Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate.
A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.
Wower
J
J
Department of Biochemistry, University of Massachusetts, Amherst 01003.
Aymie
M
M
Hixson
S S
SS
Zimmermann
R A
RA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D012259:Ribonuclease, Pancreatic; D014357:Trypsin