Nature {London) 232, 408.

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The spectra of the ferric form of most heme proteins [metmyoglobin, methemoglobin, horse radish peroxidase (EC 1.11.1.7), and ferricytochrome c at pH 1.5J are converted from high-spin (open crevice) structure to low-spin (closed crevice) form under pressure. Pressures up to 8000 kg/cm2 (780 MPa) have no effect on the spectra of high-spin ferro-and ferricytochrome c, which have a closed crevice structure at pH 7.0. Spectra of deoxy-ferromyoglobin and deoxy-ferrohemoglobin are reduced in intensity, but pressure does not change the positions of the absorption maxima. Cyanide ion prevents pressure-induced spectral changes in metmyoglobin and methemoglobin up to 8000 kg/cm2. Carbon monoxide (with a high affinity for the ferro heme iron) has a similar effect on ferromyoglobin and ferrohemoglobin. The pressure required to cause spectral changes in the heme proteins falls in the order, cytochrome c (pH 7.0) > horse radish peroxidase > myoglobin > hemoglobin. We have calculated a volume change of -50 cm:3/mol associated with the configurational change accompanying the reformation of the iron-methionine bond in cytochrome c at low pH.The effect of pressure on the complexes of hemoglobin (1-4) and myoglobin (5-7) have been the subject of considerable investigation over the last few years. These investigations have, however, been largely concerned with changes in the functional properties (1, 2), the thermodynamic properties accompanying the binding of ligands (6), and the denaturation of the protein (5). However, the Soret and visible spectra of hemoglobin and hemoglobin complexes (3, 4) and the visible spectra of metmyoglobin fluoride (7) show interesting changes when these molecules are subjected to hydrostatic pressure. We have extended these high pressure studies on metmyoglobin and methemoglobin and have investigated, in addition, the spectral changes in the visible region accompanying the pressurization of two other hemoproteins, cytochrome c and horse radish peroxidase (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7). MATERIALS AND METHODSHuman hemoglobin was prepared by the usual method (8) and oxidized to methemoglobin with 2-fold excess of potassium ferricyanide. Ferrocyanide and excess ferricyanide were removed by dialysis against 0.05 M sodium chloride at pH 6.0. Sperm whale metmyoglobin and horse heart (type III) cytochrome c were obtained from Sigma Chemical Co. and were used without further purification. Buffers used were cacodylate and Tris. Tris buffer was prepared from Trizma base (Sigma) and Trizma-HCI (Sigma) that had been dried under reduced pressure before use. All methemoglobin and metmyoglobin solutions were 0.05 M in buffer ions. Hemoglobin and myoglobin were deoxygenated by addition of sodium dithionite in a glove box previously filled with dry nitrogen.High Pressure Studies. The high pressure optical bomb used was designed by Dr. W. B. Daniels and has been described elsewhere (9).At each increment of pressure, the system was allowed 5 min to attain temperature re-equilibration. Me...

It is well known that when typical aromatic chromophores are transferred from the vapor to a solvent the spectrum usually shifts to the red. This shift is commonly understood to arise from the action of dispersion forces, which lower the energy of the excited state more than that of the ground state. As would be expected from such a theory, the shift is usually larger, the larger the refractive index of the solvent. Since pressure increases the density, and therefore the refractive index, one would expect that pressure would generally cause a further red shift of the spectrum. This is observed with benzene, chlorobenzene, naphthalene, and phenanthrene in most solvents. When the solvent is water, however, pressure causes no further red shift for the weak transitions of benzene and chlorobenzene. (The red shift in water at 1 atm is also smaller than would be expected from its refractive index.) When the solvent is perfluoro n -hexane the red shifts of the weak transitions of benzene, chlorobenzene, and naphthalene are small at 1 atm, as expected from the low refractive index of this solvent. Raising the pressure causes no further red shift at all for naphthalene and causes a shift to the blue for benzene and chlorobenzene. The weak n -π* transition of methyl nitrite shows anomalous behavior in water similar to that of benzene and chlorobenzene. Under pressure the strong transitions of naphthalene and phenanthrene in water show the normal behavior. The strong transition of naphthalene in perfluoro n -hexane shows a less anomalous pressure effect than the weak transition. The anomalous behavior of the weak transitions in water is ascribed to an interaction between the permanent dipole moments of ordered water molecules surrounding the chromophore and the static quadrupole moments of the ground and excited states of the chromophore. We have been unable to devise a simple quantitative explanation for the anomalous behavior in the fluorocarbon solvent. The small polarizability of the fluorine atom is believed to be an important factor because blue shifts in the spectra of benzene and other systems are also observed when helium or neon are present, and these atoms also have very small polarizabilities. A small polarizability of the solvent molecules would reduce the magnitudes of the dispersion interactions that are responsible for the normal red shift and would bring into prominence the contributions of repulsive forces and exchange interactions. Since the overlap of the chromophoric electron and the solvent molecules is presumably greater in the excited state than in the ground state, the repulsive interactions should raise the energy of the excited state more than that of the ground state and thus cause blue shift in the spectrum. This raises the possibility that repulsive interactions may make considerable contributions to spectral shifts in other solvents, which are normally masked by the larger red shift caused by the dispersion interaction. Most current theories of spectral shifts fail to take these repulsive contributions into effect.