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Oxygen is a toxic gas, still indispensable to aerobic life. This paper explores how normal physiology uses the physico-chemical and thermodynamic characteristics of oxygen for transforming a toxic gas into a non toxic indispensable metabolite. Plasma oxygen concentration is in the range of 10(-5) M, insufficient to sustain metabolism. Oxygen carriers, present in blood, release oxygen into plasma, thereby replacing consumed oxygen and buffering PO(2) near their P(50). They are the natural cell-bound carriers, like hemoglobin inside red cells, myoglobin inside myocytes, and artificial cell-free hemoglobin-based oxygen carriers (HBOC) dissolved in plasma. Metabolic oxygen replacement can be defined as cell-bound and cell-free delivery. Cell-bound delivery is retarded by the slow diffusion of oxygen in plasma and interstitial fluids. The 40% hematocrit of normal blood compensates for the delay, coping with the fast oxygen consumption by mitochondria. Facilitated oxygen diffusion by HBOCs corrects for the slow diffusion, making cell-free delivery relatively independent from P(50). At all oxygen affinities, HBOCs produce hyperoxygenations that are compensated by vasoconstrictions. There is a strict direct correlation between the rate of oxygen replacement and hemoglobin content of blood. The free energy loss of the gradient adds a relevant regulation of tissues oxygenation. Oxygen is retained intravascularly by the limited permeability to gases of vessel walls.

The thermal stability of the two dimers of RNase A with N- or C-terminal swapped ends is investigated by means of dissociation kinetics, differential scanning calorimetry, and circular dichroism measurements. The data indicate that the dimer characterized by the swapping of the N-terminal alpha-helices is less prone to monomerize when compared to the dimer characterized by the swapping of the C-terminal beta-strands. This finding is correlated to the structural features of the so-called open interface of the dimeric forms.

We have analyzed the time resolved fluorescence emission in the subnanosecond range of recombinant wild-type SW myoglobin and its single TRP mutants W7F and W14F. These recombinants carry a methionine at the N1 terminal end. The emission of Trp-7 in the met form of W14F showed residual lifetime components much shorter than those estimated after excitation energy transfer to the heme. We propose that in this recombinant the N1 methionine is close to Trp-7, thereby producing an extra quenching due to either collisions or electron transfer with its sulfur. When the measurements were repeated on its CO-form, the extra quenching of Trp-7 was much decreased, indicating a heme linked conformational change involving the amino terminal end of the protein. This hypothesis is supported by ligand linked conformational changes in myoglobin, reported by Ansari et al. and by Giardina et al. At neutral pH the lifetimes of W7F were consistent with estimations based on the atomic coordinates of SW myoglobin. Those of the wild-type were exactly the combination of the lifetimes of the two mutants. This suggest that the mutations did not affect the overall structure of the protein. However, in the ferric form, substitution of Trp-14 in W14F resulted in low stability at acid pH, as evident from lifetimes modifications at pH 4.8, while no modifications were produced by titrations of W7F to pH 4.5. This suggests a role of Trp-14 in the structural stability of myoglobin.

In the crystal structure of human T-state hemoglobin with a sebacyl residue cross-linking the two beta-subunit Lys82,s (DecHb), the Fe atoms of the alpha-subunit hemes are found to be oxidized with a water molecule bound. The three-dimensional structure and heme geometries were compared to those of deoxyhemoglobin and other partially and fully oxidized hemoglobins [R. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, G. Dodson, J. Mol. Biol. 228 (1992) 551]. The heme geometries of the alpha-subunits are consistent with those observed in oxidized structures. The proximal histidines of the alpha-subunits move toward the heme plane shifting the F-helix and FG-corner in a manner observed for partially oxidized human hemoglobin. This supports the hypothesis that these perturbations may precede the T- to R-state transition. Circular dichroism studies comparing DecHb and natural human hemoglobin in the deoxy and CO ligated forms confirm that the conformations of the deoxy forms are identical, but the ligated forms have slight differences in the solution structures. DecHb is found to be more resistant to autoxidation than natural hemoglobin. The time course of autoxidation of DecHb shows that it is virtually absent for the first 1500 min followed by a rapid increase. Thus, the discovery of the oxidation of the alpha-subunits in the deoxy-crystals is quite unexpected. The data confirm that ligation of the alpha-subunits precedes that of the beta-subunits. This may suggest a low ligand affinity of the alpha-diligated form of hemoglobin.

