Psoralen-sensitized photohemolysis: Dependence on pH

The effect of pH on psoralen-sensitized photohemolysis (irradiation at 366 nm, 23 W/m²) was investigated. The dose (D) dependence of the rate of photohemolysis (V) fitted V = V₀ + kD^x (where V₀ is the rate of dark hemolysis and k is a coefficient). Variation of pH did not influence the exponent x, which was about 2; however, pH had a strong influence on the k value. The lowest V was observed in the pH interval from 8.0 to 8.4. It nearly doubled as the pH was changed from 8.4 to 9.0 as well as from 8.0 to 7.4. At pH below 7.4 the hemolysis rate increased sharply (by another factor of ～4). Since psoralen does not contain acid/base groups, the effect of pH could hardly be a result of changing the photophysical properties of the sensitizer. The increase in V in the alkaline region could be attributed to more pronounced photooxidation of reduced glutathione as a substrate, while the jump at pH ～7.3 would be ascribed to titration of a psoralen photooxidation product. The latter idea was supported by the HPLC data. Psoralen was preirradiated in ethanol and mixed in the dark with phosphate buffer at different pH, after which HPLC analysis revealed several pH-dependent photoproducts; for one of them the pH titration curve closely resembled the pH profile of psoralen-sensitized hemolysis.     

Photochemical properties of Yt base in aqueous solution.

Photoreactivity of Yt base [I] has been studied in aqueous solution [pH approximately 6] saturated with oxygen. Two photoproducts (II,III], resulting from irradiation at lambda = 253.7 nm and lambda greater than or equal to 290 nm, were isolated and their structures determined. The quantum yield for Yt base disappearance [zeta dis] is 0.002 [lambda = 313 nm]. It was shown that dye-sensitized photooxidation of Yt base in aqueous solution occurs according to a Type I mechanism, as well as with participation of singlet state oxygen. Quantum yields, fluorescence decay times and phosphorescence of Yt base have been also determined.     

Antioxidant inhibition of hemolysis induced by photooxidized psoralen

Antioxidants butylated hydroxytoluene++ (ionol) and 2,2,5,7,8-pentamethyl-chromanol-6-(alpha-T-0) were shown to inhibit hemolysis induced by the ethanolic solution of photooxidized psoralen (POP). This inhibition appeared to be the same whether antioxidants were present during irradiation or were added immediately after photooxidation. Therefore antioxidants do not influence POP formation, but inhibit the interaction of POP degradation products in water with biomolecules. Possibility of POP participation in phototoxic effects of furocoumarins is discussed.     

Effect of antioxidants on the psoralen photo-oxidation process

Psoralens are capable of photosensitizing oxidation of unsaturated fatty acids due to the two-stage mechanism. During the first (light) stage psoralen solution in ethanol undergoes photooxidation under UV-irradiation (366 nm). At the second (dark) stage the addition of photooxidized psoralen (POP) to the aqueous solution of liposomes is followed by lipid oxidation. Antioxidants inhibited the UV-stage, but did not influence the dark one. Neither spectrophotometry, nor spectrofluorometry could detect photoproducts of psoralen involved in the two-stage oxidation of lipids. However, mixing of ethanol solution of POP with water resulted in the flash of chemiluminescence. The inhibition constants by antioxidants of photoproducts formation which are active in the two-stage oxidation of lipids were estimated by chemiluminescence. Stern--Volmer's constants for antioxidants: 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (DHP), 6-hydroxy-2,2,5,7,8-pentamethylchroman (chromanol--C1), water soluble sodium phenozan and butilated hydroxytoluen (ionol) appeared to be (7.4 +/- 2.2) X 10(3) M-1, (4.4 +/- 1.0) X 10(3) M-1, (3.3 +/- 0.7) X 10(3) M-1, (4.5 +/- 2.5) X 10(2) M-1, respectively. The biological importance of these two-stage oxidation photosensitized by furocoumarins is discussed.     

Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin

Photosensitized hemolysis of human erythrocytes by hematoporphyrin was suppressed by flavonols such as quercetin and rutin at submillimolar concentrations. The suppression of photohemolysis was accompanied by inhibition of lipid peroxidation by the reagents. Quercetin and rutin were photooxidized in the presence of hematoporphyrin and the photooxidation was partially suppressed by 1 mM NaN3, a quencher of singlet molecular oxygen. Flavonols were also oxidized by radicals formed during degradation of lauroyl peroxide. These results indicate that flavonols can function as antioxidants in biological systems by terminating radical chain reactions and removing singlet molecular oxygen. A pharmacological function of flavonols, decrease of the increased permeability and fragility of capillary, was discussed in relation to their antioxidative functions.     

