Fluorescent probe of ribonuclease A conformation.
The reaction of ribonuclease (RNase) with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-IAENS) yields a derivative in which one fluorescent group is covalently attached to the protein. Several arguments suggest that the chemical modification has occurred at the enzyme active site: (i) 1,5-IAENS should have the same specificity as iodoacetamide, i.e., carboxymethylate one histidine of the active site; (ii) the derivatized protein is enzymatically inactive; (iii) in the native state of the protein, the fluorescent group is (almost) completely protected from the aqueous solvent; (iv) this group has no motions other than those of the protein. The fluorescence properties of the derivatized RNase change markedly upon unfolding induced by guanidine hydrochloride (Gdn . HCl), as seen from fluorescence intensity, maximum emission wavelength, and polarization measurements. Upon Gdn . HCl-induced unfolding, the fluorescent group is transferred from a nonpolar to a highly polar environment. Dynamic fluorescence measurements show also that unfolding results in a markedly increased mobility of the fluorescent label with respect to its proteic environment. These results are compared to those of Young & Potts (1963) [Young, D. M., & Potts, J. T. (1963) J. Biol. Chem. 238, 1995--2002], who studied the fluorescence properties of a surface-labeled derivative of RNase.
D004720:Endonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000494:pharmacology; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D004720:Endonucleases; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D007460:Iodoacetamide