CHARACTERIZATION OF SITES OCCUPIED BY THE ANESTHETIC NITROUS-OXIDE WITHIN PROTEINS BY INFRARED-SPECTROSCOPY

We report here a comprehensive infrared spectroscopic study of the int eractions between the anesthetic nitrous oxide (N2O) and six proteins: lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and cyt ochrome c oxidase. Sites occupied by N2O molecules within these protei ns were characterized. Three types of hydrophobic sites were found wit hin the proteins. One with nu(3) near 2225 cm(-1) is likely to be near peptide bond carbonyls; one with nu(3) near 2219 cm(-1) may be near a benzene-like structure such as the side chains of phenylalanine and t yrosine; and the other with nu(3) near 2215 cm(-1) is likely to be in a nonpolar alkane-like environment provided by the side chains of Leu, Ile, and Val residues. The amount of N2O molecules bound to myoglobin increases as the pH decreases from 9.2 to 5.2. N2O-protein interactio ns produced no detectable changes in the ligand-binding pockets of myo globin, hemoglobin, and cytochrome c oxidase. N2O-induced secondary st ructure changes were detected only in the fully reduced cytochrome c o xidase, not in the fully oxidized oxidase and the other five proteins. N2O-indueed conformational changes in the alpha beta-interface of hem oglobin and the h2 and h3 alpha-helices of human serum albumin were de tected by monitoring the S-H stretch vibrations of cysteine residues. These findings provide direct evidence that anesthetic N2O interacts w ith proteins and occupies sites in the interior of the proteins.