Preparation and properties of three specific active derivatives of ribonuclease A obtained by methylation of methionine residues in 8 M urea.
In 8 M urea at low pH, CH3I reacts specifically with the four methionine residues of ribonuclease A, and all four residues react at the same rate. Uon removal of the denaturant, only unmodified ribonuclease and 3 of the 15 possible derivatives modified on methionine refold to regenerate activity. All the enzymatic activity is recored after chromatography on IRC-50 and the four active proteins separate from each other and from the 12 inactive derivatives, which are not eluted from the resin under the conditions used. By the use of 14CH3I, performic acid oxidation, chymotryptic digestion, and separation of the resulting peptides by ion exchange, the active species were determined to be unmodified ribonuclease, CH3Met-29-RNase, CH3Met-79-RNase, and CH3Met-29, CH3Met-79-RNase. these proteins have melting temperatures of 63, 58, 43, and 36 degrees, respectively, at pH 6.3-70. Methylation at methionine-29 or -79 has no effect on enzymatic activity. Conversely, methylation at methionine-13 or -30 prevents refolding to an active conformation at 25 degrees elution from IRC-50. These results are consistent with the positions of the four methionine residues in crystals of ribonuclease A and ribonuclease S as determined by X-ray diffraction.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007454:Iodides; D007700:Kinetics; D008715:Methionine / Q000032:analysis; D008745:Methylation; D011485:Protein Binding; D011487:Protein Conformation; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014508:Urea / Q000494:pharmacology; D014961:X-Ray Diffraction
D000596:Amino Acids; D007454:Iodides; D014508:Urea; D008715:Methionine; D012260:Ribonucleases