MODIFICATION OF α-CHYMOTRYPSIN USING A WATER-SOLUBLE PHOTO-FENTON REAGENT

Abstract— -Modification of an enzyme, α-chymotrypsin, was examined by using a water-soluble photo-Fenton reagent. By photoirradiation of the enzyme with the reagent, which can occupy a binding site of the enzyme, a tryptophan residue in the vicinity of the active site was oxidized to N -formylkynurenine. Concurrently, the catalytic properties of the enzyme were largely changed: the K _m was increased and the k _catwas decreased. The decrease in k _cat for a specific amide substrate was the most significant among the esters and amides examined. The water-soluble photo-Fenton reagent would be useful to chemically modify relatively limited regions in biomolecules.     

THERMAL GENERATION OF ELECTRONICALLY EXCITED STATES BY α-PEROXYLACTONES: ACTIVATION PARAMETERS AND EXCITATION YIELDS AS A FUNCTION OF α-DEUTERATION*

Abstract— The kinetics and the direct, the 9,10-dibromo- (DBA) and the 9,10-diphenylanthracene- (DPA) enhanced chemiluminescence of tert-butyl α-peroxylactone 1, with and without α-deuteration, was investigated in order to probe the mechanism of direct chemiexcitation by these hyperenergetic molecules. The small secondary isotope effect on the rates and activation parameters suggests that a diradical mechanism is obtained. The partitioning of the diradical intermediate into excited vs ground state product and the yield of triplet excited product are moderately (ca. 5-fold) increased by α-deuteration, but the singlet yield is unaffected. A convenient and useful chemiluminescence method for the determination of fluorescence quantum yields has been developed.     

TIME RESOLVED QUENCHING STUDY OF SERINE-195 LABELED α-CHYMOTRYPSIN

Abstract— Pyrenebutylmethylphosphonofiuoridate or pyrenepropylmethylphosphonofluoridate reacts with the serine 195 residue of α-chymotrypsin to yield a stoichiometric fluorescent derivative. The fluorescence decay of the modified enzyme was studied using a frequency-doubled, synchronously pumped picosecond rhodamine-6G laser excitation source with time-correlated single-photon-counting detection. Upon excitation above 330 nm, it could only be described adequately by a triple exponential decaying function. The exact reason for complexity of the emission has not yet been understood. Heterogeneous labeling seems irrelevant. The presence of different enzyme conformations on the single-photon-counting time-scale seems more plausible. The enzyme conformations where the pyrene label is situated close to the side groups of certain residues such as tryptophan and lysine show a much shorter fluorescence decay time resulting in non-exponential fluorescence behaviour. Dynamic fluorescence quenching experiments indicate that the pyrene label is located in a more polar region of the enzyme characterized by a negative charge. It seems that the pyrene group is too large to fit well in the hydrophobic pocket of the enzyme but is bent more towards the entrance of the cavity which is characterized by a higher polarity. Upon excitation at 296 nm, the excitation energy is transferred from the tryptophan chromophores towards the pyrene label.     

PHOTOREGULATION OF α-CHYMOTRYPSIN ACTIVITY BY ISOMERIZABLE INHIBITORS. STUDY OF THE ACTION MECHANISM*

Abstract— The N- p -phenylazophenyl-N-phenylcarbamyl chloride (PAPC) in its cis form is five times more active as inhibitor of α-chymotrypsin than in the trans form. In the present work, derivatives of PAPC have been synthesized. Each of these new compounds is photoisomerizable and is an inhibitor (in the cis and in the trans form) of α-chymotrypsin. The cis isomer is always more active than the trans. The m -methyl derivative is 17.5 times more active in the cis form than in the trans , whereas, for the para -substituted compound, this ratio is only 3.5.
Several hypotheses can explain this difference of activity between the cis and trans isomers: (1) steric hindrance towards the trans isomer, (2) lower affinity of the enzyme for the trans isomer, (3) higher reactivity of the complex formed between the enzyme and the cis form of the inhibitor. These hypotheses are discussed.     

