The role of the superoxide anion in the xanthine oxidase-induced autoxidation of linoleic acid.

A fluorescent analogue, palmitoyl-?CoA was shown to have a fluorescence lifetime (19.5 nsec.), polarization and absorption and emission characteristics useful for studying interactions with enzymes and with model membranes. The fluorescence lifetime was found to be wavelength dependent. The analogue was a better inhibitor (50% inhibition at ～ 0.2 μM) than palmitoyl-CoA (50% inhibition at 0.5 μM) when bound to mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxido reductase E.C.l.l.137). The fluorescence depolarization when bound to this enzyme was less than that observed for binding to bovine serum albumin suggesting some mobility of the chromophore while bound. The changes in polarization upon titration with phosphatidylcholine (egg) vesicles were consistent with a partition of palmitoyl-(1,N⁶etheno)CoA between vesicles and malate dehydrogenase. Such partition may have physiological consequences.     

Human aglycosyl-IgG exhibits increased hydrophobicity. Binding/fluorescence studies with 8-anilinonaphthalene-1-sulfonic acid (ANS)

Human monoclonal, aglycosyl-IgG produced in vitro in the presence of tunicamycin, was compared with its native and acid pH-altered counterparts for their respective abilities to bind the fluorescent hydrophobicity probe, 8-anilinonaphthalene sulfonate. A novel technique based on continuous-flow dynamic dialysis (Sparrow et al., 1982, Anal. Biochem. 123:255-264) allowed binding studies under non-equilibrium conditions. While the native IgG conformation exhibits two, weak ANS binding sites (ca. 10(3) l/mol), aglycosyl-IgG has one weak and one moderate affinity (least squares average Ka = 2 X 10(4) l/mol) site, and the acid conformer binds yet another two ANS molecules with moderate affinity (4 X 10(4) l/mol). Increases in affinity and in the number of sites correlate roughly with increased relative percent fluorescence by conventional fluorimetry. The fluorescence lifetime of ANS bound to altered IgGs is about 10% longer (T2 = 15 nsec by time-resolved fluorimetry) than that for native IgG. All populations also exhibit a rapid decay component (T1 = 3 nsec) analogous to that seen for ANS in 50% aqueous dioxane. Results are discussed in relation to structural role(s) for IgG-linked heterosaccharides.     

An extended series of fluorescent sulfhydryl reagents useful in energy transfer measurements

Synthesis of four new fluorescent sulfhydryl reagents is described. All are isomers of the previously synthesized N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonic acid (1,5-I-AEDANS) and its 1,8-isomer (1,8-I-AEDANS). Three of these new probes (1,4-Br-AEDANS, 2,8-Br-AEDANS, and 2,6-I-AEDANS) carry a single sulfonic acid residue and the fourth (3-(2,7)-Br-AEDANS) carries two sulfonic acid residues. The excitation and emission spectrum of each of these probes is distinct when covalently attached to bovine serum albumin. In addition, they all show a single fluorescent lifetime in the range of 8.0 to 20.8 nsec. This extended range of fluorescent isomers can thus be useful for selecting approprlate energy donors in fluorescence energy transfer experiments.     

Proof of nanosecond timescale relaxation in apomyoglobin by phase fluorometry

Apomyoglobin was labeled with the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). Apparent phase shift and demodulation lifetimes of bound TNS were measured at various emission wavelengths. The lifetimes increased with increasing wavelength. Similar results were obtained for TNS in the viscous solvent glycerol at 10°C but not for TNS in vitrified or fluid solvent. The wavelength-dependent lifetimes suggest apomyoglobin is relaxing around the TNS molecule during its fluorescent lifetime. Importantly, the apparent phase lifetimes exceeded the apparent modulation lifetimes on the long wavelength side of the emission for TNS in apomyoglobin at 3°C and for TNS in glycerol at 10°C. This result proves the increasing lifetimes are a result of an excited state reaction during the lifetime of the excited state and are not a result of heterogeneity in the fluorescence emission. From the lifetimes on the short wavelength side of the emission the relaxation time of apomyoglobin was estimated to be 18 nsec.     

Binding of 1,N6-ethanoadenosine triphosphate to actin

G-actin is known to bind one molecule of ATP. Its polymerization to F-actin is accompanied by the splitting off of the terminal phosphate of the bound nucleotide. We have found that the fluorescent 1,N⁶-ethanoadenosine triphosphate (?ATP) can substitute for ATP in G-actin and that G-actin containing bound ?ATP possesses essentially full polymerizability. The binding of this ATP analog has been studied by following the inactivation of the ?ATP·G-actin complex. The binding constant (4?5.7 × 10⁶ M^?1) obtained in the absence of EDTA is about 50% of that for ATP, while the binding constant obtained in the presence of EDTA (0.9?3.0 × 10⁵ M^?1) is comparable to those for ATP and ADP. These findings suggest that ?ATP can be used as a structural probe for actin. The fluorescence lifetime of ?ATP bound to G·actin is 36 nsec. The rotational relaxation time of ?ATP·G-actin is near 60 nsec. at 20°C.     

