[Purification and characterization of the respiratory inhibitor RF from rabbit reticulocytes].

In the stroma-free hemolysate of rabbit reticulocytes there exists a respiratory inhibitor (RF) of protein nature. It could be purified by the following procedure: (NH4)2SO4-precipitation leads to DEAE-Sephadex A-50-chromatography leads to isoelectric focusing leads to gelfiltration on Sephadex G-200. In addition to the activity the identity of the inhibitor could be proved immunologically at all steps of the purification. A molecular weight of about 80,000 was determined by gelelectrophoresis in the SDS-mercaptoethanol-system. The protein has an IP of 5.55--5.65 and consists of one polypeptide chain. This could be shown both by SDS-gelelectrophoresis and by estimation of only one N-terminal amino acid residue (glycine). Only after oxidation with performic acid the amino acid analysis of the protein gives reproducible values. The sum of weight-% is about 80 (without Tyr). The polarity of the protein with 41 mol-% agrees with the polarity of soluble globular proteins investigated by other authors. Acid hydrolysis without preceeding performic acid oxidation gives a sum of amino acid residues of only 35--45 weight-%, the glycoprotein nature of the inhibitor could be shown by staining the SDS-gels with Schiff's reagents and by carbohydrate determination with anthrone reagent.