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Papers by Jane Vanderkooi

Probing the Active Site of Trypsin with Rose Bengal: Insights into the Photodynamic Inactivation of the Enzyme¶

Photochemistry and Photobiology, 2004

In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds c... more In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds competitively to the enzyme, and it can be used as a probe of the active site of the enzyme. On the basis of the emission wavelength, the binding site of trypsin is relatively polar and is similar to that of acetone in its polarity. The triplet state of rose bengal is quenched by trypsin. This quenching may be caused by the tryptophan and tyrosine residues that are in the near vicinity of the trypsin active site. This quenching can compete with the formation of singlet oxygen from the excited triplet state of rose bengal. We demonstrate that the singlet oxygen involved in the photoinactivation of trypsin is produced by the free rose bengal in solution and the bound dye is incapable of producing singlet oxygen. This explains the lack of correlation between photoinactivation efficiency and sensitizer binding capability previously reported by Wade and Spikes.

Reactions of excited triplet states of metal-substituted myoglobins

Proceedings of SPIE, May 1, 1990

The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compa... more The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compared. Both metal derivatives exhibit long triplet state lifetimes at room temperature, but whereas the Pd derivative showed exponential decay and an isosbestic point in the transient absorption spectra, the decay of the Zn derivative was nonsingle exponential and the transient absorption spectra showed evidence of more than one excited state species. No difference was seen in triplet quenching by oxygen for either derivative, indicating that differences in the polypeptide chain between the two derivatives are not large enough to affect oxygen penetrability. Quenching was also observed by anthraquinone sulfonate. In this case, the possibility of long-range transfer by an exchange mechanism is considered.

Archives of Biochemistry and Biophysics, Dec 1, 1988

The luminescence of the CO adduct of two isozymic tyrosinases isolated from Agaricus bispora, an ... more The luminescence of the CO adduct of two isozymic tyrosinases isolated from Agaricus bispora, an edible white mushroom, has been studied. At room temperature the emission appears as a single smooth peak centered at 530 nm with FWHM of 2700 cm-' and a lifetime of 36 ps. The lifetime and wavelength of the emission are virtually unchanged on lowering the temperature from 298 to '77°K. Solvent composition affects the wavelength of emission minimally. The emission is quenched by oxygen but not by a series of substrate analogs, inhibitors, or Lewis bases. The emission further appeared independent of aggregation state of the enzyme or isozyme type. A comparison of these data is made with those obtained by other researchers for the tyrosinase from Neurospora crassa and for several hemocyanins. The comparison supports the hypothesis that regulation of enzymatic activity does not take place within the coordination sphere of the copper atom observed. In addition, it suggests that the 550-to 560-nm emissions previously observed may not be considered characteristic of all CO derivatives of coupled binuclear copper proteins.

Photoreduction of membrane bound cytochrome c by excited-state phencthiazine

Biochemical and Biophysical Research Communications, Apr 1, 1976

Abstract Ferricytochrome c can be reduced in a photochemical reaction by excited state phenothiaz... more Abstract Ferricytochrome c can be reduced in a photochemical reaction by excited state phenothiazine. This reaction is observed between phenothiazine which is solubilized by phospholipid artificial membranes and cytochrome c which is adsorbed to the membrane surface. Under conditions when cytochrome c is not bound to the phospholipid, the rate of reduction by phenothiazine is greatly reduced. The phosphorescence of phenothiazine is quenched in the presence of cytochrome c , implying that the excited triplet state interacts with cytochrome c . Oxygen inhibits the reaction since possibly, as a paramagnetic species, it increases intersystem crossing of the excited states of phenothiazine. On the basis of molecular models the proximity between the iron of ferricytochrome c and phenothiazine is estimated to be over 20 A.