Fluorescence studies of 1,N6-ethenoadenosine triphosphate bound to G-actin: the nucleotide base is inaccessible to water

When 1,N6-ethenoadenosine triphosphate (epsilon-ATP) is free in solution, its fluorescence is collisionally quenched by iodide ion, by methionine, by tryptophan, and by cysteine. None of these quenches the fluorescence of epsilon-ATP bound to G-actin. Thus, the ethenoadenine base is bound in a region of the protein which is inaccessible to collisions with these reagents. Since we have previously shown that the fluorescence of epsilon-ATP is quenched by water, the long lifetime of epsilon-ATP bound to G-actin (36 nsec, vs 27 nsec for epsilon-ATP in water) indicates that the bound nucleotide base is inaccessible to collisional quenching by water molecules.     

Fluorescence lifetime reference standards for the range 0.189 to 115 nanoseconds

A review of the modern literature reveals that the values for quinine fluorescence lifetime are in good agreement, the mean value being 18.91±0.56 nsec. By virtue of some very unusual properties, quinine appears suitable for use as a lifetime reference standard for any value from 0.189 to 18.9 nsec, with an expected accuracy of ±3% throughout this range. Cl^?, not normally considered a quenching agent, quenches quinine emission at diffusion-controlled rates. The Stern-Volmer plot was unique in that the strict lincarity, indicating pure collisional quenching, was maintained even when fluorescence was > 99% quenched. Thus, solutions of quinine-NaCl can be made up having lifetimes known to great accuracy. Similarly, γ-pyrenebutyrate solutions containing KI are suitable standards for the range up to 115 nsec. The compositions of such solutions have been calculated and tabulated. It is argued that the lifetimes of these solutions are at least as reliable as any of the hundreds of lifetimes which have been reported in the literature. Several important applications of such lifetime standards are discussed.     

Energy transfer in bacterial photosynthesis

Four possible explanations are offered to account for low fluorescence increase observed for purple bacteria under transition from active to inhibited photosynthesis. The increase observed is inconsistent with high (1.0) yield of primary photosynthetic process of P₈₉₀ photooxidation. The dependences of fluorescence yield and lifetime on the portion of active reaction centres have been analysed for each case. Experimental investigation carried out favours the existence of background fluorescence together with fluorescence, whose quantum yield correlates with the reaction centre functional state. The important conclusion is made that lifetime of photosynthetic fluorescence is much lower than 1 nsec and energy is transferred to the reaction centres by a mechanism other than inductive-resonance.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: Lifetime of the Excited State In Vivo: I. Chlorophyll a in Algae, at Room and at Liquid Nitrogen Temperatures; Rate Constants of Radiationless Deactivation and Trapping

Using a mode-locked laser (λ, 632.8 nm), fluorescence decay of chlorophyll (Chl) a in the green alga Chlorella pyrenoidosa, the red alga Porphyridium cruentum, and the blue-green alga Anacystis nidulans was measured by the phase-shift method under conditions when photosynthesis was not operative (3-(3,4-dichlorophenyl)-1,1-dimethylurea [DCMU] poisoning, or cooling to 77°K). In the presence of 10^-5 M DCMU, the lifetime of Chl a fluorescence (τ) at room temperature is about 1.7 nsec in Chlorella, 1.0 nsec in Porphyridium, and 0.7 nsec in Anacystis. At 77°K, τ is 1.4 nsec (for fluorescence at about 685 nm, F-685) and 2.3 nsec (for F-730) in Chlorella, 0.9 nsec (F-685) and 1.2 nsec (F-730) in Porphyridium, and 0.8 nsec (F-685 and F-730) in Anacystis. From the above measurement, and the assumption that τ₀ (the intrinsic fluorescence lifetime) for Chl a in all three algae is 15.2 nsec, we have calculated the rate constants of radiationless transition (that includes energy transfer to weakly fluorescent system I) processes competing with fluorescence at room temperature to be about 5 × 10⁸ sec^-1 in Chlorella, 9 × 10⁸ sec^-1 in Porphyridium, and 13 × 10⁸ sec^-1 in Anacystis. At 77°K, this rate constant for Chl a that fluoresces at 685 nm remains, in the first approximation, the same as at room temperature. From the τ data, the rate constant for the trapping of excitation energy is calculated to be about 1.2 × 10⁹ sec^-1 for Chlorella, 2 × 10⁹ sec^-1 for Porphyridium, and 2 × 10⁹ sec^-1 for Anacystis. The efficiency of trapping is calculated to be about 66% (Chlorella), 68% (Porphyridium), and 60% (Anacystis). (It is recognized that variations in the above values are to be expected if algae grown under different conditions are used for experimentation.) The maximum quantum yield of Chl a fluorescence for system II (λ, 632.8 nm), calculated from τ measurements, is about 10% in Chlorella, 6-7% in Porhyridium, and 5% in Anacystis under conditions when photosynthesis is not operative; the values at 77°K appear to be very close to those with DCMU added at room temperature. ø for F-730 at 77°K, however, is somewhat higher than for F-685. The predicted quantum yields of fluorescence for Chl a in intact cells (both systems I and II) at low intensities of 632.8 nm light are about 2-3, 1-2, and 1% for Chlorella, Porphyridium, and Anacystis, respectively.     

