PHOTOPHYSICAL STUDIES ON THE BINDING OF TETRASULFONATOPHENYLPORPHYRIN TO LENS PROTEINS

Previous studies have shown that mesotetra(p-sulfonatophenyl)porphine (TPPS) binds to lens proteins. This characteristic should increase the residence time of the sensitizer in the lens and therefore enhance the probability of inducing photooxidative damage to that tissue in vivo. Subsequent in vivo studies have verified that contention. The present studies were performed to determine the effect of such binding on the spectroscopy and photophysics of the porphyrins. It was found that the binding of TPPS (1) quenches the fluorescence of lens proteins, (2) causes a shift in the ground state absorption spectra, fluorescence excitation spectra and the triplet excited state spectrum of TPPS to longer wavelengths and (3) results in an increase in the triplet state lifetime of TPPS. In the presence of the isolated crystallins the average triplet lifetime increases in the following order: gamma less than beta less than alpha.     

PHOTOCHEMICAL MODIFICATIONS OF lac REPRESSOR: EFFECT OF EFFECTORS BINDING ON TRYPTOPHAN PHOTOOXIDATION

Abstract— UV irradiation of lac repressor modifies the fluorescence of the protein and its binding to the inducer and to the operator. It has been previously shown that the total loss of fluorescence is due to photooxidation of, on average, one of the two tryptophyl residues of each protomer. The present work explains this observation by showing that N-formylkynurenine formed at one site is responsible for the quenching of fluorescence of the other tryptophan via an energy transfer process. Consequently, no photoreaction occurs for the second tryptophyl residue. Photodamage of the two tryptophyl residues (in position 201 and 220) of each protomer were assayed by spectrofluorometric titration in the pH range from 8.5 to 5. For repressor alone, both residues are equally photodamaged. In the presence of the inducer isopropyl-β-D-thio-galactoside, IPTGt, residue 220 is completely protected, and tryptophan 201 is slightly more exposed to photooxidation. In the presence of antiinducer, residue 220 is only partially protected. Our results are discussed in terms of conformational changes triggered by the two types of ligands.     

THE EFFECT OF FLUORIDE ON BINDING and PHOTODYNAMIC ACTION OF PHTHALOCYANINES WITH PROTEINS

Fluoride inhibits chloroaluminum phthalocyanine tetrasulfonate (AlPcS)-induced photohemolysis when added to dye loaded cells prior to light exposure. The mechanism by which F- exerts this effect was studied by measuring the binding of phthalocyanine (Pc) to various proteins in the absence and presence of F-. Parallel measurements were made of the photodynamic action under these conditions. Fluoride reduced the binding to proteins of AlPcS and CoPcS. The binding of CuPcS, ZnPcS and H2PcS was not affected. When bound to bovine serum albumin and exposed to light, H2Pc, ZnPc and AlPcCl were bleached at a biphasic rate. Only the photobleaching of AlPcCl was affected by F-. The effect of F- was to inhibit the initial rapid phase without affecting the slower phase. In the presence of D2O only the second phase of photobleaching was enhanced, in the absence or presence of F-. No effect of F- was observed on tryptophan photooxidation or glyceraldehyde-3-phosphate dehydrogenase photoinactivation by AlPcS. Crosslinking of spectrin monomers photosensitized by AlPcS was inhibited by F- in parallel with the reduced binding of dye to the protein. It is concluded that F- exerts its effect by complexing with metal ligands of Pc. As a result, the dye may be released from the protein or the binding mode may be changed in such a way that effective photochemistry is prevented. Primary photophysical processes of Pc most probably are not affected by F-.     

PHOTOCHEMICAL INTERACTIONS BETWEEN NITROXIDE FREE RADICALS AND PHENOTHIAZINES IN SOLUTION

Abstract— Ultraviolet irradiation of photosensitive molecules like phenothiazine derivatives leads to the formation of short lived free radicals which are able to reduce stable nitroxide free radicals generally used as spin labels. The measurement of the electron spin resonance signal decay of nitroxides offers a tool for studying the photochemical reaction of phenothiazine derivatives in solution at room temperature, in a 10^-5 to 10^-2 M concentration range. Analysis of the reaction mechanism shows that the paramagnetic nitroxide is an efficient quencher of the phenothiazine triplet state; this reaction was used to demonstrate the influence of the solvent, quenching by oxygen and the role of the chemical structure of six phenothiazine derivatives on their photoreactivity in solution.     

