Photooxidation of methionine with immobilized methylene blue photooxidizer

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37°C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4°C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94% while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

Photooxidation of methionine with immobilized methylene blue as photooxidizer.

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.     

Nature of tryptophan photooxidation products in lysozyme in the presence of methylene blue]

One out of six trytophan residues in two lysozyme modification, obtained under lysozyme photooxidation in the presence of methylene blue, is found to be oxidized to N'-formylkinurenine (in one modification) and to kinurenine (in the other modification). The transition of one modification into another via detaching of N'-formyl group by soft acid hydrolysis has shown that one and the same tryptophan residue is oxidized in both products, Possible mechanism of tryptophan oxidation to the products mentioned is discu-sed on the basis of the hypothesis on signlet mechanism of lysozyme photooxidation in the presence of methylene blue.     

Selective photooxidation of methionine, tyrosine and tryptophane using thionine and toluidine blue as sensitizers (author's transl)

Thionine and toluidine blue were used as sentizers on photooxidation processes of methionine, tryosine and tryptophane. They were more effective than methylene blue. Methionine was photooxidized to sulfoxide and tryptophane to kinurenine. A tyrosine-sensitizer addition compound was postulated. Dye concentration, pH, temperature and EDTA presence conditions were determined on each one of the modification reactions. Methionine at acid pH was selectively modified. On the basis of obtained results and published references, a direct interaction of singlet oxygen with methionine and tryptophase and the excited dye with tyrosine was respectively discussed.     

Visible light-induced photooxidation of glucose sensitized by riboflavin

We conducted this study to evaluate the oxidation of glucose induced by visible light in the presence of sensitizers such as methylene blue and flavins (i.e., flavin mononucleotide and riboflavin). The concentration of the sensitizers was similar to that of flavin in parenteral nutrients. The photooxidation of glucose sensitized by flavin mononucleotide or riboflavin was greater than that which was observed in the presence of methylene blue, whereas the isotopic effect of deuterium oxide (D(2)O) was enhanced more substantially in the presence of methylene blue than in the presence of flavins. These results show that methylene blue exerts its action through singlet oxygen and that at a high substrate concentration (as was used in this work) flavin mononucleotide and riboflavin act preferentially as type I sensitizers. In the flavin photosensitized processes, the presence of hydrogen peroxide, superoxide anion, and hydroxyl radical was demonstrated. The photooxidation of glucose is favored by an increase in pH, and it also depends on the energy absorbed by the system. By using a specific reagent for glucose (i.e., o-toluidine), it was possible to quantify the photoconversion of glucose. The results obtained in this work should be considered in the management of glucose-containing parenteral nutrients that are exposed to visible light in the presence of a multivitamin complex containing flavin mononucleotide.     

Kinetics of photoinactivation and photooxidation of mitomycin C in the presence of riboflavin

The kinetic relation between the photoinactivation and photooxidation of mitomycin C in the presence of riboflavin was investigated. The photoinactivation was tested for lambda-phage induction in Escherichia coli K-12 (lambda) cells and colony formation of E. coli Bs-1 cells. Mitomycin C lost its phage-inducing and antibiotic activities when the antibiotic was irradiated in vitro with visible light in the presence of riboflavin. The loss of phage-inducing activity followed a Stern-Volmer type equation with respect to the dose of irradiation, and the inactivation constant was evaluated to be 0.96X10(-4)m2/J. The initial rate of decay of mitomycin C by photooxidation in the presence of riboflavin obeyed first order kinetics, and its cross section was estimated to be 2.51X10(-6)m/J independent of the intensity of incident light. The cross section for photooxidation was found to be proportional to the inactivation constant. These results suggest that the photoinactivation of mitomycin C is caused by its photooxidation. In order to rationalize this conclusion, a mechanism of photooxidation was proposed and reactions in vivo of the photoproduct were discussed in relation to the inactivation.     

Ethoxyformylation and photooxidation of histidines in transferrins

The chemical reactivity of histidines in ovotransferrin and human serum transferrin was studied utilizing two different reactions. Upon dye-sensitized photooxidation of ovotransferrin and ethoxyformylation of human serum transferrin and ovotransferrin, losses in histidine and iron-binding activity were observed. All of the histidines in both apoproteins could be ethoxyformylated by the use of 170 to 400 molar excesses of reagent resulting in complete loss in activity. The histidines of human serum transferrin showed a greater reactivity toward the reagent than did those of ovotransferrin. The binding of each iron protected two histidines from ethoxyformylation, and in both cases the proteins remained completely active. First-order losses in histidine and iron-binding activity were observed when ovotransferrin was irradiated in the presence of methylene blue. Comparison of the first-order rates indicates the loss of two histidines per binding site accounts for the inactivation of the protein. However, iron binding did not protect ovotransferrin from photoinactivation as expected. Evidence from both modification technqiues indicates: (1) Histidines are essential for iron-binding activity. (2) There are two essential histidines in each binding site. The advantages of using two modification reactions, ethoxyformylation and photooxidation, in the study of the functional role of histidines in proteins are demonstrated in this work.     

Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization

Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified.When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter.Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers.Photooxidized fibrinogen has affinity for thrombin-activated fibrinogen-Sepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.     

Evidence for histidine as another functional group of δ-aminolevulinic acid dehydratase from beef liver

δ-Aminolevulinic acid dehydratase (EC 4.2.1.24) was obtained in highly purified form from beef liver. Upon photooxidation of the enzyme in the presence of methylene blue as a sensitizer led to a loss of the enzymatic activity according to pseudo-first order kinetics. The pronounced pH dependence (pk value of 6.8) of the photooxidation rate and the results of amino acid analysis suggested that the inactivation was largely due to the modification of the histidine residue. The finding of the enzyme with little activity in the presence of diethylpyrocarbonate was consistent with such a speculation. On the basis of these results, it can be postulated that the histidine residue seems to play an important role in the enzymatic activity of δ-aminolevulinic acid dehydratase.     

Probing the topography of proteins in solution by photosensitized oxidation. The catalytic region of papain

The thiol function of the single cysteinyl residue at the active site of papain was selectively conjugated with either the dinitrophenyl or the fluorescein thiocarbamyl group. Absorption, fluorescence and circular dichroism studies showed that, in both cases, the introduction of the chromophoric moiety caused no significant alterations of the spatial geometry characteristic of native papain. Irradiation of the dinitrophenyl derivative by visible light resulted in the specific photo-oxidation of histidine-159 and trytophan-177, which appeared to be the only potentially photo-oxidizable amino acids adjacent to the labelling group; their distance from the thiol function of cysteine-25 was evaluated to be about 5 Å. These two residues do not appear to make an essential contribution to the structural stability of papain, since the oxidative modification of their side chains induced only limited modifications of the over-all conformation of the protein. On irradiation of the fluorescein-papain complex, the preferential photooxidation of tryptophan-177, histidine-159 and tryptophan-26 took place; in parallel, there was a drastic collapse of the tertiary structure of the protein molecule. It is concluded that tryptophan-26 is oriented in a direction different from that of tryptophan-177 and histidine-159; hence, this residue is probably not directly involved in the catalytic function of the enzyme. However, the intactness of its side chain is critical for maintaining the native three-dimensional structure of papain.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. II. pH and inhibitor-induced conformational transitions affecting histidine-48 and one tyrosine residue of ribonuclease A.

The microenvironment of histidine-48 of bovine pancreatic ribonuclease A was investigated by proton magnetic resonance spectroscopy (1H NMR) using partially deuterated enzyme in which resolution of the C(2)-H resonance of histidine-48 was simplified. The NMR titration curves at 100 and 250 MHz of histidine-48 of ribonuclease A are discontinuous both for the enzyme alone in 0.3 M chloride and for its complex with cytidine 3'-phosphate. This suggests that titration of histidine-48 occurs only as the result of a slow conformational transition. The sum of the peaks corresponding to histidine-48 in the acid-stable and base-stable forms of the enzyme is less than one proton in the transition region, which indicates that there exists at least one intermediate conformational form of the enzyme. The transition from the acid-stable form to an intermediate form has a pHmid of 5.6, and the transition from an intermediate form to the base-stable form has a pHmid of 6.9. In ribonuclease S and in ribonuclease A in the presence of 0.3 M acetate, the titration curve of histidine-48 is continuous, and the area of the peak is uniform throughout the titration. Proton NMR difference spectra at 100 and 250 MHz reveal a pH-induced conformational change with a pHmid of 5.7 that affects the chemical shift of a single tyrosine residue. This conformational transition is absent in ribonuclease S and is altered in ribonuclease A by the presence of either acetate or cytidine 3'-monophosphate. It is postulated that the same conformational transition is responsible for both the tyrosine perturbation and the disappearance of the histidine-48 peak observed in the acid-stable form of the enzyme. It is proposed that the perturbed tyrosine is tyrosine-25. The transition with pHmid 5.6 is attributed to dissociation of aspartic acid-14, and the transition with pHmid 6.9 is assigned to dissociation of histidine-48. A peak in the aromatic region that moves upfield on addition of the competitive inhibitor cytidine 3'-monophosphate is assigned to a tyrosine, and evidence is presented that this tyrosine is tyrosine-25. Inhibitor binding appears to induce a conformational change in the histidine-48/tyrosine-25 region which is remote from the active site.     

