D2O Buffer Research Articles

Water and nonelectrolyte permeability in brain synaptosomes isolated from normal and uremic rats.

Water and nonelectrolyte permeabilities of synaptosomes isolated from the brain of normal and uremic rats were measured by stopped-flow light scattering. Intensity of scattered light (550 nm) increased linearly with decreasing synaptosome size in the range of normal size to 30% of normal size. In response to a 250-mM inwardly directed gradient of an impermeant solute (mannitol or sucrose), there was a 100- to 500-ms time course of increased light scattering resulting from osmotic water efflux. In response to an inwardly directed urea gradient, light scattering first increased (water efflux) and then decreased (urea influx) over a 2- to 10-s time course. Based on an average synaptosome surface-to-volume ratio of 86,000 cm-1, determined by electron microscopy, the permeability coefficient for osmotic water transport (Pf) is 4.5 X 10(-3) cm/s and for urea transport (Purea) is 1.5 X 10(-6) cm/s (23 degrees C); temperature-dependent studies gave an activation energy for Pf of 18 (greater than 16 degrees C) and 3 kcal/mol (less than 16 degrees C) and for Purea of 9.8 kcal/mol (10-55 degrees C). Osmotic water and urea transport were not inhibited by 2 mM p-chloromercuribenzene sulfonate, 120 microM phloretin, and 10 microM phenylurea or by exposure to high-energy radiation (0-10 Mrad). Diffusional water permeability coefficient is approximately 4 X 10(-4) cm/s based on the time course of light scattering after mixture of synaptosomes in D2O buffer with isosmotic H2O buffer. Water and urea transport properties of synaptosomes isolated from uremic rats were not significantly different from those of normal rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Optical properties and small-angle neutron scattering of bovine heart mitochondrial oligomycin sensitivity conferring protein.

The mitochondrial oligomycin sensitivity conferring protein (OSCP) was isolated from beef heart mitochondria according to Senior (1979) [Senior, A.E. (1979) Methods Enzymol. 55,391-397] with a supplementary step of Sephadex chromatography for more extensive purification. The ultraviolet and fluorescence spectra of OSCP were consistent with the presence of tyrosyl residues and the absence of tryptophanyl residues. From the circular dichroism spectrum of OSCP, 43% alpha-helical structure was calculated; the dichroism spectra of OSCP in H2O and D2O were identical. A molecular weight (Mr) of 22 000 for OSCP was determined by sodium dodecyl sulfate gel electrophoresis at different concentrations of the polyacrylamide gel. The radius of gyration (Rg) and the shape of OSCP in H2O and D2O were studied by small-angle neutron scattering. The experimentally determined Rg value of OSCP in H2O was 24 +/- 1 A, and its Mr was 25 000 +/- 3000. Comparison of the experimental Rg value with that expected for a compact globular protein of the same molecular weight (17 A) led to the conclusion that OSCP is a considerably elongated molecule protein with an axial ratio higher than 3. In D2O buffer, the Rg value was higher than that in H2O, a situation in contrast with that observed for most globular hydrophilic proteins; this might be due to a preferential location of the positively charged lysine residues.

Phosphorus-31 NMR of the Intact Crystalline Lens: I. The Living Lens Spectrum. II. The Spectroscopic Effects of Deuterium Oxide Incubation

Abstract Spectra of intact bovine and canine crystalline lenses in vitro at 37°C were generated using phosphorus-31 nuclear magnetic resonance (NMR). Lens incubation in a pure D2O buffer results in a progressive narrowing of the tissue P-31 NMR signals.

Hematoporphyrin photosensitization of serum albumin and subtilisin BPN'.

Abstract—The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (1O2) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen‐saturated, D2O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non‐exponential and mimick a second‐order process in the initial stages. The rate of HP photobleaching was 30‐fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that 1O2 was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen‐saturated, D2O buffer at pH 8.0. The reactivity of HSA was approximately 2‐fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Å2 for the inactivation of subtilisin BPN' by 1O2 and 20 Å2 for the oxidation of tryptophan in BSA.