THE α-CHYMOTRYPSIN EFFECT ON VARIOUS 9-AMINOACRIDINE FLUORESCENT PROBE EMISSION

Abstract The influence of a-chymotrypsin upon the fluorescence of various 9-aminoacridine derivatives was examined. Fluorescence is quenched in the presence of this protein. Moreover, quenching is suppressed by the coexistence of hydrocinnamic acid, a competitive inhibitor of the enzyme activity. This leads us to conclude that there is a fluorescent complex formation between protein and fluorophores. Then, the apparent association constants were calculated and the florescence lifetimes of "free" and "bound" 9-aminoacridine were determined by phase-modulation fluorometry.
It is deduced that 9-aminoacridine compounds are probably located in the enzymatic active site and that the fluorescence quenching is not due to specific interactions with amino acid residues but is a consequence of the characteristics of this binding site, in particular its hydrophobicity and polarity.
Alternatively, when these fluorescent probes are located in the vicinity of some aromatic amino acids (histidin, tryptophan and tyrosin), then the production of non fluorescent complex takes place. It is concluded that 9-aminoacridine compounds can be used for the determination of the type of amino acid residues in the binding sites of proteins.     

PHOTOREGULATION OF α-CHYMOTRYPSIN ACTIVITY IN ORGANIC MEDIA: EFFECTS OF BIOIMPRINTING

Abstract α-Chymotrypsin exhibits photoswitchable activities in an organic solvent after covalent modification of the protein backbone with thiophenefulgide active ester (2). The thiophenefulgide-modified α-chymotrypsin exhibits reversible photoisomerizable properties between states (3)-E and (3)-C. The modified α-chymotrypsin, where nine lysine residues are substituted by thiophenefulgide units, retains 60% of the activity of the native enzyme. The activities of thiophenefulgide-modified α-chymotrypsin toward esterification of N -acetyl-L-phenylalanine (4) by ethanol in cyclohexane are controlled by the configuration of the attached photoisomerizable component and by prior bioimprinting of the protein backbone with the reaction substrate (4). The esterification of (4) in cyclohexane using bioimprinted (3)-C is two-fold faster than in the presence of (3)-E. In the presence of a nonbioimprinted enzyme, esterification of (4) by (3)-C is five-fold faster than with (3)-E. The activity of bioimprinted (3)-E toward esterification of (4) is 4.5-fold higher than that of nonbioimprinted (3)-E. Switchable cyclic esterification of (4) is accomplished by sequential photoisomerization of the thiophenefulgide-modified α-chymotrypsin between states (3)-C and (3)-E.     

ANALYSIS OF FLUORESCENCE KINETICS AND ENERGY TRANSFER IN ISOLATED α SUBUNITS OF PHYCOERYTHRIN FROM Nostoc sp.

Abstract— The fluorescence decay profiles, relative quantum yield, and transmission of the phycoerythrin a subunit, isolated from the photosynthetic antenna system of Nostoc sp., were measured using single picosecond laser excitation. The fluorescence decay profiles were found to be intensity independent for the intensity range investigated (4 × 10¹³ and 4 × 10¹⁵ photons-cm-² per pulse). The decay profiles were fitted to a model assuming both chromophores absorb and fluoresce. The inferred total deactivation rates for the two chromophores, in the absence of energy transfer and when the effects of the response time of the streak camera and the finite pulse width are properly included, are 1.0 × 10¹⁰s' and 1.0 × 10⁹ s ¹ for the s and f chromophores. respectively, whereas the transfer rate between the two fluorophorcs is estimated to be 1.0 × 10¹⁰ s⁻¹ giving a s→ f transfer rate on the order of (100 ps)⁻¹. Steady-atate polarization measurements were found to be equal to those calculated using the rate parameters inferred from the kinetic model fit to the fluorescence decays. The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is suggestive of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated α subunits for the intensity range investigated (4 × 10¹³ to 4 × 10¹⁵ photons-cm ² per pulse).     