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.     

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.     

Photosensitized transformation of adrenochrome

The methylene blue-sensitized photooxidation of adrenochrome was studied by steady-state kinetics. The buffered, aqueous system was irradiated with light longer than 600 nm, wavelengths at which only the sensitizer absorbs. During irradiation, disappearance of adrenochrome and the formation of adrenochrome-melanin was observed. Calculated rate constants were determined on the basis of spectroscopic measurements. It was found that the observed transformation reaction steps are pH dependent. The participation of two types of photosensitized mechanism has been evidenced. Type II, singlet oxygen mechanism, predominates at pH below 9, whereas above pH 9, Type I applies. We observed the so-called "isotope effect" and a decrease of photooxidation rate in the presence of azide ion, a well-known singlet oxygen quencher, indicating the participation of singlet oxygen.     

Studies of a reactive histidine of dyphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart.

Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart was inactivated at neutral pH by bromoacetate and diethyl pyrocarbonate and by photooxidation in the presence of methylene blue or rose bengal. Inactivation by diethyl pyrocarbonate was reversed by hydroxylamine. Loss of activity by photooxidation at pH 7.07 was accompanied by progressive destruction of histidine with time; loss of 83% of the enzyme activity was accompanied by modification of 1.1 histidyl residues per enzyme subunit. The pH-rate profiles of inactivation by photooxidation and by diethyl pyrocarbonate modification showed an inflection point around pH 6.6, in accord with the pK_a for a histidyl residue of a protein. Partial protection against inactivation by photooxidation or diethyl pyrocarbonate was obtained with substrate (manganous isocitrate or magnesium isocitrate) or ADP; the combination of substrate and ADP was more effective than the components singly. As demonstrated by differential enzyme activity assays between pH 6.4 and pH 7.5 with and without 0.67 mm ADP, modification of the reactive histidyl residue of the enzyme caused a preferential loss of the positive modulation of activity by ADP. The latter was particularly apparent when substrate partially protected the enzyme against inactivation by rose bengal-induced photooxidation.     

Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.

EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity. Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo and in vitro. In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ. However, a significant level of phosphatase activity remained when histidine-243 was replaced with certain amino acids, such as tyrosine. In addition, the phosphatase activity of a previously identified kinase- phosphatase+ mutant was not abolished by the replacement of histidine-243 with asparagine. These data indicated that although conserved histidine-243 is important for the phosphatase activity, a histidine-243-P intermediate is not required. Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in close contact with aspartate-55 (OmpR) for both OmpR phosphorylation and dephosphorylation. Phosphotransfer occurs from histidine-243-P to aspartate-55 during phosphorylation, but water replaces the phosphorylated histidine side chain leading to hydrolysis during dephosphorylation.     

Effect of near-infrared and blue laser light on vero E6 cells SARS-CoV-2 infection model

Photobiomodulation therapy (PBMT) employing laser light has been emerging as a safe strategy to challenge viruses. In this study the effect of blue and near-infrared (NIR) laser light was assessed in an in vitro model of SARS-CoV-2 infection. PBMT at blue wavelength inhibited viral amplification when the virus was directly irradiated and then transferred to cell culture and when cells already infected were treated. The NIR wavelength resulted less efficacious showing a minor effect on the reduction of the viral load. The cells receiving the irradiated virus or directly irradiated rescued their viability to level comparable to not treated cells. Virion integrity and antigenicity were preserved after blue and NIR irradiation, suggesting that the PBMT antiviral effect was not correlated to viral lipidic envelope disruption. Our results suggested that PBMT can be considered a valid strategy to counteract SARS-CoV-2 infection, at least in vitro.     

The functional site of placental anticoagulant protein: essential histidine residue of placental anticoagulant protein

Placental anticoagulant protein (PAP) rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH 7.9 and 8 degrees C. Photooxidized PAP failed to bind the phospholipid vesicle. It seemed unlikely that the protein underwent a change in molecular size during the photooxidation on the basis of its behavior in electrophoresis and gel filtration. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged. The peptide, SHLRKV, was included in the functional site of PAP and still showed an anticoagulant activity. On the other hand, the peptide which substituted histidine by alanine, SALRKV, no longer showed the activity. It was shown that the histidine residue is involved in Ca2+ or the phospholipid binding site of the protein.