Absorption anisotropy and emission anisotropy measurements in poly(vinyl) alcohol (PVA) films of different porphyrin derivatives are reported. Wavelength dependent absorption anisotropy in oriented PVA films, and wavelength dependent excitation spectrum of emission anisotropy of fluorescent porphyrin derivatives in isotropic PVA films indicate the presence of multiple transition moments with different well-defined orientation. Comparison of linear dichroism and orientation behavior in stretched PVA films of deuteroporphyrin III (C2V symmetry) and its iron derivative reveals significant out-of-plane transition moment components. A considerable participation of out-of-plane polarized absorption components is also observed for metal derivatives of non-symmetrical protoporphyrin IX. It appears that central metal substitutions in porphyrin rings do not produce 'circular' degeneration of electronic transition moments. Instead, the presence of metal induces absorption components orthogonal to the porphyrin plane.

We used time-resolved fluorescence in the pico- to nanosecond time range to monitor the presence of tetramers, dimers and monomers in carbonmonoxyhemoglobin (COHb) solutions and to investigate how their distributions change under different experimental conditions. Comparison of fluorescence lifetime computed from the atomic coordinates of COHb (Vasquez et al., 1996) with those experimentally measured allowed identification of molecular species present in the hemoglobin solution. It was possible to observe modification of the distribution of tetramers, dimers, monomers and species with disordered hemes produced by different experimental conditions. Protein concentration affected the detectable lifetimes, indicating increasing amounts of dimers and monomers at low protein concentrations, while the amount of inverted hemes was not modified. Titration with up to 1 M NaCl modified only the extent of dissociation of hemoglobin into dimers, without affecting heme inversion and monomer formation. Hyperbaric pressure increased the amounts of dimers and monomers. This is the first time that monomeric subunits of hemoglobin have been detected at neutral pH in the normal system.

Recent developments of ultrafast fluorimeters allow measuring time-resolved fluorescence on the picosecond time scale. This implies one is able to monitor lifetimes and anisotropy decays of highly quenched systems and of systems that contain fluorophores having lifetimes in the subnanosecond range; both systems that emit weak signals. The combination of weak signals and very short lifetimes makes the measurements prone to distortions which are negligible in standard fluorescence experiments. To cope with these difficulties, we have designed a new optical cell for front-face optics which offers to the excitation beam a horizontal free liquid surface in the absence of interactions with optical windows. The new cell has been tested with probes of known lifetimes and anisotropies. It proved very useful in detecting tryptophan fluorescence in hemoglobin. If only diluted samples are available, which cannot be used in front-face optics, regular square geometry can still be utilized by inserting light absorbers into a cuvette of 1 cm path length.

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.

When an allosteric macromolecular system is capable of existing in two conformations, both of which may be converted into energy-storing forms by the binding of a substrate or by the absorption of radiant energy, then a kinetic process may occur, such as an enzymic conversion of the substrate into products, which liberates energy and selectively depletes one or more of the forms of the macromolecule. Upon a continuous supply of energy, a steady state, or pseudoequilibrium, is reached during which the selective depletion of molecular species results effectively in a directional flow of energy through the system. This perturbs the distribution of the various molecular species. This effect may simulate both positive and negative binding cooperativity, and mimic the presence of multiple binding sites with different affinities even in monomeric, monovalent systems. Specific model systems are presented analogous to the transport of Ca2+ by sarcoplasmic reticulum and the allosteric behavior of phosphorylase b.

This paper presents data on the effect of phosphates and polyphosphates on the formation of hemoglobin S fiber, and on the Bohr effect of hemoglobin S samples whose concentration was high enough (near 5 mM) in order to form fibers upon deoxygenation. The experiments were performed in 0.2 M Bistris or Tris buffers at 30 degrees C in the presence and absence of inositol hexakisphosphate and of 2,3-diphosphoglycerate. Alternatively, 0.2 M phosphate buffers were used without addition of effectors. Under these conditions, few fibers were formed in Tris or Bistris buffers, while extensive fiber formation occurred in the presence of phosphates and polyphosphates. In all cases, increasing pH strongly inhibited fiber formation. At pH 7.5 and above, fibers were not formed in our samples. In the presence of phosphates and polyphosphates fiber formation reduced the oxygen affinity of hemoglobin S with respect to either hemoglobin A or soluble hemoglobin S under similar experimental conditions. The fiber-polyphosphate complexes showed a larger Bohr effect than that in hemoglobin A. In the presence of inositol hexakisphosphate fiber-forming solutions of hemoglobin S liberated as much as six protons per tetramer upon oxygen binding. The increased liberation of protons was probably due to a higher affinity of the effectors for the fibers of hemoglobin S. Very likely the higher affinity was supported by a conformational change of hemoglobin S specific for the fibers.