ABSORPTION AND FLUORESCENCE SPECTRA OF CHLOROPHYLL-a IN POLAR SOLVENTS AS A FUNCTION OF TEMPERATURE

Abstract— Absorption and fluorescence spectra of chlorphyll-a in aqueous alcohol were determined as a function of temperature between 173° and 293°K. From a comparison of these spectra with ones obtained in pure methanol, it is suggested that changes in molecular association occur within aggregates of chlorophyll-a in aqueous-alcoholic solution induced by variation of the dielectric constant. The latter varies over a wide range in aqueous alcohol as a function of temperature.     

TRYPTOPHAN FLUORESCENCE LIFETIMES AS A FUNCTION OF EXCITATION WAVELENGTH*

Abstract— –Fluorescence decay times of aqueous dilute solutions (?20 µM) of L-tryptophan have been determined using the phase shift technique as well as single photon-counting coupled with synchrotron radiation (ACO at Orsay and SPEAR at Stanford). Decay times were obtained as a function of the excitation wavelength (in the spectral region 220–320 nm) monitoring emission of λ> 320 nm (in certain specified cases, λ> 360 nm). We have found that, at neutral pH and 20°C. fluorescence decays are single exponentials and independent of the excitation wavelength; under these conditions we find τ= 3.1 ± 0.1 ns.     

THE MOLECULAR CHAPERONE FUNCTION OF α-CRYSTALLIN IS IMPAIRED BY UV PHOTOLYSIS

Buffer solutions of the lens protein γ-crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63°C or to UV radiation at 308 nm. When α-crystallin was added to the protein solutions in stoichiometric amounts, heat or UV irradiation did not cause turbidity, or turbidity developed much less rapidly than in the absence of α-crystallin. Hence, normal α-crystallin functioned as a molecular chaperone, providing protection against both UV and heat-induced protein aggregation. When α-crystallin was preirradiated with UV at 308 nm, its ability to function as a chaperone vis-a-vis both UV and heat-induced aggregation was significantly impaired, but only at relatively high UV doses. A major effect of preirradiation of α-crystallin was to cause interpeptide crosslinking among the αA₂ and αB₂ subunits of the α-crystallin macromolecule. In our experiments α-crystallin was exposed to UV doses, which resulted in 0, 50 and 90% crosslinking as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated α-crystallin. The results are consistent with the notion that UV irradiation of α-crystallin results in loss of chaperone binding sites.     

USEFUL PHOTOLUMINESCENCE PROBES OF MICELLAR SYSTEMS—CYCLIC AZOALKANES AS FLUORESCENCE ACCEPTORS AND 1,5-DIMETHYLNAPHTHALENE AS A FLUORESCENCE DONOR

Abstract— Certain cyclic azoalkanes are shown to exhibit substantial fluorescence intensity and attractive absorption parameters in detergent solutions. Quenching of the fluorescence of detergent solutions of 1,5-dimethylnaphthalene by these bicyclic azoalkanes has been investigated and is found to provide a useful system to study properties of micellar systems.     

UPTAKE AND BIOCONVERSION OF α-TOCOPHERYL ACETATE TO α-TOCOPHEROL IN SKIN OF HAIRLESS MICE

Abstract— The photoprotective effect of topically applied α-tocopheryl acetate (vitamin E acetate), a stable derivative of α-tocopherol (vitamin E), and its possible bioconversion to the active antioxidant species (α-tocopherol) was examined in skin tissue of female hairless mice (HRS/J) exposed to UV-B irradiation. Our results indicate that topically applied α-tocopheryl acetate is absorbed into and retained by skin tissue. Furthermore, skin tissue from UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments contained significantly higher levels (P < 0.001) of α-tocopheryl acetate than non-UV-B-irradiated mice that received identical daily topical α-tocopheryl acetate treatments. Finally, free α-tocopherol levels in skin also were significantly increased (P < 0.00 1) by topical applications of α-tocopheryl acetate and skin levels of free α-tocopherol were significantly greater (P < 0.001) in UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments than in non-UV-Birradiated animals. These results suggest that UV-B irradiation enhances both the absorption of α-tocopheryl acetate and its bioconversion to free α-tocopherol.     