Inhibition of psoralen sensitized photohemolysis by a marigold (Bidentis tripartita) extract]

Hemolysis induced by psoralen and UV-A radiation (PUVA-hemolysis) was significantly inhibited by the addition of Bidentia tripartita extract. The rate of hemolysis was reduced both when the extract was present during irradiation, or added after PUVA-treatment. The inhibition effect was more pronounced when the extract was present during irradiation.     

Relationship of hemolysis buffer structure, pH and ionic strength to spontaneous contour smoothing of isolated erythrocyte membranes

Isolated human erythrocyte membranes crenate when suspended in isotonic medium, but can use MgATP to reduce their net positive curvature, yielding smooth discs and cup forms that eventually undergo endocytosis. An earlier report from this laboratory (Patel, V.P. and Fairbanks, G. (1981) J. Cell Biol. 88, 430-440), has described a phenomenon of ATP-independent shape change in which ghosts prepared by hemolysis and washing in synthetic zwitterionic buffers crenated at 0 degree C, but underwent conversion to smooth discs and cups when warmed in the absence of MgATP. We have further explored the effect of the hemolysis condition on the requirement for ATP in ghost shape change. 25 hemolysis buffers were applied at 10 mM (pH 7.4, 0 degree C). Eight anionic buffers with relatively high ionic strength (e.g., phosphate and diethylmalonic acid (DMA] yielded ghosts requiring ATP for shape change, while two cationic buffers (Bistris and imidazole) and ten synthetic zwitterionic buffers (e.g., Tricine and Hepes) with lower ionic strength produced ghosts that smoothed spontaneously at 30 degrees C. Hemolysis at intermediate ionic strength yielded mixed populations in which spontaneous smoothing was expressed in all-or-none fashion. Maximal ATP-independent shape change was induced by hemolysis at pH 7.3-7.7, while ATP was required after hemolysis at pH less than or equal to 7.1 even when the ionic strength at hemolysis was low. Ghosts requiring ATP could be converted to ATP independence by washing at low ionic strength, but ATP independence could not be reversed readily by washing at high ionic strength. Exposure to low ionic strength at pH greater than 7.1 presumably changes membrane organization in a way that alters the temperature dependence of tensions within the bilayer or skeleton of the composite membrane.     

Psoralen sensitized thermoactivated photodamage to leukocyte membranes]

Psoralen sensitized photodamage of rat peritoneal exudate cells was investigated. Irradiation of cells induced latent lesions in membranes which during thermal activation at the post-irradiation stage were transformed into permeability channels for trypan blue. The effect linearly increased with fluence of irradiation which indicates one hit production of thermolabile psoralen photoproducts in the membranes.     

Calcium binding constituents of the organic shell matrix from the freshwater snail Biomphalaria glabrata

The Ca2+ binding of an EDTA-free water-soluble (SM) and -insoluble (IM) organic matrix of the freshwater snail Biomphalaria glabrata was investigated, using a 45Ca2+ autoradiography after SDS-electrophoretical separation and a calcium binding assay. Electrophoresis of the SM showed a considerable amount of Alcian blue and Stains all positive material, regarded as glycosaminoglycans (GAGs) or proteoglycans (PGs). This part of the SM was slightly positive after 45Ca2+ autoradiography at pH 6.8. The Ca2+ binding increased, raising the pH to 7.4 and 8.0 and was especially strong when simulating the real conditions of the extrapallial space with a carbonate buffer of pH 7.4. The Ca2+ binding assay of the IM showed the same pH-dependency that was observed in the SM. The titration of the IM with Ca2+ at pH 8.0 lead to a dissociation constant of 7.5 x 10(-5) M. While Mg2+ displaced 45Ca2+ in the same way as nonradioactive Ca2+, an approximately 400-fold amount of Na+ was necessary to reduce the binding of 45Ca2+ to 50%. The Ca2+ binding of the organic matrix from the B. glabrata shell appears to be a process of low specificity, medium affinity and high pH-dependency. Apparently, acidic carbohydrate-rich PGs are the only calcium binding constituents of the organic shell matrix.     

Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence

Three identical deoxyoligonucleotide third strands with a 3′-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell β-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.     

Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa

Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans. P. aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide. Here, the biochemical characterization of recombinant P. aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported. Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue. Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule. Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C. However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent. Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay). Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle. As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P. aeruginosa from endogenous and environmental cyanide.     

