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Biomedical subjects

B Chance

Publications and source records attributed to B Chance.

At least 415 records · Page 23Linked to original sources
PubMed1980

Noninvasive measurements of pyridine nucleotide fluorescence from the cornea.

The autofluorescence of reduced pyridine nucleotides (NADH and NADPH) and oxidized flavoproteins within the rabbit cornea were noninvasively measured as a function of depth. This was accomplished by combining a corneal specular microscope with a time-shared spectrofluorometer. When either 8 mM sodium pentobarbital or sodium sulfide, known inhibitors of mitochondrial respiration were applied to cornea, the autofluorescence at 440 nm (excited at 366 nm) increased and that at 540 nm (excited at 460 nm) decreased. No autofluorescence was measurable following destruction of the cellular membranes by freezing and leaching of the cellular constituents. The 440 nm autofluorescence is from reduced pyridine nucleotides, whereas the 540 nm autofluorescence is from the oxidized flavoproteins. The time course of the pyridine nucleotide autofluorescence after the application of the pentobarbital to either the endothelial or epithelial bathing solutions made it possible to measure the diffusion properties of this drug through the cornea. The method used is useful studying the diffusion and effects of metabolically active drugs upon the cornea.

Animals↗
PubMed1979

The reaction of cytochrome oxidase with oxygen in the fission yeast Schizosaccharomyces pombe 972h-. Studies at subzero temperatures and measurement of apparent oxygen affinity.

1. Cytochrome alpha 3 in whole-cell suspensions of the fission yeast Schizosaccharomyces pombe reacted in the reduced form with CO to give a photodissociable CO complex with absorption maxima at 429, 543 and 591 nm in CO-liganded reduced-minus-reduced difference spectra. 2. Other CO-bound haemoproteins, cytochromes P-420 and P-450, were not photodissociated under the conditions employed. 3. Measurements of the rates of reassociation of CO with cytochrome alpha 3 after flash photolysis over the temperature range from -101 to -109 degrees C gave a value for Eact. of 28.6 kJ/mol. 4. Between -94 and -106 degrees C, O2 reacted with cytochrome oxidase in intact cells to give an oxygenated intermediate (compound A). 5. At -70 degrees C compound A was converted into a second spectrally distinct intermediate (compound B). 6. Electron transport, indicated by the oxidation of cytochromes alpha + alpha 3 and cytochrome c, did not occur until the temperature was raised to -50 degrees C. 7. At room temperature cytochfome oxidase was oxidized to 50% of its steady-state concentration by 0.35 microM-O2.

Ascomycota↗
PubMed1979

The reaction of cytochrome omicron in Escherichia coli with oxygen. Low-temperature kinetic and spectral studies.

1. The reactions of cytochrome omicron in intact cells of aerobically grown Escherichia coli with O2 and CO have been studied at low temperature. 2. Flash photolysis of CO-liganded cells in the presence of O2 and at temperatures between -79 and -102 degrees C results in the oxidation of kinetically heterogeneous beta-type cytochromes (including cytochrome omicron), but not of cytochrome d. 3. The reaction of reduced cytochrome omicron with O2 involves O2 binding to give intermediate(s) with spectral characteristics similar to that of the reduced oxidase-CO complex. Observation in the alpha-region suggests that unexplained ligand dissociation accompanies the initial O2 binding. 4. At temperatures below -98 degrees C, an 'end point' in the reaction is reached; further reaction and oxidation of cytochrome omicron occurs on raising the temperature. 5. There is a linear relationship between the rate of formation of the oxygen compound and the O2 concentration up to 0.5 mM. The second-order constant for its formation (k+1) is 0.91 M-1.S-1 at -101 degrees C. The reaction is not readily reversible, the value of k-1 being 1.4 X 10(-5) S-1 and the kd 1.5 X 10(-5) M. 6. The energy of activation for this reaction at low temperatures is 29.9kJ (7.1 kcal)/mol. 7. The reaction with O2 is distinguished from that with CO by the markedly lower velocity and high photolytic reversibility of the latter. 8. Comparisons are drawn between the intermediate(s) in the O2 reaction of cytochrome omicron in E. coli and those identified in other bacteria and in the reaction of cytochrome aa3 with O2.

Carbon Monoxide↗
PubMed1979

Spectroscopic forms of carbonmonoxi-cytochrome oxidase.

A systematic study of the errors of low-temperature recording of kinetics of the cytochrome oxidase-CO reaction had identified the classic devitrification process of Keilin & Hartree [(1950) Nature (London)165, 504-505]. The methodology described here minimizes this effect, and the computation methods afford appropriate ways of detecting a residual effect. Thus it has been possible to identify that absorption difference spectra and kinetics of the reaction of fully reduced or half-reduced cytochrome oxidase with CO indicate only one spectroscopic form of the respective carbonmonoxi-cytochrome oxidase.

Animals↗
PubMed1979

Chemiluminescence of Acanthamoeba castellanii.

