Abstract
A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sensitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein. The purified protein binds heme with a higher affinity (Kd 0.15 microM) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the increase of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose members include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a pI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally designated it heme-binding protein.
Highlights
A low molecular weight protein purified from rat amino acid composition,and abundance identify it as belongliver cytosol was observed to bind heme with an affin- ing to the Z class of liver cytosolic proteins [3,4,5,6,7,8,9,10,11,12]
Judging by the total amount of protein elutedfrom oleic acid-agarose,these higher molecular weight proteins should constitute more than 30% of the totalcytosolic fatty acid-binding proteins
The purified protein had an %fold higher affinity for heme than for oleic acid, the fatty acid for which FABP has supposedly the highest affinity [4,6].This was determined fluorometrically by competition studies between ANS and oleic acid, as shown in Fig. 5, and by difference absorption spectroscopy wherea 30-fold excesosf oleic acid was required to displace bound heme.Still, our binding constant for oleic acid is higher than thatreported by Ketterer et ai. [4]for the basic formof their aminoazodye-bindingprotein A, and that
Summary
Materials crease of its tissue levels in response to the administration of hypolipidemic agents, the proteinwas identified as being related to proteinosf the Z class, whose members include fatty acid binding protein and sterol car-. The abbreviations used are: FABP, fatty acid-binding protein; HBP, heme-binding protein; EGTA, ethylene glycolbis(/3-aminoethyl ether)-N,N,N’,N’-tetraaceticacid; PMSF, phenylmethylsulfonyl fluoride; ANS, 8-anilino-1-naphthalenesulfonicacid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SCP, sterol carrier protein. Binding Studies which was eluted with 10-20 ml of distilled water and concentrated Fluorescence spectroscopy was used to determine the interaction to 0.5 ml in a Micro-ProDiCon while dialyzing against Na phosphate of purified HBP with heme, protoporphyrin IX and its zinc and buffer, pH 7.4, with 20% glycerol. Competitive binding was determined in the preseonf ceexcess ANS (ANS:protein molar ratio about50) by measuring the decrease in the fluorescence of the HRP-ANScomplex at 460 nm after each addition of ligand solution (the excitationwavelength was 390 nm).
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