FEBS Letters, Oct 4, 1982

Site-selected fluorescence spectra of porphyrin derivatives of heme proteins

Biochemistry, Dec 1, 1985

The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesopor... more The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesoporphyrin derivatives of horseradish peroxidases A and C, leghemoglobin, and myoglobin were examined as a function of temperature and excitation wavelength. At room temperature, the emission spectra were unresolved and were independent of excitation wavelength. At low temperature (4.2 K), the spectra depended upon excitation wavelength: using narrow-band excitation into the high-energy side of the 0-1 and 0-0 bands gave unresolved emission spectra whereas excitation into the low-energy side produced quasi-line spectra. The resolved spectra were different for the five proteins and further varied with pH, indicating chromophore-protein interactions. The spectra are interpreted in terms of site selection and phonon interactions.

Biochemistry, Apr 1, 1974

Steady-state and time-resolved fluorescence anisotropy of 12-(9-anthroyl)stearic acid was examine... more Steady-state and time-resolved fluorescence anisotropy of 12-(9-anthroyl)stearic acid was examined in the presence of red blood cell membranes and phospholipid dispersions containing various amounts of cholesterol. The fluorescence emission spectrum, quantum yield, and fluorescence lifetime of AS are changed little by the presence of cholesterol. The rotational correlation time, 4 for 12-(9-anthroyl)stearic acid fluorescence anisotropy at 37" is 7.8 x 10-9 sec in normal red blood cells and 8.5 X low9 sec in blood cells containing twice the normal complement of cholesterol. A similar increase in 4 was observed in dipalmitoyllecithin vesicles containing high contents of cholesterol. The energy of activation for rotation of 12-(9-anthroyl)stearic acid is lowered by the A lthough there is no universally accepted model for membrane architecture, there is much evidence to indicate that many properties of membranes can be accounted for by a dynamic, fluid lipid bilayer structure (Gitler, 1972; Singer and Nicholson, 1972). Techniques used to detect the dynamic properties of the membrane include spin-label (

Applied Spectroscopy, Jul 1, 2011

The effect of high pressure on the OH stretch of dilute HOD in D 2 O was examined using high pres... more The effect of high pressure on the OH stretch of dilute HOD in D 2 O was examined using high pressure FTIR. It was found that at pressures directly above the ice VI to ice VII transition, ice VII displays a splitting in the OH absorption indicative of differing hydrogen bonding environments. This result is contrary to published structures of ice VII in which each OH oscillator should experience an identical electronic environment. The anomalous band was found to decrease in absorbance and finally disappear at ~43.0 kbar. In addition, the pressure response of the amide I′ and II′ bands of three small model peptides was examined. Analysis of these bands' response to increased pressure indicates significant side chain dependence of their structural rearrangement, which may play a role in the composition of full length proteins of barophilic organisms.

Biochimica Et Biophysica Acta - Biomembranes, 1977

Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether a... more Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.

Biochimica et biophysica acta, Sep 1, 1977

The phospholipids from cells grown on elaidate at 3'7°C were greatly enriched in trans-18 : 1 in ... more The phospholipids from cells grown on elaidate at 3'7°C were greatly enriched in trans-18 : 1 in both the acyl chains (79%) and alk-1-enyl chains (86%). The phospholipids showed a relatively narrow melting transition beginning at 24°C with both probes and ending at 12" C with TEMPO partitioning and at 15" C with diphenylhexatriene fluorescence polarization. Membranes from elaidate-grown cells also gave a relatively narrow inflection in diphenylhexatriene polarization studies, but the mid-point was 5°C higher than that obtained with the phospholipids. These studies indicate that the changes observed in the polar head group and glycerol acetal derivative composition of cells grown on oleate, partially compensate for the greatly increased proportions of cis unsaturated and cyclopropane acyl and alk-1-enyl chains in the phospholipids.

Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes

Biochemistry, Jun 1, 1990

Proceedings of SPIE, Apr 1, 1992

ABSTRACT

Influence of the pH on the pocket field of cytochrome c type proteins

Chemical Physics, Dec 1, 1997

Abstract We performed hole burning Stark effect experiments on a cytochrome c type protein. From ... more Abstract We performed hole burning Stark effect experiments on a cytochrome c type protein. From the behavior of the holes in an external electric field conclusions on local electric fields in the heme pocket can be drawn. We varied the pH value in order to study the influence of different groups on the pocket field: (a) titratable amino acid residues, (b) the porphyrin side chains (especially the propionates) and (c) the two axial ligands His18 and Met80. Surprisingly, a variation of the pH between 4.1 and 12.7 has no influence on the Stark effect. However, at a pH-level

Biospectroscopy, 1999

Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers... more Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately ␣-helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is ϳ 3 kcal.