Synthesis and properties of a new fluorescent analog of ATP: Adenosine-5′-triphosphoro-γ-1-(5-sulfonic acid) napthylamidate

An analog of ATP has been synthesized which contains the fluorophore, 1-aminonapthalene-5-sulfonate attached via a γ-phosphoamidate bond. This analog is strongly fluorescent (quantum yield = 0.63) with an emission maximum at 460 nm; the excited state lifetime is 20 nsec. It is a substrate for DNA-dependent RNA polymerase of $\text{E. coli}$ and wheat germ RNA polymerase II. It is also a substrate for $\text{E. coli}$ valyl t-RNA synthetase, venom phosphodiesterase, and potato apyrase. Cleavage of the α-β phosphoryl bond as a result of RNA synthesis or by venom phosphodiesterase produces a 15 nm red shift in the fluorescence emission spectrum. This property should make this nucleotide useful for studies of the mechanisms of enzymatic reactions involving cleavage of the α-β phosphoryl bond.     

Fluorescence lifetime distributions of diphenylhexatriene-labeled phosphatidylcholine as a tool for the study of phospholipid-cholesterol interactions.

Fluorescence lifetimes of 1-palmitoyl-2-diphenylhexatrienylpro-pionyl-phosphatidylc hol ine in vesicles of palmitoyloleoyl phosphatidylcholine (POPC) (1:300, mol/mol) in the liquid crystalline state were determined by multifrequency phase fluorometry. On the basis of statistic criteria (chi 2red) the measured phase angles and demodulation factors were equally well fitted to unimodal Lorentzian, Gaussian, or uniform lifetime distributions. No improvement in chi 2red could be observed if the experimental data were fitted to bimodal Lorentzian distributions or a double exponential decay. The unimodal Lorentzian lifetime distribution was characterized by a lifetime center of 6.87 ns and a full width at half maximum of 0.57 ns. Increasing amounts of cholesterol in the phospholipid vesicles (0-50 mol% relative to POPC) led to a slight increase of the lifetime center (7.58 ns at 50 mol% sterol) and reduced significantly the distributional width (0.14 ns at 50 mol% sterol). Lifetime distributions of POPC-cholesterol mixtures containing greater than 20 mol% sterol were within the resolution limit and could not be distinguished from monoexponential decays on the basis of chi 2red. Cholesterol stabilizes and rigidifies phospholipid bilayers in the fluid state. Considering its effect on lifetime distributions of fluorescent phospholipids it may also act as a membrane homogenizer.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Measurement of the fluorescence lifetime of chlorophyll a in vivo

New measurements have been made of fluorescence lifetime (τ) of chlorophyll a in the algae Chlorella pyrenoidosa, Porphyridium cruentum, Anacystis nidulans, and in spinach chloroplast. τ-values of 0.6 and 0.7 nsec were obtained with green plants. Anacystis and Porphyridium gave a τ of 0.5 nsec. The previously described two stage decay of fluorescence in vivo in these organisms could not be confirmed. This observation could have been caused by a second wave of light emission from the exciting hydrogen lamp (not detected in earlier work). The lifetimes found in this study (calculated, as before, by the method of convolution integrals) were close to those found by other observers for “low” excitation intensities; the value first reported from this laboratory (1.0-1.7 nsec) may have corresponded to “high” excitation intensity.     

Effect of cholinergic ligands on the lipids of acetylcholine receptor-rich membrane preparations from torpedo californica

Ion permeation, triggered by ligand-receptor interaction, is associated with the primary events of membrane depolarization at the neuromuscular junction and synaptic connections. To explore the possible sites of ion permeation, the long-lived fluorescent probe pyrene (fluorescence lifetime ～400 nsec) has been inserted into the lipid phase of acetylcholine receptor-rich membrane (AcChR-M) preparations from Torpedo californica. The pyrene probe is susceptible to both fluidity and permeability changes in the lipid bilayer. These changes are detected by variations in the rate of decay of the excited singlet state of pyrene after pulsation with a 10-nsec ruby laser flash. Variations of these lifetimes in the membrane preparations alone or in the presence of quenchers show that binding of cholinergic agonists and antagonists, neurotoxins, and local anesthetics to AcChR-M produces varying effects on the properties of the pyrene probe in the lipid phase. It is concluded that binding of cholinergic ligands to the receptor does not significantly alter the fluidity or permeability of the lipids in the bilayer in contact with pyrene. On the other hand, local anesthetics do affect these properties.     

