Haemoglobin (Hb) is the tetrameric protein molecule that in vertebrate blood transports oxygen from the lungs to the tissues. This function depends on four subunits in the molecule binding cooperatively so that their affinity for oxygen increases as the level of oxygenation increases. X-ray analysis has shown that deoxyhaemoglobin, which has a low oxygen affinity, and oxyhaemoglobin, which has a high oxygen affinity, differ principally in their subunit or quaternary structures, referred to as the T and R states, respectively. As it switches from the T state to the R state during oxygenation, Hb increases its oxygen affinity. However, the structural pathway between deoxy- and oxy-haemoglobin is not known, principally because there has been no accurate structural knowledge of the intermediate states. We report here the crystal structure of T state human Hb in which the alpha chains are oxygenated and the beta subunits are oxygen-free. In this crystal the Hb appears to be in an intermediate state between the unliganded T state and the liganded R state. There is also evidence that the Hb molecule operates by loading and unloading the beta haems and thus the alpha-oxy, beta-deoxy Hb crystal may represent a physiologically important state.

As part of our ongoing development of the CMIA nonisotopic immunoassay method, in which the tracers are metal carbonyl complexes and detection is by Fourier transform infrared spectroscopy, we examined the potential use as tracers of the complexes CpFe(CO)2(5,5-diphenylhydantoin) 2d and CpFe(CO)(PPh3)(5, 5-diphenylhydantoin) 3. The present study involved the synthesis of a series of hydantoin complexes (2a-2d), in particular that of the derivative of 5,5-diphenylhydantoin 2d. The structure of 2d was confirmed by X-ray crystallography. The infrared analysis, establishing the position and intensity of the characteristic metal-carbonyl peaks of complexes 2d and 3 in the 1850-2200 cm-1 region, shows that measurement of the absorbance values of these characteristic peaks will permit quantitative analysis in the picomole range, the norm for routine use in immunoassay and thus suitable for use as CMIA tracers. Cross-reaction rates of these tracers with anti-DPH specific antibodies show that 2d and 3 are both recognized by anti-DPH antibodies (cross-reaction rates 43 and 20%, respectively). In developing a CMIA of DPH with these tracers, it was found that 3, with a single, intense band at 1977 cm-1, had very promising IR characteristics for use in multiassay CMIA, but probably owing to its relatively weak affinity for the antibodies, it was not possible to develop a CMIA for DPH using this tracer. Complex 2d, however, showed better recognition by the antibodies, and using this complex as a tracer, it was possible to develop a particularly sensitive monoassay of DPH by the CMIA method.