Biophysical Journal, Sep 1, 1998

The photoexcited metastable triplet state of Mg 2ϩ-mesoporphyrin IX (MgMPIX) or Mg 2ϩ-protoporphy... more The photoexcited metastable triplet state of Mg 2ϩ-mesoporphyrin IX (MgMPIX) or Mg 2ϩ-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg 2ϩ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8-110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MgPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is Ͼ Ͼ150 cm Ϫ1 , suggesting symmetry breaking from the anisotropy of internal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527-16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q 0,0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg 2ϩ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.

Biochemistry, Jun 27, 1989

Room temperature phosphorescence techniques were used to study the structural and dynamic feature... more Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine a-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 f 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide < nitrite < acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be-3.2 X lov4. The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 OC. Arrhenius plots exhibited discontinuities at 26 and 29 OC for the short-and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations 'This work was supported by an NIH Cell and Molecular Biology

Biospectroscopy, 1997

Pro, and hexafluorovaline) and some of their metabolites (a-ketoglutarate, oxalacetate, pyruvate,... more Pro, and hexafluorovaline) and some of their metabolites (a-ketoglutarate, oxalacetate, pyruvate, succinate, citrate, and acetate) were determined in the infrared (IR) region from 1300 to 1700 cm 01 under conditions that are appropriate for biological studies (i.e., in phosphate-buffered D 2 O solution). The strongest transition in this region is n a OCO , with an extinction coefficient Ç 1 mM 01 cm 01 , and an emphasis was made to demonstrate use of this transition for enzymatic assays and to study proteins. To these ends, these relevant features were demonstrated. The value for n a OCO is a function of the residue p K: the higher the frequency, the lower the p K of the carboxylic acid. The high extinction of n a OCO permits detection of carboxyl groups in parvalbumin, a protein that is rich in Asp and Glu. The IR profiles for the amino acids and their metabolite products are sufficiently characteristic so that IR can be used to monitor enzymatic reactions involving amino acids. We show that transaminase reactions, which interconvert amino and keto acids, can be monitored by IR.

Spectral Splitting in the α (Q_0,0) Absorption Band of Ferrous Cytochrome c and Other Heme Proteins

Biochemistry, 1996

ABSTRACT

Surface of Cytochromec: Infrared Spectroscopy of Carboxyl Groups

Biochemistry, Dec 1, 1997

The carboxylate groups of organic acids give strong absorption in the infrared between approximat... more The carboxylate groups of organic acids give strong absorption in the infrared between approximately 1550 and 1650 cm-1. For acetate and chloroacetate derivatives, the infrared (IR) frequency of the carboxylate antisymmetric stretching mode (v(a)OCO) is related to the square root of the pK of the acid, with a shift of approximately 20 cm-1 to higher frequency for a pK drop in the range 5-3. It follows that v(a)OCO may respond to conditions on the protein surface. In this paper, the IR amide I' and carboxylate absorptions of cytochrome c from horse, yeast, and tuna are compared with model compounds such as Val-Glu and microperoxidase-11, the 11 amino acid fragment of horse cytochrome c containing the covalently bound heme. For microperoxidase-11, the contribution from all four carboxylates can be accounted for and the 1567 cm-1 absorption is assigned to the heme propionates. For the proteins, the carboxylate absorption band is inhomogeneous, i.e., there is a distribution of frequencies. Both the amide I' and carboxylate bands are sensitive to protein conformation as shown by their different pH, salt, and redox dependence.

Biophysical Journal, Jun 1, 1995

The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc ... more The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 104, 342 x 104, and 353 x 104 cm-1, respectively, and are temperature invariant over the temperature ranges studied. An El value at 4 K of 73 x 1 04 cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrin derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the El value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2) zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt care interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-' is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The El value is approximately 60 x 104 cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of-40 cm-1 is estimated. In contrast to those for the metal cytochromes, the El value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.