1. Chemiluminescence of Acanthomoeba castellanii in the presence of O2 was of similar intensity in organisms harvested early or late during exponential growth [when cyanide (1 mM) stimulates or inhibits respiration respectively]. 2. Cyanide (up to 1.5 mM) stimulated photoemission in both types of organism by 250--300 photons/s per 10(7) cells above the value observed under aerobic conditions. 3. 'Dibromothymoquinone' (2,5-dibromo-6-isopropyl-3-methyl-p-benzoquinone) (up to 80 microM) further increased chemiluminescence. 4. Similar responses were also demonstrated in whole homogenates and in subcellular fractions; 36% of the chemiluminescence was provided by a fraction sedimenting at 100000g-min, and 20% in that fraction that was non-sedimentable at 200000g-min. 5. Mitochondrial substrates (succinate, 2-oxoglutarate, NADH) in the presence or absence of ADP and Pi or peroxisomal substrates (glycollate, urate or ethanol) gave no increases in light emission by whole homogenates or in any of the fractions. 6. It is suggested that reactions responsible for production of chemiluminescence are those primarily producing superoxide anions and leading to lipid peroxidation and singlet-oxygen formation. Photoemission enhancement and superoxide dismutase inhibition showed similar cyanide concentration-dependencies.

Amoeba↗
PubMed1979

The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii.

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of -18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2--9.8.

Azotobacter↗
PubMed1979

Microsecond luminescence emission from copper cytochrome c.

The luminescence of Cu-substituted cytochrome c exhibits temperature-dependent decay modes and spectral shifts at temperatures below 80 K. By comparison with the data from metal-substituted porphyrins, we determined intramolecular rate parameters. The observed tripdoublet decay time is 13 +/- 1 mus at 77 K and the quartet decay time is 12 +/- 5 mus below 30 K. We also investigated the luminescence emission from Cu cytochrome c in the presence of cytochrome c depleted mitochondria and in the presence of soluble cytochrome c oxidase; upon binding, the intensity and decay time of the emission are altered. The results indicate an interaction between cytochrome c and its mitochondrial binding site which depends on the electronic state of cytochrome c.

Copper↗
PubMed1979

Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.

The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.

Animals↗
PubMed1979

The nature of the copper atoms of cytochrome c oxidase as studied by optical and x-ray absorption edge spectroscopy.

X-ray absorption edge spectroscopy has been used to study the copper of 1--2 mM cytochrome c oxidase in the resting oxidized, mixed-valence, and fully reduced states. A comparison was made of this protein with copper complexes and with natural and artificial copper proteins. Spectra were obtained with synchrotron radiation from the SPEAR storage ring using highly sensitive fluorescence detectors. Temperatures of -80 to -120 degrees C were employed further to improve the stability of the samples and to avoid the possibility of either auto- or photon-induced reduction of the materials, which might have occurred in previous studies. In order to characterize the valence states of the Cu and Fe components, the samples were monitored by infrared and visible spectroscopy before and after irradiation by the X-ray beam. The combination of the optical and X-ray absorption techniques has afforded a deconvolution of the four species of copper in the various states of cytochrome c oxidase and the tentative assignment of Cu alpha, the copper redox coupled to the heme alpha of cytochrome alpha, as a highly covalent type of copper and Cu alpha 3, the copper of cytochrome alpha 3, as a more ionic 'blue' type I copper. The implications of these findings upon the mechanism of action of cytochrome oxidase are briefly outlined.

Absorptiometry, Photon↗
PubMed1979

Compound C2, a product of the reaction of oxygen and the mixed-valence state of cytochrome oxidase. Optical evidence for a type-I copper.

Compound C2 is a product of the reaction of O2 and the mixed-valence state of cytochrome oxidase. The mixed-valence state of membrane-bound cytochrome oxidase is obtained at -24 degrees C, by using either ferricyanide or yeast peroxidase complex ES as oxidants, and the configurations of oxidized haem a and its associated copper (a3+Cua2+) and of reduced haem a3 and its associated copper (ac3+.CO.Cua3+) are obtained. The mixed-valence-state cytochrome oxidase mixed with O2 at -24 degrees C and flash-photolysed at -60 to -100 degrees C reacts with O2 and initially forms an oxy compound (A2) similar to that formed from the fully reduced state (A1). Thereafter the course of the reaction differs from that obtained in the fully reduced state, and absorbance increases are observed at 740--750 nm and 609 nm and a decrease at 444 nm, with no increase in absorbance at 655 nm. One possible attribution of the absorbance increases is to charge-transfer interaction between the iron of haem a3 and the copper associated with haem a3, Cua3(2+), having properties of a type-I 'blue' copper. A possible attribution of the decrease in absorbance at 444 nm is to liganding of a3(2+). A related explanation is that the 609 nm absorbance involves a charge-transfer interaction of both iron and copper as a mixed-valence binuclear complex, Cua3, having properties of a non-blue copper. Intermediates in addition to Compound C2 are not yet identifiable by chemical or spectroscopic tests. The kinetic and equilibrium properties of Compound C2 are described.