This paper shows that analysis of sedimentation equilibrium data, searching for average molecular weights, gives quantities which are dependent on the total protein concentration of the samples, while knowledge of the molecular weight of the monomeric subunits allows a more meaningful search for the concentrations of the individual polymeric components of the system. From these the various average molecular weights can be construed, and the various dissociation equilibrium constants evaluated. Also, in this paper considerations are proposed on the meaning of nonideality terms in associating systems and possible ways for estimating them. As an example the proposed procedures have been applied to measurements of sedimentation equilibrium in carbonmonoxyhemoglobin.

Human hemoglobin and its isolated beta-subunits were denatured by addition of HCl so as to reach final pH values ranging from 2.0 to 3.2. The beta-subunits were alkylated in both the beta 93 and beta 112 cysteines; this treatment makes the beta-subunits monomeric. The kinetics of acid denaturation of the two proteins was followed spectropolarimetrically in the millisecond time range, measuring the changes in circular dichroism at 225 nm. At all pH values, in both systems, the decay of ellipticity could be simulated by two exponentials. The initial ellipticity values of the solutions, obtained by extrapolation at zero time, were those expected for the native proteins. The rates of denaturation were lower in the hemoglobin system than in the isolated monomeric beta-subunits. The data suggest that in the tertiary structure of hemoglobin and beta IAA there are different domains which unfold at different rates upon exposure to acid.

Continuing our investigation of the relationships between internal motions and functional properties of soluble and membrane-bound proteins we have explored the lifetimes and correlation times associated with the fluorescence emission of fluorescein-labeled Ca2+-dependent ATPase of sarcoplasmic reticulum. The emission was characterized by two lifetime components near 1.8 and 4.1 ns, probably due to exposure of the probe to environments of different polarities. The time-dependent anisotropy showed the presence of two correlation times near 0.8 and 6.6 ns. The shorter correlation time was due to motions of the probe around its point of attachment on the surface of the protein. The longer correlation time indicated the presence of internal motions of the protein. Both lifetimes and correlation times were insensitive to temperature between 2 and 10 degrees C. They were also insensitive to addition and removal of 100 microM free Ca2+.

The rate of reaction of the terminal valines of the alpha- and beta-chains of hemoglobin with 1-fluoro-2,4-dinitrobenzene was followed spectrophotometrically at 353 nm. The variation with pH of the rate of dinitrophenylation of these groups was measured for both carbonmonoxy- and deoxyhemoglobin. In carbonmonoxyhemoglobin the results indicated a pK near 6.7 and 7.7 for the amino terminal groups of the two kinds of subunits, and were attributed to the alpha- and beta-chains respectively. Removal of ligands produced an increase of 0.1 in both pK values and a decrease of 40% of the pH-independent kinetic constant for dinitrophenylation of the beta-subunits. These modifications are due to the conformational changes associated with ligand binding in the system. In phosphate buffers the contribution to the Bohr effect of the amino terminal residues of either chains is negligible.

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.

Perturbation of sarcoplasmic reticulum ATPase with the nonionic detergent C12E8 is modulated by the amount of free Ca2+ present in the solvent prior to the addition of detergent. CD measurements show that the enzyme exists in solution in two different conformations that react differently with the detergent. They probably represent the free enzyme, and its complex with Ca2+. On this assumption, titrations with increasing amounts of Ca2+ produced data superimposable on curves obtained measuring Ca2+ bound to sarcoplasmic reticulum vesicles.

We have used time-resolved fluorescence spectroscopy for following the unfolding of apomyoglobin in urea and guanidine hydrochloride (GdnHCl). The data have been compared with those obtained using classical techniques such as CD and steady-state emission spectroscopy. Both the average intensity of the lifetimes and the size of the librational cone of the fluorophores, as measured by time-resolved fluorescence, increased with denaturant concentration and their changes largely preceded the modifications detectable with CD and the shift of the maximum of emission spectra. The data indicate that the changes in the local environments of the tryptophans were completed when the global modification monitored by CD and the emission spectra was still minimal. This suggests that an initial event in the denaturation of apomyoglobin is localized at the tryptophan residues. The correlation times of native apomyoglobin showed the rotational diffusion characteristics of a rigid rotor. In 3.6 M GdnHCl and 7.5 M urea, where the secondary structure is practically absent, the correlation times of the two systems became very short, as expected from the motion of a flexible polymer. In GdnHCl, under conditions of partial unfolding, it was not possible to detect the presence of native totally folded molecular species.

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.