Probing the Active Site of Trypsin with Rose Bengal: Insights into the Photodynamic Inactivation of the Enzyme¶

Photochemistry and Photobiology, 2004

In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds c... more In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds competitively to the enzyme, and it can be used as a probe of the active site of the enzyme. On the basis of the emission wavelength, the binding site of trypsin is relatively polar and is similar to that of acetone in its polarity. The triplet state of rose bengal is quenched by trypsin. This quenching may be caused by the tryptophan and tyrosine residues that are in the near vicinity of the trypsin active site. This quenching can compete with the formation of singlet oxygen from the excited triplet state of rose bengal. We demonstrate that the singlet oxygen involved in the photoinactivation of trypsin is produced by the free rose bengal in solution and the bound dye is incapable of producing singlet oxygen. This explains the lack of correlation between photoinactivation efficiency and sensitizer binding capability previously reported by Wade and Spikes.

Reactions of excited triplet states of metal-substituted myoglobins

Proceedings of SPIE, May 1, 1990

The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compa... more The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compared. Both metal derivatives exhibit long triplet state lifetimes at room temperature, but whereas the Pd derivative showed exponential decay and an isosbestic point in the transient absorption spectra, the decay of the Zn derivative was nonsingle exponential and the transient absorption spectra showed evidence of more than one excited state species. No difference was seen in triplet quenching by oxygen for either derivative, indicating that differences in the polypeptide chain between the two derivatives are not large enough to affect oxygen penetrability. Quenching was also observed by anthraquinone sulfonate. In this case, the possibility of long-range transfer by an exchange mechanism is considered.

Archives of Biochemistry and Biophysics, Dec 1, 1988

The luminescence of the CO adduct of two isozymic tyrosinases isolated from Agaricus bispora, an ... more The luminescence of the CO adduct of two isozymic tyrosinases isolated from Agaricus bispora, an edible white mushroom, has been studied. At room temperature the emission appears as a single smooth peak centered at 530 nm with FWHM of 2700 cm-' and a lifetime of 36 ps. The lifetime and wavelength of the emission are virtually unchanged on lowering the temperature from 298 to '77°K. Solvent composition affects the wavelength of emission minimally. The emission is quenched by oxygen but not by a series of substrate analogs, inhibitors, or Lewis bases. The emission further appeared independent of aggregation state of the enzyme or isozyme type. A comparison of these data is made with those obtained by other researchers for the tyrosinase from Neurospora crassa and for several hemocyanins. The comparison supports the hypothesis that regulation of enzymatic activity does not take place within the coordination sphere of the copper atom observed. In addition, it suggests that the 550-to 560-nm emissions previously observed may not be considered characteristic of all CO derivatives of coupled binuclear copper proteins.

Photoreduction of membrane bound cytochrome c by excited-state phencthiazine

Biochemical and Biophysical Research Communications, Apr 1, 1976

Abstract Ferricytochrome c can be reduced in a photochemical reaction by excited state phenothiaz... more Abstract Ferricytochrome c can be reduced in a photochemical reaction by excited state phenothiazine. This reaction is observed between phenothiazine which is solubilized by phospholipid artificial membranes and cytochrome c which is adsorbed to the membrane surface. Under conditions when cytochrome c is not bound to the phospholipid, the rate of reduction by phenothiazine is greatly reduced. The phosphorescence of phenothiazine is quenched in the presence of cytochrome c , implying that the excited triplet state interacts with cytochrome c . Oxygen inhibits the reaction since possibly, as a paramagnetic species, it increases intersystem crossing of the excited states of phenothiazine. On the basis of molecular models the proximity between the iron of ferricytochrome c and phenothiazine is estimated to be over 20 A.

FEBS Letters, Oct 4, 1982

Site-selected fluorescence spectra of porphyrin derivatives of heme proteins

Biochemistry, Dec 1, 1985

The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesopor... more The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesoporphyrin derivatives of horseradish peroxidases A and C, leghemoglobin, and myoglobin were examined as a function of temperature and excitation wavelength. At room temperature, the emission spectra were unresolved and were independent of excitation wavelength. At low temperature (4.2 K), the spectra depended upon excitation wavelength: using narrow-band excitation into the high-energy side of the 0-1 and 0-0 bands gave unresolved emission spectra whereas excitation into the low-energy side produced quasi-line spectra. The resolved spectra were different for the five proteins and further varied with pH, indicating chromophore-protein interactions. The spectra are interpreted in terms of site selection and phonon interactions.