α-OXIDASE ACTIVITY IN PLANTS AS PROMOTOR OF ELECTRONIC ENERGY

Abstract The a-oxidase activity of higher plants acting on long chain fatty acids generates the lower aldehyde in the ground state; however if chlorophyll or chioroplasts are present the chlorophylls are excited most likely by a chemically initiated electron exchange (CIEEL) luminescence process with the putative a-peroxylactone intermediate. When the aldehyde is substituted for the acid, the lower aldehyde appears in the triplet state. The chiral discrimination observed in the quenching by D- and L-tryptophan of the chlorophyll sensitized emission indicates that the triplet aldehyde is generated within the enzymatic preparation and transfers energy while still bound to the enzyme.
Chlorophylls in chioroplasts are excited by addition of a long chain fatty acid or aldehyde. The mechanism, however, is unknown.     

COMPARISON OF THE PHOTOTOXICITY OF α-TERTHIENYL WITH THAT OF A SELENIUM AND OF AN OXYGEN ANALOGUE

Abstract— Two analogues of α-terthienyl, namely 2, 5di(2'-thienyl) selenophene and 2, 5di(2'-thienyl) furan have been prepared and their phototoxicities toward several microorganisms have been compared. Dose response studies with Escherichia coli indicate that α-terthienyl is more active than these analogues. α-Terthienyl was shown to be the most effective of the three compounds in the photoinactivation of yeast alcohol dehydrogenase. Diagnostic tests showed the participation of singlet oxygen in the photosensitization to different extents by these three thiophenes.     

GLOBAL ANALYSIS OF FLUORESCENCE DECAY: APPLICATIONS TO SOME UNUSUAL EXPERIMENTAL AND THEORETICAL STUDIES

The methodology for the simultaneous analysis of fluorescence decay curves is shown to create the possibility for some new types of fluorescence decay experiments. These new experiments include the use of the T-format in anisotropy experiments without the need to match the timing characteristics of photomultiplier tubes. Decay surfaces obtained at multiple timing calibrations can be analyzed simultaneously to resolve widely differing decay rates. Methodologies are described for the discrimination of associative from nonassociative behavior for multiexponential anisotropy decay. A systems theory approach to the analysis of excited state reactions is described.     

ON THE LUMINESCENCE OF α-DIKETONES OF THE STEROID SERIES

Abstract—The absorption and luminescent properties of two steroids [16-ketoestrone (I), 5α-androstane-3β-ol-16,17-dione (II)] are compared. The behaviour of these compounds which differ only in their A-ring is significantly different with regard to solvent dependence. The transfer of excitation energy in compound (I) from the phenolic to the diketonic chromophore is studied and compared with data from analogous estrogens. The results obtained in this study indicate a significant long range (presumably conformational) influence of the steroid A and D rings on each other.     

PHOTOPEROXIDATION OF CHOLESTEROL IN HOMOGENEOUS SOLUTION, ISOLATED MEMBRANES, AND CELLS: COMPARISON OF THE 5α- AND 6β-HYDROPEROXIDES AS INDICATORS OF SINGLET OXYGEN INTERMEDIACY

Singlet oxygen (1O2) can react with cholesterol (Ch) to give three possible ene-addition hydroperoxides: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), and 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH). The rates of dye-sensitized photogeneration and also the fates of 5 alpha-OOH and 6 beta-OOH in membrane bilayers have been studied and compared. Irradiation of unilamellar [14C]Ch/phospholipid vesicles in the presence of aluminum phthalocyanine tetrasulfonate or merocyanine 540 resulted in formation of 5 alpha-OOH and 6 beta-OOH, as determined by high performance liquid chromatography with radiochemical or electrochemical detection. The initial rate of 6 beta-OOH formation was 30-35% that of 5 alpha-OOH in a variety of liposomal systems. However, after a lag, 5 alpha-OOH invariably decayed via allylic rearrangement to 7 alpha-OOH (also known to be a free radical product), whereas 6 beta-OOH accumulated in unabated fashion until Ch depletion became limiting. Photooxidation of Ch in an isolated natural membrane (erythrocyte ghost) or in L1210 leukemia cells gave similar results. When the reaction was carried out in pyridine or methanol, the rate of 6 beta-OOH formation relative to 5 alpha-OOH was reduced by approximately half, with essentially no isomerization of the latter to 7 alpha-OOH.(ABSTRACT TRUNCATED AT 250 WORDS)     