Red fluorescent protein DsRed: Parametrization of its chromophore as an amino acid residue for computer modeling in the OPLS-AA force field

Topology of the neutral form of the DsRed fluorescent protein chromophore as a residue of [(4-cis)-2-[(1-cis)-4-amino-4-oxobutanimidoyl]-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl]acetic acid was calculated with OPLS-AA force field. Use of this topology and molecular dynamics simulation allows calculating the parameters of proteins that contain such residue in their polypeptide chains. The chromophore parameters were obtained by ab initio (RHF/6-31G**) quantum chemical calculations applying density functional theory (B3LYP). Using this chromophore, we have calculated the molecular dynamics trajectory of tetrameric fluorescent protein DsRed in solution at 300 K (4 nsec). Correctness of the chromophore parametrization was revealed by comparison of quantitative characteristics of the chromophore structure obtained from the molecular dynamic simulations of DsRed protein with the quantitative characteristics of the chromophore based on the crystallographic X-ray data of fluorescent protein DsRed (PDB ID: 1ZGO, 1G7K, and 1GGX), and also with the quantitative characteristics of the chromophore obtained by quantum chemical calculations. Inclusion of the neutral form of DsRed protein chromophore topology into the OPLS-AA force field yielded the extended force field OPLS-AA/DsRed. This force field can be used for molecular dynamics calculations of proteins containing the DsRed chromophore. The parameter set presented in this study can be applied for similar extension in any other force fields.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Events: Lifetime of the Excited State In Vivo: II. Bacteriochlorophyll in Photosynthetic Bacteria at Room Temperature

Lifetime of the excited state (τ) of bacteriochlorophyll (BChl) in photosynthetic bacteria, measured with a mode-locked argon laser (oscillating at 488 nm; mode locked at 56 MHz) as light source, ranged from 0.3 to 2.5 nsec. These τ values are reported with a precision of ±0.1 nsec. The value of τ at high exciting light intensity (I) was two to three times that at low intensity. For young cultures of green bacterium Chloropseudomonas ethylicum, τ ranged from 0.5 (low I) to 1.0 nsec (high I); for those of the purple bacterium Rhodospirillum rubrum, from 0.4 (low I) to 1.0 nsec (high I); and for those of the BChl b-containing Rhodopseudomonas viridis, from 1.0 (low I) to 2.5 nsec (high I). These data provide information regarding the efficiencies of the photochemical process in these bacteria. Quantum yield (ø) of BChl fluorescence, calculated from ø = τ/τ₀ (where τ₀ is the intrinsic lifetime of fluorescence), ranges from 2-6% at low intensities to 6-14% at high intensities.     

Heterogeneity in the structural dynamics of Sulfolobus solfataricus beta-glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.     

Probing the optical properties and luminescence mechanism of a UV‐emitting SrBPO5:Ce3+ phosphor

Ce‐doped (1 × 10^?5 to 3.0 mol%) SrBPO₅ phosphors were synthesized using a conventional solid‐state reaction route at 1273 K in an air atmosphere. Phase and morphology of the samples were studied from powder X‐ray diffraction (XRD) patterns and scanning electron microscope (SEM) micrographs, respectively. The band gap energies of the pure and Ce‐doped SrBPO₅ phosphors were calculated from the recorded diffuse reflectance spectra. Photoluminescence (PL) and Ce³⁺ lifetime were recorded at 300 and 77 K. Photoluminescence lifetime measurements revealed two‐lifetime values for Ce³⁺ at both 300 K (17 and 36 nsec) and 77 K (12 and 30 nsec), suggesting the presence of two different environments around Ce³⁺. Time‐resolved emission spectroscopy (TRES) studies confirmed the presence of Ce³⁺ in two different environments. In addition, SrBPO₅:Ce exhibited intense UV emission, signifying its possible use as an efficient sensitizer for solid‐state lighting applications. The effect of γ‐irradiation on PL was also determined. Thermally stimulated luminescence (TSL) glow curves of the γ‐irradiated phosphor, along with trap parameters, dose–response, and the possible TSL mechanism were also investigated. Positron annihilation lifetime spectroscopy was carried out to probe defects present in undoped and Ce‐doped SrBPO₅.     

Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.