Biochemistry, Apr 1, 1974

Steady-state and time-resolved fluorescence anisotropy of 12-(9-anthroyl)stearic acid was examine... more Steady-state and time-resolved fluorescence anisotropy of 12-(9-anthroyl)stearic acid was examined in the presence of red blood cell membranes and phospholipid dispersions containing various amounts of cholesterol. The fluorescence emission spectrum, quantum yield, and fluorescence lifetime of AS are changed little by the presence of cholesterol. The rotational correlation time, 4 for 12-(9-anthroyl)stearic acid fluorescence anisotropy at 37" is 7.8 x 10-9 sec in normal red blood cells and 8.5 X low9 sec in blood cells containing twice the normal complement of cholesterol. A similar increase in 4 was observed in dipalmitoyllecithin vesicles containing high contents of cholesterol. The energy of activation for rotation of 12-(9-anthroyl)stearic acid is lowered by the A lthough there is no universally accepted model for membrane architecture, there is much evidence to indicate that many properties of membranes can be accounted for by a dynamic, fluid lipid bilayer structure (Gitler, 1972; Singer and Nicholson, 1972). Techniques used to detect the dynamic properties of the membrane include spin-label (

Applied Spectroscopy, Jul 1, 2011

The effect of high pressure on the OH stretch of dilute HOD in D 2 O was examined using high pres... more The effect of high pressure on the OH stretch of dilute HOD in D 2 O was examined using high pressure FTIR. It was found that at pressures directly above the ice VI to ice VII transition, ice VII displays a splitting in the OH absorption indicative of differing hydrogen bonding environments. This result is contrary to published structures of ice VII in which each OH oscillator should experience an identical electronic environment. The anomalous band was found to decrease in absorbance and finally disappear at ~43.0 kbar. In addition, the pressure response of the amide I′ and II′ bands of three small model peptides was examined. Analysis of these bands' response to increased pressure indicates significant side chain dependence of their structural rearrangement, which may play a role in the composition of full length proteins of barophilic organisms.

Biochimica Et Biophysica Acta - Biomembranes, 1977

Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether a... more Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.

Biochimica et biophysica acta, Sep 1, 1977

The phospholipids from cells grown on elaidate at 3'7°C were greatly enriched in trans-18 : 1 in ... more The phospholipids from cells grown on elaidate at 3'7°C were greatly enriched in trans-18 : 1 in both the acyl chains (79%) and alk-1-enyl chains (86%). The phospholipids showed a relatively narrow melting transition beginning at 24°C with both probes and ending at 12" C with TEMPO partitioning and at 15" C with diphenylhexatriene fluorescence polarization. Membranes from elaidate-grown cells also gave a relatively narrow inflection in diphenylhexatriene polarization studies, but the mid-point was 5°C higher than that obtained with the phospholipids. These studies indicate that the changes observed in the polar head group and glycerol acetal derivative composition of cells grown on oleate, partially compensate for the greatly increased proportions of cis unsaturated and cyclopropane acyl and alk-1-enyl chains in the phospholipids.

Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes

Biochemistry, Jun 1, 1990

Proceedings of SPIE, Apr 1, 1992

ABSTRACT

Influence of the pH on the pocket field of cytochrome c type proteins

Chemical Physics, Dec 1, 1997

Abstract We performed hole burning Stark effect experiments on a cytochrome c type protein. From ... more Abstract We performed hole burning Stark effect experiments on a cytochrome c type protein. From the behavior of the holes in an external electric field conclusions on local electric fields in the heme pocket can be drawn. We varied the pH value in order to study the influence of different groups on the pocket field: (a) titratable amino acid residues, (b) the porphyrin side chains (especially the propionates) and (c) the two axial ligands His18 and Met80. Surprisingly, a variation of the pH between 4.1 and 12.7 has no influence on the Stark effect. However, at a pH-level

Biospectroscopy, 1999

Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers... more Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately ␣-helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is ϳ 3 kcal.