THE PHOTODYNAMIC EFFECT OF ROSE BENGAL ON PROTEINS OF THE MITOCHONDRIAL INNER MEMBRANE

Photodynamic action promoted by Rose Bengal was evaluated in solutions of unsaturated fatty acids or histidine, and on beef heart submitochondrial particles. Rose Bengal-promoted photooxidation of histidine was mainly due to the opening up of the imidazole ring by singlet oxygen. Photosensitization of polyunsaturated fatty acids (PUFA) resulted in oxygen consumption and thiobarbituric acid-reactive substances (TBARS) formation, the extent of which was linearly related to the increasing degree of unsaturation. Photosensitization of submitochondrial particles caused oxygen consumption and TBARS production. These processes involved two different reaction components: during the first, most of the mitochondrial proteins were inactivated, the most sensitive being succinate dehydrogenase and cytochrome c. The values for the rate ratios of [TBARS] formation/[O2] consumption for the first and second phase were 0.36 and 1.32%, respectively, pointing to a larger contribution of lipid peroxidation during the second phase. The calculation of the rate constants for reaction of singlet oxygen with mitochondrial proteins suggests that singlet oxygen is more reactive towards proteins than to PUFA. The biological role of this selectivity is discussed in terms of the mitochondria as one of the first targets for photosensitized reactions.     

CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED PHOTOHEMOLYSIS*

Abstract— Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3ß-hydroxy-5α-hydroperoxy-△⁶-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.     

THE EFFECT OF LIPOSOMES'' MEMBRANE COMPOSITION ON THE BINDING OF THE PHOTOSENSITIZERS HPD AND PHOTOFRIN II

Abstract— Photofrin II (PF-II) is the commercial name of the active photosensitizer which is used in photodynamic therapy of cancer. The effect of the composition of lipid membranes on the binding of PF-II was studied and compared to hematoporphyrin derivative (Hpd), which is a complex mixture of porphyrins and from which PF-II is separated. We find that increasing the content of cholesterol in the bilayer decreases the partitioning of PF-II into the bilayer, similar to what we have found earlier with Hpd. However, inserting DMPC or DPPC into the membrane, which was shown to decrease the binding of Hpd, causes the opposite trend with PF-H. A membrane fluidizer such as benzyl alcohol also has different effects on the membrane binding of Hpd and PF-II. The rate of binding of PF-II to a lipid membrane is about 10 times lower than that of Hpd. These results as well as I^- quenching of the fluorescence of the two porphyrins indicate that PF-II is immersed less homogeneously and deeper in the bilayer than Hpd. The unique additive-dependent binding of PF-II to lipid membranes calls for care in using Hpd as a model photosensitizer.     

STUDIES ON DNA SEQUENCE SPECIFIC BINDING PROTEINS FOR 5'''' FLANKING UPSTREAM REGION OF CPSI GENE

Two specific carbamyl phosphate synthetase Ⅰ gene binding nuclear proteins (M. W. 109kD and 74 kD) have been determined in the rat liver by the protein blotting technique (South-western blot assay). The result shows that they are not present in the normal rat spleenand F-26 rat hepatoma cell. The Ba131 nuclease deletion in the CPSI gene 5' upstream regionproves that the binding sites for 109 kD and 74 kD are respectively located in the regions of- 38 bp to - 4 bp and - 113 bp to -38 bp. The binding proteins may be the liver-specific onesof the CPSI gene, which are related to hepatocyte differentiation and hepatocarcinogenesis.     

竹红菌乙素对人红细胞膜蛋白荧光猝灭机理的研究

本文利用人红细胞膜蛋白内源荧光(主要指色氨酸),经加入不同的竹红菌乙素之后,引起了荧光猝灭现象,根据荧光猝灭原理分别从吸收光谱,荧光光谱,稳态变温实验和瞬态荧光等技术分析了上述的猝灭过程,实验证明:该猝灭是以动态碰撞过程为主要作用机理,据此作者提出了乙素可以用于生物膜体系的蛋白质荧光猝灭剂的理由。     

BINDING OF GLYCOLATE OXIDASE TO PEROXISOMAL MEMBRANE AS AFFECTED BY LIGHT

The stability of the bond of glycolate oxidase to the peroxisomal membrane obtained from etiolated barley leaves, (Hordeum vulgare L. var Dvir) is affected by the pH of the homogenization medium. Peroxisomes isolated at pH 4 or 10 retain glycolic oxidase, while peroxisomes isolated at pH 69 loose this enzyme during centrifugation. Preillumination of whole seedlings, detached leaves or even homogenates changes the effect of pH on the retention of glycolate oxidase-attachment which is strengthened at pH 9–10 and becomes very loose at pH 4–7. White and red light are responsible for the effect described. Far red light reverses this effect of red light. Illumination induces a change in the isoelectric point (IpH) of peroxisomes without affecting the IpH of soluble enzymes. This red light-induced change on IpH of microbodies is not reversed by far red light. The possible effects of light on peroxisomal membrane properties and on modulation of glycolate oxidase binding are discussed.     

