Application of 19F NMR spectroscopy to a study of carbon monoxide binding to human hemoglobin modified at Cys-beta 93 with the S-trifluoroethyl residue.
The S-2,2,2-trifluoroethyl residue (-SCH2CF3) has been incorporated into human hemoglobin, Hb4(SH)2, as a reporter group at Cys-beta 93 using a sequence of disulfide interchange reactions [F. C. Knowles (1981) Anal. Biochem. 110, 19-26]. The 19F NMR spectrum at 235.2 MHz of carboxyhemoglobin (COHb)4(SSCH2CF3)2 was a band 50 Hz wide at half peak height. Conversion of the carbon monoxide derivative to the ligand-free form was accompanied by a downfield shift of 125 Hz (0.53 ppm). Weighed aliquots of solutions of Hb4(SSCH2CF3)2 and (COHb)4(SSCH2CF3)2 were mixed, yielding solutions of known fractional saturation with carbon monoxide. An independent estimate of F of these samples was derived from the amplitudes of the resonance intensities in the 19F NMR spectra. These independent methods for determination of the value of F were not uniformly in agreement. In the presence of inositol hexaphosphate the estimate of F derived from 19F NMR spectra was considerably less than the actual value. The discrepancies between the two independent methods for determining F can be explained by a preferred order of binding of carbon monoxide to the alpha-chains. The preference for binding to the alpha-chains was abolished by removing phosphates. A model for transmission of cooperative effects in hemoglobin was presented which accounted for the characteristic shape of the hemoglobin-oxygen dissociation curves as well as the chain heterogeneity revealed by 19F NMR experiments.
D000495:Allosteric Site; D001665:Binding Sites; D002248:Carbon Monoxide / Q000097:blood*; D005461:Fluorine; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation
D006454:Hemoglobins; C040587:trifluoroethyl-cysteine-beta93 hemoglobin; D005461:Fluorine; D002248:Carbon Monoxide