Biophysical Journal, Sep 1, 1998

The photoexcited metastable triplet state of Mg 2ϩ-mesoporphyrin IX (MgMPIX) or Mg 2ϩ-protoporphy... more The photoexcited metastable triplet state of Mg 2ϩ-mesoporphyrin IX (MgMPIX) or Mg 2ϩ-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg 2ϩ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8-110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MgPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is Ͼ Ͼ150 cm Ϫ1 , suggesting symmetry breaking from the anisotropy of internal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527-16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q 0,0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg 2ϩ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.

Biochemistry, Jun 27, 1989

Room temperature phosphorescence techniques were used to study the structural and dynamic feature... more Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine a-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 f 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide < nitrite < acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be-3.2 X lov4. The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 OC. Arrhenius plots exhibited discontinuities at 26 and 29 OC for the short-and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations 'This work was supported by an NIH Cell and Molecular Biology

Biospectroscopy, 1997

Pro, and hexafluorovaline) and some of their metabolites (a-ketoglutarate, oxalacetate, pyruvate,... more Pro, and hexafluorovaline) and some of their metabolites (a-ketoglutarate, oxalacetate, pyruvate, succinate, citrate, and acetate) were determined in the infrared (IR) region from 1300 to 1700 cm 01 under conditions that are appropriate for biological studies (i.e., in phosphate-buffered D 2 O solution). The strongest transition in this region is n a OCO , with an extinction coefficient Ç 1 mM 01 cm 01 , and an emphasis was made to demonstrate use of this transition for enzymatic assays and to study proteins. To these ends, these relevant features were demonstrated. The value for n a OCO is a function of the residue p K: the higher the frequency, the lower the p K of the carboxylic acid. The high extinction of n a OCO permits detection of carboxyl groups in parvalbumin, a protein that is rich in Asp and Glu. The IR profiles for the amino acids and their metabolite products are sufficiently characteristic so that IR can be used to monitor enzymatic reactions involving amino acids. We show that transaminase reactions, which interconvert amino and keto acids, can be monitored by IR.

Spectral Splitting in the α (Q_0,0) Absorption Band of Ferrous Cytochrome c and Other Heme Proteins

Biochemistry, 1996

ABSTRACT

Surface of Cytochromec: Infrared Spectroscopy of Carboxyl Groups

Biochemistry, Dec 1, 1997

The carboxylate groups of organic acids give strong absorption in the infrared between approximat... more The carboxylate groups of organic acids give strong absorption in the infrared between approximately 1550 and 1650 cm-1. For acetate and chloroacetate derivatives, the infrared (IR) frequency of the carboxylate antisymmetric stretching mode (v(a)OCO) is related to the square root of the pK of the acid, with a shift of approximately 20 cm-1 to higher frequency for a pK drop in the range 5-3. It follows that v(a)OCO may respond to conditions on the protein surface. In this paper, the IR amide I' and carboxylate absorptions of cytochrome c from horse, yeast, and tuna are compared with model compounds such as Val-Glu and microperoxidase-11, the 11 amino acid fragment of horse cytochrome c containing the covalently bound heme. For microperoxidase-11, the contribution from all four carboxylates can be accounted for and the 1567 cm-1 absorption is assigned to the heme propionates. For the proteins, the carboxylate absorption band is inhomogeneous, i.e., there is a distribution of frequencies. Both the amide I' and carboxylate bands are sensitive to protein conformation as shown by their different pH, salt, and redox dependence.

Biophysical Journal, Jun 1, 1995

The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc ... more The photoactivated metastable triplet states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 104, 342 x 104, and 353 x 104 cm-1, respectively, and are temperature invariant over the temperature ranges studied. An El value at 4 K of 73 x 1 04 cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrin derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the El value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2) zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt care interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-' is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The El value is approximately 60 x 104 cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of-40 cm-1 is estimated. In contrast to those for the metal cytochromes, the El value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.