IRREVERSIBLE BINDING OF NITROFURANTOIN TO SKIN AND TO PLASMA PROTEINS OF UV-A EXPOSED RATS

Abstract— Allergic reactions to the widely applied urinary tract disinfectant nitrofurantoin (NFT) often concern the skin. They are probably mediated by a NFT-protein adduct. which has been suggested to result from incomplete nitro-reduction during metabolism. In this study it was investigated whether photoactivation of NFT in rats, upon exposure to UV-A, leads to formation of adducts between the drug and biomacromolecules. Rats were given p.o. doses of [¹⁴C]-NFT, and some were exposed to UV-A. More irreversible binding was found in skin of the back (both in dermis and in epidermis). tail, ears, eyes, plasma proteins, and spleen of light-exposed rats compared to those kept in the dark. This photobinding increased (up to ∼0.6 nmol/mg protein =∼25 nmol/g tissue) with dose and light-intensity. When rats were kept at 32°C, instead of 22°C during NFT/UV-A treatment, even more irreversible binding was observed; this is probably caused by an increased dermal blood flow. Under these conditions, irreversibly bound radioactivity was even found in kidney, lung and liver. Irreversible binding in these inner organs is explained by systemic distribution of radioactivity, photobound to plasma proteins. Photodecomposition of NFT in the skin, followed by reaction of photoproducts with (cellular) biomacromolecules may explain some of the skin rashes and other (allergic) reactions reported for this drug.     

UV INACTIVATION OF ENZYMES IN SUPRAMOLECULAR COMPLEXES OF BIOLOGICAL MEMBRANES. THE PHENOMENON OF PHOTOCHEMICAL ALLOTOPY

Abstract. The photosensitivity of erythrocyte acetylcholinesterase (AChE) is different in its free and membrane-bound states. The modification of the structure of membraneous lipids by phospholipases A₂, C and D or by cholesterol depletion is accompanied by a change in AChE photosensitivity. UV light was demonstrated to induce cooperative structural transitions in the erythrocyte membrane. This follows from the data obtained by circular dichroism and solubilization in detergents. In contrast to free AChE, UV light acts on the membraneous enzyme as a mixed inhibitor (simultaneous change in V _max and K _m). The anomalous behaviour of membrane-bound enzyme, termed the phenomenon of photochemical allotopy, is associated with a modification of the structure within the microenviron-ment of the residual AChE. The phenomenon depends on membrane integrity, and disappears after treatment of erythrocyte ghosts with ultrasound, trypsin, phospholipases and neuraminidase and remains unchanged in cholesterol-depleted membranes. The nature and localization of events responsible for this phenomenon are discussed.     

THE RESOLUTION OF CHLOROPHYLL a/b BINDING PROTEINS BY A PREPARATIVE METHOD BASED ON FLAT BED ISOELECTRIC FOCUSING

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.     

A NEW BIOLOGICAL TARGET OF FUROCOUMARINS: PHOTOCHEMICAL FORMATION OF COVALENT ADDUCTS WITH UNSATURATED FATTY ACIDS

Abstract— Furocoumarins, potent skin therapy and tanning agents, form covalent adducts in a photochemical reaction with unsaturated fatty acids. These adducts and the chemical kinetics of their formation have been characterized by chromatography, isotopic tracers, electronic absorbance and fluorescence spectroscopy and mass spectrometry. Adduct formation does not require oxygen. The quantum yield of adduct formation in ethanol or methanol-water solutions is comparable to the quantum yield for formation of furocoumarin-thymine adducts in DNA.     