Calcium ion binding by tobacco mosaic virus

Calcium ion titrations were performed on solutions of tobacco mosaic virus using a calcium-specific ion-exchange electrode. Scatchard analyses were used to obtain the number of calcium ion binding sites per protein subunit (n) and the apparent stability constant for complex formation (beta' Ca). These experiments were performed on unbuffered solutions, in either water or 0.01 M-KCl, to allow a determination of the number of hydrogen ions released per calcium ion bound (chi). The results indicate that near neutrality, the virus particle possesses two calcium ion binding sites per subunit having apparent stability constants greater than 10(4) M-1. The results are interpreted as if these two sites are non-identical and titrate independently. The higher affinity site for the virus in water has a value of log beta' Ca, which varies from about 8.5 at pH 8.5 to about 3.9 at pH 5.0, and for the virus in 0.01 M-KCl has a value that varies from about 6.2 at pH 8.0 to about 3.7 at pH 5.5. The higher affinity site for the virus in water binds up to two competing hydrogen ions, one with an apparent pKH value greater than 8.5 and the other with a value that varies from 6.0 at pH 5.5 to 7.3 at pH 8.0. For the virus in 0.01 M-KCl, only the competing hydrogen ion binding with an apparent pKH value greater than 8.5 remains. The results could be interpreted as indicating that the electrical charge on the virus particle has a constant value in the pH range 5.5 to 8.0 despite the fact that hydrogen ion titration curves for the intact virus particle indicate that the charge should vary from about -1 per subunit at pH 5.5 to about -4 at pH 8.0.     

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily

Thiols represent preferential targets of peroxynitrite in biological systems. In this work, we investigated the mechanisms and kinetics of the reaction of peroxynitrite with the dithiol dihydrolipoic acid (DHLA) and its oxidized form, lipoic acid (LA). Peroxynitrite reacted with DHLA being oxidation yields higher at alkaline pH. The stoichiometry for the reaction was two thiols oxidized per peroxynitrite. LA formation accounted for approximately 50% DHLA consumption at pH 7.4, probably reflecting secondary reactions between LA and peroxynitrite. Indeed, peroxynitrous acid reacted with LA with an apparent second-order rate constant (k(2app)) of 1400 M(-1) s(-1) at pH 7.4 and 37 degrees C. Nitrite and LA-thiosufinate were formed as reaction products. Surprisingly, the k(2app) for peroxynitrite-dependent DHLA oxidation was only 250 M(-1) s(-1) per thiol, at pH 7.4 and 37 degrees C. Testing various low-molecular-weight thiols, we found that an increase in the thiol pK (pK(SH)) value correlated with a decrease of k(2app) for the reaction with peroxynitrite at pH 7.4. The pK(SH) for DHLA is 10.7, in agreement with its modest reactivity with peroxynitrite.     

Proton block of rat brain sodium channels. Evidence for two proton binding sites and multiple occupancy

The acid titration function of bilayer-incorporated batrachotoxin (BTX)- modified sodium channels was examined in experiments in which the pH was decreased symmetrically, on both sides of the membrane, or asymmetrically, on only one side. In an attempt to minimize interpretational ambiguities, the experiments were done in 1.0 M NaCl (buffered to the appropriate pH) with channels incorporated into net neutral bilayers. When the pH was decreased symmetrically (from 7.4 to 4.5), the small-signal conductance (g) decreased in accordance with the predictions of a simple (single-site) titration function with a pK of approximately 4.9. As the pH was decreased below 6.5, the single- channel current-voltage (i-V) relation became increasingly rectifying, with the inward current being decreased more than the outward current. When the pH was decreased asymmetrically (with the pH of the other solution being held constant at 7.4), the titration behavior was different for extra- and intracellular acidification. With extracellular acidification, the reduction in g could still be approximated by a simple titration function with a pK of approximately 4.6, and there was a pronounced rectification at pHs < or = 6 (cf. Woodhull, A. M. 1973. Journal of General Physiology. 61:687-708). The voltage dependence of the block could be described by assuming that protons enter the pore and bind to a site with a pK of approximately 4.6 at an apparent electrical distance of approximately 0.1 from the extracellular entrance. With intracellular acidification there was only a slight reduction in g, and the g-pH relation could not be approximated by a simple titration curve, suggesting that protons can bind to several sites. The i-V relations were still rectifying, and the voltage-dependent block could be approximated by assuming that protons enter the pore and bind to a site with a pK of approximately 4.1 at an apparent electrical distance of approximately 0.2 from the intracellular entrance. Based on the difference between the three g-pH relations, we conclude that there are at least two proton binding sites in the pore and that they can be occupied simultaneously.     

