ligands are terminal-ring analogues of commercial nonbiomimetic Cibacron blue 3GA (CB3GA) and parent Vilmafix blue A-R (VBAR). The biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing immobilised CB3GA and VBAR, were evaluated for their ability to purify mitochondrial malate dehydrogenase (mMDH) from bovine heart. All but two biomimetic-dye adsorbents displayed higher purifying ability for MDH, compared to nonbiomimetic-dye adsorbents. Furthermore, immobilised anthraquinone-dyes were able to discriminate between the mitochondrial and the cytoplasmic MDH isoenzymes, binding only to the former. One immobilised biomimetic-dye (BM5), bearing as biomimetic terminal moiety 4-aminophenyloxanylic acid, showed the highest purifying ability. This affinity adsorbent was exploited in the purification of mMDH from unpretreated bovine heart extract in one-step. The procedure afforded mMDH at 54% overall yield and of specific activity approx. 1300 U mg-1 (25 degrees C), using step-elution with a mixture containing 0.1 mM beta-nicotinamide adenine dinucleotide (NAD+) and 1.5 mM sulphite. Commercial analytical-grade bovine heart mitochondrial MDH, when assayed under identical conditions, gave a specific activity not exceeding 950 U mg-1. The well-known adsorbent Cibacron blue 3GA-agarose exhibited 8% lower recovery and 25% lower purification for mMDH. The product obtained from the procedure based on the BM5-adsorbent was free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase, and since it has also shown high specific activity, it should be suitable for analytical applications.
Labrou
N E
NE
Department of Agricultural Biotechnology, Agricultural University of Athens, Greece.
Clonis
Y D
YD
D000096:Acetone; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D014227:Triazines
D004396:Coloring Agents; D014227:Triazines; D000096:Acetone; C012842:Cibacron Blue F 3GA; D008291:Malate Dehydrogenase