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Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.

Proton magnetic resonance (PMR) spectroscopy has proved to be an important tool in structural studies of biological macromolecules (Roberts and Jardetzky, 1970; Casy, 1971; Jardetzky and Wade-Jardetzky, 1971; Dwek, 1973; James, 1975; Wuthrich, 1976; Thomas, 1976). The magnetic fields currently available can provide resonance frequencies of 360 MHz or higher, and with the spectral dispersion available under these conditions, PMR is often the method of choice for examining small proteins and peptides in solution. In these situations it is usually possible to assign many of the signals observed to specific amino acids and thus have available a number of spectroscopic probes into the structure of the molecule; we take “structure” in this context to include not only sequence information but also details of conformational motions.

p-Trifluoromethylbenzenesulfonyl-α-chymotrypsin, an analog of tosylchymotrypsin, has been prepared and shown to be stable enough to permit fluorine nuclear magnetic resonance experiments. Up to four distinct trifluoromethyl resonances can be observed for the modified protein at 94.1 MHz even when the enzyme derivative is prepared from protein which has been purified by several methods. The resonances observed appear to represent proteins which are grossly similar as regards molecular size and the ability to bind and hydrolyze substrates, but nonetheless distinctive enough in the active-site region to produce appreciable chemical-shift effects.

A fluorine NMR study of cyanomethemoglobin prepared from hemoglobin isolated from rabbits maintained on a diet containing DL-p-fluorophenylalanine is described. The results indicate that substitution of fluorophenylalanine occurs essentially randomly at all phenylalanine positions of the alpha- and beta-globin chains; a set of hybrid hemoglobins in which only the alpha- or only the beta-chains contain the fluorinated amino acid was prepared and used to ascertain the fluorine NMR signals arising from each chain. The temperature and pH dependences of chemical shifts, spin-lattice relaxation times, 19F(1H) nuclear Overhauser effects, and the effect of chemical modification of the beta-93 sulfhydryl groups were examined. When considered in light of presently available X-ray structures of human and horse hemoglobins, the available data permit a tentative assignment of most signals to particular fluorophenylalanine/phenylalanine positions in the globin sequences.