PHOTOCHEMICAL INTERACTION OF FUROCOUMARINS WITH BROMODEOXYURIDINE AND POLYDEOXY-NUCLEOTIDES CONTAINING BROMODEOXYURIDINE: ITS BIOLOGICAL IMPLICATIONS

Abstract— The photoreaction of 5-bromodeoxyuridine (BUdR) exposed to 360 nm light in the presence of the furocoumarins, 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (&MOP), was studied and compared to those of thymidine. BUdR reacted with furocoumarins, producing cyclobutane-containing adducts, as does thymidine. Furocoumarins reacted also with BUdR-containing polymer, poly(dA-BUdR) in the double stranded form, at a rate similar to that of thymidine-containing polymer, poly(dA-dT). Polyamines, which slow the photoreactions of TMP with DNA, had no effect on its binding to the two former polynucleotides. It is suggested that because of the similar photoreactions of BUdR and thymidine with furocoumarins, this combination could be used to elucidate the mechanism by which BUdR sensitizes biological systems. In Escherichia coli some sensitization by BUdR of TMP plus 360 nm light killing was observed. It is therefore concluded that at least part of the sensitization of bacteria by BUdR to UV and ionizing radiation is caused by interference with the repair processes. Since no such sensitization was observed in a uvr B mutant, BUdR apparently impairs the efficiency of the excision resynthesis pathway of repair.     

SYNTHESIS OF SULFONES OF 4H-1,4-BENZOTHIAZINES AND PHENOTHIAZINES

Conversion of 5/7-chloro-4H-1,4-benzothiazines and 1/3-chlorophenothiazines into sulfones is reported. The 5/7-chloro-4H-1,4-benzothiazines were synthesized by the condensation and oxidative cyclization of 2-amino 3/5-chlorobenzenethiol with β diketones in DMSO. The phenothiazines have been synthesized via Smiles rearrangement by the reaction of 2-amino-3/5-chlorobenzenethiol with halonitrobenzenes. 4H-1,4-Benzothiazine and phenothiazine sulfones have been prepared by the oxidation of benzothiazines and phenothiazines with 30% hydrogen peroxide in glacial acetic acid. The structure of all the synthesized compounds has been confirmed by IR and NMR spectral studies.     

MODEL STUDIES ON THE PHOTOCHEMICAL PRODUCTION OF LENTICULAR FLUOROPHORES

Abstract With aging, human lens proteins accumulate fluorophores having blue and green emissions. Model studies were undertaken to determine the role of 3-hydroxykynurenine (3-HK) and its glucoside (3-HKG) in the photochemical production of those fluorophores. Experiments were carried out using 10⁻³ M 3-HK solutions in the presence or absence of glycine (1 M ), which was used to mimic the environment of the lens. The solutions were photolyzed (transmission above 295 nm) for various periods of time while the loss of starting material and the formation of fluorescent photoproducts (blue emission at 470 nm, and green emission at 520 nm) were monitored using fluorescence and UV-visible spectroscopy and thin-layer and high-pressure liquid chromatography analysis. Several parameters were varied such as oxygen tension and the addition of the free radical scavenger, penicillamine. The photolytic loss of 3-HK in the absence of glycine occurred approximately 5-10 times faster than in its presence. Conversely, blue and green fluorophores formed in irradiated solutions containing glycine but not with the photolysis of 3-HK alone. The blue fluorophore was formed first and appeared then to be photochemically converted to the green one, with the rate of formation of the latter increasing with an increase in UV dosage or oxidizing conditions. The addition of penicillamine drastically reduced the photochemical formation of both fluorophores.
Both the blue and green fluorophores appear to result from the photochemically induced covalent attachment of 3-HK to glycine. In the human lens, these reactions can explain the age-related loss of 3-HKG with the concomitant formation of fluorophores covalently attached to lens proteins, probably via the amino group of lysine.     

DIRECT PHOTO-AFFINITY LABELING OF CYCLIC NUCLEOTIDE BINDING PROTEINS WITH GUANOSINE-3'',5''-MONOPHOSPHATE

Abstract— Radioactivity from [³H]-guanosine-3',5'-monophosphate was shown to become stably linked to proteins in testis extracts during ultraviolet irradiation. Adenosine-3',5'-monophosphate competitively inhibited incorporation into these proteins. Guanosine-3',5'-monophosphate had a lower affinity for binding sites than did adenosine-3',5'-monophosphate. Similar peptides were photo-labeled with [³H]-guanosine-3',5'-monophosphate and [3H]-adenosine-3',5'-monophosphate. Guanosine-3',5'-monophosphate was more efficiently incorporated than was adenosine-3',5'-monophosphate. Extracts from a number of tissues incorporated both cyclic nucleotides photochemically.