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

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.