ON THE ACRIDINE AND THIAZINE DYE SENSITIZED PHOTODYNAMIC INACTIVATION OF LYSOZYME—SINGLET OXYGEN SELF‐QUENCHING BY THE SENSITIZERS

Abstract— The quantum yield of the photodynamic inactivation of lysozyme increases in the sequence acridine orange, methylene blue, proflavine and acriflavine (1:5:6:12). At least up to protein concentrations of 0.1 mM, singlet oxygen is exclusively responsible for the inactivation of the enzyme. For methylene blue, acriflavine and proflavine the quantum yields decrease considerably with increasing dye concentrations. From measurements in H2O and D2O buffer solutions it was concluded that in the case of methylene blue the effect is mainly caused by the quenching of singlet oxygen [rate constant (3–4) × 108M−1 s−1]. For the acridine sensitizers both singlet oxygen and dye triplet quenching processes have to be taken into consideration. It has been found that all sensitizers act as competitive inhibitors of the enzymatic reaction of lysozyme. However, the dye‐protein interaction near the active center cannot be responsible for the observed dye self‐quenching effect.

The involvement of 1O2 in the inactivation of mixed function oxidase and peroxidation of membrane lipids during the photosensitized oxidation of liver microsomes.

Abstract— Dye sensitized photooxidation of rat liver microsomes results in inactivation of mixed function oxidase and peroxidation of the membrane lipids. Both the flavoenzyme NADPH: cytochrome P450 reductase and cytochrome P450 components of the mixed function oxidase were inactivated. Singlet oxygen was responsible for these effects as the inactivation was markedly enhanced if the microsomes were in D2O buffer during the irradiation. Singlet oxygen quenchers or traps also protected the microsomes against enzyme inactivation and lipid peroxidation. Antioxidants, whilst preventing the lipid peroxidation, did not protect cytochrome P450 from inactivation which suggests that lipid peroxidation was not responsible for cytochrome P450 inactivation.

Substituent effects on the rates of formation of pyridinium ylides

Pyridinium ylides are formed by deprotonation of the 2- and 6-positions of 3-substituted 1-methylpyridinium ions in D2O buffer solutions at 75·0° in reactions catalysed by deuterioxide ion. The reactivity of the 2-position was determined for 10 substituted ions; eight ions gave kinetic results for the 6-position. Dual substituent parameter equations employing inductive and resonance effects correlate rate constants very well.

Stability characteristics of deuterated myoglobin.

AbstractPurified sperm whale myoglobin was deuterated by being exposed to pD 3.5 in D2O buffer for 1 hr, then raised to pD 10.6 for an additional hour, and finally brought to neutrality in a D2O environment. Control myoglobin was similarly treated in H2O. The specific rotation at 233 mμ and/or the absorbance in the Soret region were used to follow the helix‐coil transition of myoglobin subjected to denaturation by acid, alkali, urea and guanidine. Deuterated and control myoglobin had similar 50% transition points in the four denaturing media studied (acid: pH 4.4, pD 4.9; alkali: pH 9.4, pD 10.0; urea, 7.2M; guanidine, 2.5M). The shift toward the alkaline side of 0.5 or 0.6 units of the transition induced by either acid or alkaline denaturation reflects only the weakened acidity of ionizable groups in deuterium systems. Deuterated myoglobin in 3.25M guanidine had a 20% faster denaturation rate than that of control. Acid, urea, and guanidine denaturation curves showed fairly steep transitions, taken as indicative of a one‐step process involving only two states (native and denatured molecules). Supporting this conclusion was the fact that plots of absorption and polarimetry measurements of the helix‐coil transition induced by either acid or guanidine could be superimposed.

13 C NMR spectra of lecithin vesicles and erythrocyte membranes.

UNLIKE proton nuclear magnetic resonance (NMR) spectroscopy, 13C NMR has the potential advantages in studies of macromolecular systems of a wider range of chemical shifts (∼200 p.p.m.) and of reduced resonance broadening in structures with restricted molecular motion. Thus we have been able to observe 13C NMR spectra of unsonicated lecithin and erythrocytes, which do not yield high resolution proton spectra. We also report T1 relaxation times for 13C nuclei in the polar headgroup of the lecithin molecule and at both ends of the alkyl chains, in sonicated vesicles in D2O buffer and in deuterochloroform. The results are consistent with increasing chain motion towards the terminal methyl group of the alkyl chains for both systems, in agreement with relaxation data for stearic acid derivatives specifically labelled with 19F nuclei in lecithin vesicles1, and with the results of similar spin label experiments of Hubbell and McConnell2.