Photooxidation of histidine and tryptophan residues of papain in the presence of methylene blue.

Papain [EC 3.4.22.2] was photooxidized using methylene blue as a sensitizer. The photooxidzed enzyme lost its caseinolytic activity and had significantly decreased histidine and tryptophan contents. The tyrosine content was the same before and after the photooxidation. The SH content of the photooxidized enzyme, as determined after reduction with dithiothreitol, was also unchanged. The loss of histidine was always slower than the loss of enzymatic activity, being less than one residue per molecule even when the enzymatic activity was completely lost. However, the inactivation and the oxidation of a histidine residue were pH-dependent in a similar fashion in the pH range of 5.0-8.0, the pH profiles conforming to theoretical titration curves with apparent pKa values of 6.6 and 6.7, respectively. The fact that the ionization of a histidine residue in papain has a normal imidazole pKa value is entirely in accord with the finding for stem bromelain [EC 3.4.22.4] (Murachi, T., Tsudzuki, T., & Okumura, K. (1975) Biochemistry 14, 249-255), and is of great significance in relation to the mechanism of catalysis by these enzymes.     

Redox titration of the primary electron acceptor of Photosystem I in spinach chloroplasts

The midpoint potential of the primary electron acceptor of Photosystem I in spinach chloroplasts was titrated using the photooxidation of P₇₀₀ at −196 °C as an index of the amount of primary acceptor present in the oxidized state. The redox potential of the chloroplast suspension was established by the reducing power of hydrogen gas (mediated by clostridial hydrogenase and 1,1′-trimethylene-2,2′-dipyridylium dibromide) at specific pH values at 25 °C. Samples were frozen to −196 °C and the extent of the photooxidation of P₇₀₀ was determined from light-minus-dark difference spectra. This titration indicated a midpoint potential of −0.53 V for the primary electron acceptor of Photosystem I.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.     

Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding

His12 and His119 are critical for catalysis of RNA cleavage by ribonuclease A (RNase A). Substitution of either residue with an alanine decreases the value of k(cat)/K(M) by more than 10(4)-fold. His12 and His119 are proximal to the scissile phosphoryl group of an RNA substrate in enzyme-substrate complexes. Here, the role of these active site histidines in RNA binding was investigated by monitoring the effect of mutagenesis and pH on the stability of enzyme-nucleic acid complexes. X-ray diffraction analysis of the H12A and H119A variants at a resolution of 1.7 and 1.8 A, respectively, shows that the amino acid substitutions do not perturb the overall structure of the variants. Isothermal titration calorimetric studies on the complexation of wild-type RNase A and the variants with 3'-UMP at pH 6.0 show that His12 and His119 contribute 1.4 and 1.1 kcal/mol to complex stability, respectively. Determination of the stability of the complex of wild-type RNase A and 6-carboxyfluorescein approximately d(AUAA) at varying pHs by fluorescence anisotropy shows that the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to 4.0. At pH 4.0, replacing His12 with an alanine residue decreases the stability of the complex with 6-carboxyfluorescein approximately d(AUAA) by 2.3 kcal/mol. Together, these structural and thermodynamic data provide the first thorough analysis of the contribution of histidine residues to nucleic acid binding.     

PH-dependence of detergent-induced hemolysis and vesiculation of erythrocytes

The influence of pH of the medium on the parameters of detergent-induced fast hemolysis and vesiculation of human erythrocytes was studied. In the range of pH 6.3-7.2 neither the extent nor the rate of the vesiculation induced by 25 microM sodium dodecyl sulfate (SDS) changed. However, a decrease of pH from 8.0 to 5.8 strongly modified both the extent and the rate of the hemolysis induced by SDS. Within the range of pH 8.0-6.4, the effect can be ascribed to the increase of the positive charge of the membrane. This could lead to the accumulation of the membrane-bound anion detergent and, hence, to the change of the hemolysis parameters. Non-charged detergent Triton X-100 did not display any pH-dependence. At pH between 6.4 and 5.8 the extent and rate of hemolysis changed in a complicated manner. The kinetic curves of hemolysis could be approximated by a single exponential within the pH range between 8.0 and 7.2. Upon further reduction of pH, a second exponential component, with a larger time constant, appeared in the kinetic curves. At 5.8 < pH < 7.2, the contribution of the "fast" hemolysis dropped virtually to zero, with pK about 6.0. This points to a structural transition of the membrane, possibly involving histidine. We suggest that the parameters of the detergent-induced hemolysis are sensitive to the changes of the charge and structural state of erythrocyte membrane.