Die Kooperativität bei der CO2-Hb-Bindung: Messungen mit Hilfe der 13C-NMR-Spektrometrie / The Cooperativity of the CO2 Hb Binding: Measurement by 13C -NMR Spectrometry

Abstract

The detailed structure of the CO2 Hb binding curve is of considerable physiological interest, because the carbamino derivates, which are formed by the reaction of CO2 with Hb, are respon­sible for the well known Haldane effect. Under physiological conditions CO2 binds to the four terminal NH2-groups of the protein chains of Hb and it is generally assumed, that there exists no cooperativity between these groups. To examine whether this assumption is correct or not, we have measured the CO2Hb binding curve over a wide PCO2-range (from 0 kPa to about 70 kPa) by using 13C-NMR spectrometry. Morrow and coworkers (1976) have shown that carbamino-Hb causes a specific NMR signal. The quantitative analysis of the NMR spectra were done according to the method of internal standard. By analyzing mixtures of known composition, we were able to show that this method is working. So we had to use the following equation: Z is the CO2 saturation of Hb, I is the intensity of the carbamino signal, and IHb is the intensity of the carbons of Hb, which are involved in the amide binding. (I/IHb)max is (I/IHb) at high PCO2, where Hb is completely saturated by CO2. For our experiments we used solutions of human desoxygenated Hb, which were equilibrated with a mixture of N2 and CO2. The CO2 contained 90% 13C-isotope. The pH was adjusted by titration to 7.2. The NMR measurements were mainly done with a Varian XL 100 NMR spec­trometer. The CO2 Hb binding curve, we have measured, is S-shaped and has an average Hill value (n̅) of 2.1. This result is quite striking with respect to the findings of other groups. A Hill value of 2.1 proves that CO2 like O2 binds to Hb in a cooperative manner. The discrepancy to the results of other groups may be explained by the fact that these authors measured the CO2 Hb binding curve only at comparatively low PCO2. In our opinion one can only get reliable n-values from the whole CO2 Hb binding curve.