The isolation and partial characterization of ribonuclease A from Bison bison.
1. Bison ribonuclease was isolated from pancreas glands of Bison bison by acid extraction, (NH(4))(2)SO(4) fractionation, affinity chromatography on Sepharose-5'-(4-aminophenylphosphoryl)uridine 2',3'-phosphate and ion-exchange chromatography on Bio-Rex-70. 2. The selectivity of the affinity column towards bison ribonuclease in heterogeneous protein solutions was greatly improved by employing piperazine buffers at pH5.3, which decreased non-specific interactions of other proteins. Rapid desorption from the affinity column was obtained with sodium phosphate buffer (pH3). 3. Bison ribonuclease has a total amino acid content very similar to ox ribonuclease. Inactivation of bison ribonuclease with iodoacetic acid leads to the formation of 0.62 residues of pi-carboxymethylhistidine and 0.36 residues of tau-carboxymethylhistidine. The amino acid composition of peptides isolated from diagonal peptide ;maps' and also of peptides isolated after pH1.6 and 2.4 two-dimensional high-voltage electrophoresis of a digest of bison ribonuclease labelled with pyridoxal 5-phosphate indicates that there is complete homology between ox and bison ribonucleases. 4. The Schiff-base attachment site of pyridoxal 5-phosphate was identified as lysine-41 by NaBH(4) reduction followed by peptide isolation.
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D001188:Artiodactyla / Q000378:metabolism*; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000032:analysis; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates; D008239:Lysine; D008762:Methylhistidines; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D010455:Peptides; D010710:Phosphates; D010879:Piperazines; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012260:Ribonucleases / Q000302:isolation & purification*; D014500:Uracil Nucleotides
D002021:Buffers; D007461:Iodoacetates; D008762:Methylhistidines; D010455:Peptides; D010710:Phosphates; D010879:Piperazines; D014500:Uracil Nucleotides; D011732:Pyridoxal Phosphate; D012260:Ribonucleases; D002918:Chymotrypsin; D008239:Lysine; D000645:Ammonium Sulfate