Fluorescence and the structure of proteins. XXII. Fluorescence of aminotyrosyl residues formed in ribonuclease A.
1. Tyrosyl residues on ribonuclease A were nitrated with tetranitromethane and then reduced to aminotyrosyl residues. By variation of reaction conditions and degree of exposure of tyrosyl residues it was possible to convert from 1 to all 6 tyrosyl to aminotyrosyl residues. 2. At the lower levels of 1-3 aminated tyrosyl residues/molecule the change in conformation seemed minor and 70% of the enzymatic activity was retained. When the three buried tyrosyl residues or all six residues were aminated only 5% of the enzymatic activity was retained. 3. Titration data, susceptibility to urea denaturation, and fluorescence characteristics indicated that some of the aminotyrosyl residues were buried in the interior and others were exposed on the surface of the protein. On the basis of the activation/emission wavelengths it was possible to distinguish buried (288/320 nm) and exposed (288/365-395 nm) aminotyrosyl residues as well as exposed tyrosyl residues (275-305 nm). 4. The modification of specific tyrosyl residues on a protein to aminotyrosyl residues appears to have some promise for observation of changes in environment of the residues that accompany various conformation changes by monitoring the fluorescence.
D000588:Amines; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D012260:Ribonucleases; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000032:analysis; D014508:Urea
D000588:Amines; D014443:Tyrosine; D014508:Urea; D012260:Ribonucleases