Separation of the photophysical aspects of the sub-picosecond (sub-ps) time-resolved resonance Raman signal from contributions due to conformation has been achieved by comparing deoxyhemoglobin (Hb) in the T state with (carbonmonoxy)hemoglobin (HbCO), deoxy-beta 4 (beta 4 CO) (All R state), and monomers deoxymyoglobin and (carbonmonoxy)myoglobin (MbCO) [beta 4 consists of a tetramer of four beta-subunits and shows no cooperativity]. In all photolyzed species, Hb*(CO), Mb*(CO), and beta 4*(CO), the iron-histidine out-of-plane mode (vFe-His), indicative of heme doming, achieves 90% of its full intensity in 1 ps. The frequency of this mode (223-228 cm-1) is shifted significantly relative to equilibrium deoxy-Hb (210-216 cm-1) in the T state, but not with respect to either equilibrium deoxy-Mb or deoxy-beta 4. A correlation between the +12 cm-1 bandshift of vFe-His and the -2 cm-1 shift of the electron density marker band (v4 at 1370 cm-1) relative to T-state deoxy-Hb is shown to hold on all time scales, including the sub-picosecond time scale. Photolyzed Hb*(CO) consists of R-state or weakly interacting tetramers on the picosecond time scale and is shown to have properties similar to those of photolyzed Mb*(CO) and beta 4*(CO) on the picosecond time scale. These results establish that heme doming occurs as an ultrafast reaction to ligand dissociation and that heme doming is the primary event in the sequence of conformational changes leading to the cooperative R-->T transition.

Optical difference spectrophotometry has been applied for measuring the early part of the 0 2 dissociation curve of human hemoglobin (Hb); i.e., below 1 0 2 saturation levels or log b(Z -y ) ] from -3.0 to -2.0, where y = fractional 0 2 saturation. Two matched cuvettes, sealed on glass tonometers of known volume, were filled with the same deoxygenated Hb solution (65 pM in tetramer) under atmospheric pressure of pure argon. The ~O Z values were calculated after the addition to the sample tonometer of accurate volumes of 02/argon mixtures the composition of which was varied by using a precise gas-mixing pump. 4-5 points of 0 2 saturation below y = 0.01 were thus measured, which allowed for the calculation of AI, the first association constant of OZ to the tetrameric Hb. We have compared the effects of protons, chloride ions, C02 and bisphosphoglycerate (Pz-glycerate) on the pH dependence (alkaline Bohr effect) of A1 and p50. At low chloride concentrations (0.005 M) the alkaline Bohr effect at A1 was identical to that measured at p50, indicating that exactly one-fourth of the Bohr protons were released at the first oxygenation step. At higher chloride concentration (0.15 M) A l was decreased and ~7 5~ was increased as was the Bohr effect. C 0 2 additions (pC0z 38 Torr, 5050 Pa, at I = 0.15 M) lead to a decreased A1 and increased p50 but to a large decrease of the alkaline Bohr effect. The effect of COZ was predominant at A1, indicating a large release of carbamino adducts from the tetrameric Hb at the first step of ligation. Pz-glycerate decreased A1 and increased p50 values. The alkaline Bohr effect was enhanced at p50 by Pz-glycerate but lowered at A1. These results are compared to those already published by other workers. The variations of the pH dependence of Al in the presence of effectors indicate that A1 should be considered as a phenomenological parameter refering to one of the possible structures that the H b tetramer can take in the unliganded state. They indicate also that the whole H b molecule feels the first oxygenation step, which itself implies structural changes within the quaternary structure.The complete recording of the whole 0 2 binding curve of human hemoglobin has provided new and important information on its functional behaviour both on its homotropic and heterotrophic reactions with ligands. These curves have allowed the estimation of the four association constants (Ai) which can be uBed to describe the stepwise process of O2 binding to the tetrameric hemoglobin [I, 21. Among these four constants only the first ( A 1 ) and the fourth ( A 4 ) may be determined directly, A2 and A3 being calculated from mathematical analyses of the whole binding curves. Besides the few direct measurements performed Abbreviations. Hb, hernoglobin; Bistris, [bis(2-hydroxyethyl)-amino]tris(hydroxymethyl)inethane ; NazPz-glycerate, 2,3-bisphosphoglycerate (Na salt).Definitions. A l (Torr-I), the first intrinsic equilibrium constant of the reaction of the tetrameric hemoglobin molecule with 0 2 and free of statist...

The effects of hypoxic hypoxia on renal hemodynamics and metabolism have been studied in anaesthetized mechanically ventilated rabbits. Acute hypoxa (FIO2 = 0.10, PaO2 = 35 torr) induces at constant mean arterial pressure a 45% decrease in RBF, GFR, TNa and RVO2 whereas free water clearance increases. These alterations were still apparent 50 min after resuming normal arterial oxygenation. In order to assess the role of the stimulation of catecholamine release in these observations, two other sets of experiments were performed: 1) the animals were ventilated with the same hypoxic gas mixture but after alpha adrenergic blockade (phentolamine: 0.2 mg - kg - min-1 i.v.), 2) hypoxia was induced by ventilating the animals with CO (FICO = 0.002) at constnat PaO2. Increase in renal vascular resistance and reduction of renal O2 uptake were still observed. This indicates that adrenergic stimulation cannot fully explain the renal vasoconstriction encountered in hypoxia. The role of a local vasoactive factor, especially that of the renin angiotensin system is discussed. The apparent O2 cost of Na reabsorption was not greatly modified by any type of hypoxia and the Na: O2 ratio remained close to the value observed in normoxic animals. This indicates that the kidney may adapt to hypoxia by reducing its O2 demand keeping unaltered its tubular function and basal O2 needs.

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