Animals↗
PubMed1979

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.

Antimycin A↗
PubMed1979

Kinetics of the potential-sensitive extrinsic probe oxonol VI in beef heart submitochondrial particles.

The interaction of the potential-sensitive extrinsic probe oxonol VI with beef heart submitochondrial particles has been investigated under time resolved and equilibrium conditions. The time course of the probe absorption spectrum red shift induced by ATP or NADH injection into a suspension of submitochondrial particles in a dye solution is biphasic, consisting of a faster process described by a second-order rate law with k2 approximately 3 x 10(5) M-1 sec-1. For the ATP pulse experiments, the slower process follows first-order kinetics with k1 approximately 0.3 sec-1. In oxygen pulse experiments to an anaerobic dye-particle system, the slower process is not significantly developed due to rapid depletion of the oxygen, but the faster process follows second-order kinetics with the same rate of the oxygen, but the faster process follows second-order kinetics with the same rate constant as for the ATP and NADH cases. Evidence for permeation of the submitochondrial particle membrane by oxonol VI has been obtained; the slower process is interpretable as describing the permeation of the membrane bilayer. The results of the time-resolved work are consistent with a mechanism involving a redistribution of the dye from the bulk phase to the particle membrane. The value of the second-order rate constant for passive binding of the dye to submitochondrial particles is not compatible with a mechanism proposed to explain the microsecond probe response times in bilayer and excitable membrane experiments nor are such rapid signals observed in the oxonol VI-submitochondrial particle system.

Adenosine Triphosphate↗
PubMed1979

The behavior of oxonol dyes in phospholipid dispersions.

The interaction of a class of oxonol dyes with sonicated phospholipid vesicles was followed optically. The spectra of vesicle-associated dyes resemble those found for the dyes in organic solvents, indicating that the oxonols occupy a hydrophobic region of the membrane. At equilibrium the affinity of the oxonols for the vesicles depends on the structure of the dye, the physical and chemical composition of the vesicles, and the ionic strength of the medium. The oxonols occupy soybean lipid vesicles to a level of 147.9 +/- 17.1 nmol/mg lipid with a dye membrane dissociation constant of 3.33 +/- 0.54 muM. The interaction of the oxonols with soybean lipid vesicles is biphasic. The fast phase has a second order rate constant of 9.04 +/- 0.36 x 10(6)M(-1) s(-1) and the number of "fast" binding sites, 68 +/- 8 nmol/mg lipid, was determined from the ratio of the second order rate constants obtained with lipid and with dye in excess. The dissociation of oxonols from soybean lipid vesicles is also biphasic, and the fast process has a rate constant of 17 +/- 2 s(-1), yielding a dissociation constant for the fast sites (k(-1)/k(2)) of 1.88 +/- 0.15 muM. The slow phases of oxonol association with, and release from, soybean lipid vesicles are not second order and have half times of between 0.2 and 5 min, depending on the physical and chemical composition of the membrane lipids. The amplitudes of the slow phases are sensitive to the composition of the aqueous media on each side of the vesicle membranes, which suggests that the slow processes represent the permeation of the membrane by the oxonols. The importance of the properties of the oxonol dyes in the interpretation of their behavior in natural membranes is discussed.

Alkenes↗
PubMed1979

Rapid ATP assays in perfused mouse liver by 31P NMR.

31P NMR was used to continuously monitor ATP and inorganic phosphate levels in perfused mouse liver. Under "optimal" conditions, the time resolution of the technique was approximately 1 min. In the absence of any metabolic perturbations the ATP level remained constant for at least 2 hr and decreased by only approximately 20% in 18 hr. Both ATP and inorganic phosphate levels responded to alterations in the oxygen supply to the liver. The half-time for this response was approximately 1 min, and the response to short periods of hypoxia or ischemia was partially reversible. The addition of insulin caused only a minor decrease in the ATP level but significantly decreased the rate of response of ATP and phosphate levels to hypoxia and ischemia.

Adenine Nucleotides↗
PubMed1979

Fluorescence mapping of mitochondrial redox changes in heart and brain.

Fluorescence techniques may be utilized to map changes in the distribution of mitochondrial redox states in heart and brain during ischemic or hypoxic stress. The basis of these techniques is the intrinsic fluorescence of reduced NADH and oxidized flavoprotein in mitochondria which respond to changes in critical oxygen supply. Ischemic areas in rabbit hearts induced by coronary ligation were detected and mapped based on the increase in NADH fluorescence in the ischemic zone. The width of the jeopardized normoxic tissue surrounding the ischemic area (less than 50--350 mu) was measured by combination of fluorescein angiography and NADH fluorescence. Areas of increased NADH fluorescence in gerbil brains after carotid artery ligation or induction of spreading depression were mapped in a similar manner. Intraoperative monitoring of flavoprotein fluorescence from human cerebral cortex after superficial temporal artery middle cerebral artery (STA-MCA) anastomoses demonstrated increased rates of cortical oxidative metabolism after the surgical procedures.

Animals↗