FLUORESCENCE RELAXATION KINETICS AND QUANTUM YIELD FROM THE PHYCOBILISOMES OF THE BLUE-GREEN ALGA NOSTOC SP. MEASURED AS A FUNCTION OF SINGLE PICOSECOND PULSE INTENSITY*

Abstract— A detailed experimental study of the effect of intensity of a 6 ps excitation pulse on the decay kinetics and yield from phycobilisomes (PBsomes) is presented. The fluorescence from the c-phycoerythrin (PE) emission from PBsomes was found to decay as a single exponential with a time of 31 ± 4ps for an excitation intensity <10¹⁴ photons/cm² per pulse. The risetime of the c-phycocyanin (PC) and allophycocyanin (APC) emission from PBsomes was found to be 34 ± 13 ps. Therefore, at low excitation intensities, the energy transfer time between the constituent phycobiliproteins, PE and PC, is measured to be 34 ± 13ps from the fluorescence decay time of PE and the fluorescence risetime of the PC and APC emission. The fluorescence yield from the PE emission component in PBsomes was found to be intensity dependent for excitation intensities >10¹⁴ photons/cm². The decrease in yield with increased intensity in this case occurred at a higher intensity than in the isolated phycobiliprotein PE. The fluorescence yield of the PC and APC emission component was also found to decrease markedly with increasing excitation intensity. This is in contrast to the case of the isolated phycobiliprotein APC which showed only a slight quenching of the fluorescence. The higher quenching observed for the APC emission in the PBsome evidences the higher effective absorption of APC via energy transfer from PE to PC and APC.     

BIODISTRIBUTION AND PDT EFFICACY OF A KETOCHLORIN PHOTOSENSITIZER AS A FUNCTION OF THE DELIVERY VEHICLE

Abstract C8KC is a new ketochlorin photosensitizer that must be formulated with an emulsifier because of its poor water solubility. In this report, we compare properties of Cremophor EL (CRM) and Tween 80 as delivery vehicles for C8KC. Unlike Tween 80, CRM altered the physical properties of both human and mouse plasma lipoproteins, resulting in decreased electrophoretic mobility of the individual lipoproteins along with the formation of a l ipoprotein degradation product: a phospholipid fraction of low buoyant density. In human plasma, where there was sufficient low-density lipoprotein (LDL) for a distinction to be made, CRM caused a shift in binding of a ketochlorin from albumin to LDL and the degraded lipoprotein fraction. In mice bearing the RIF tumor, the use of CRM for drug formulation was associated with longer plasma and tissue persistence of C8KC, and enhanced photodynamic therapy (PDT) efficacy. These results indicate the importance of both sensitizer and vehicle as determinants of PDT efficacy.     

9, 10-DICYANOANTHRACENE-SENSITIZED PHOTOOXYGENATION OF α,α'-DIMETHYLSTILBENES. MECHANISM AND KINETICS OF THE COMPETING SINGLET OXYGEN AND ELECTRON TRANSFER PHOTOOXYGENATION REACTIONS

Abstract— The 9, lodicyanoanthracene-sensitized photooxygenation of 2-methyl-2-butene and (+)-limonene proceeds via the singlet oxygen pathway in carbon tetrachloride as well as in acetonitrile, although the fluorescence of the sensitizer in acetonitrile is quenched by these olefins in an electron transfer quenching mechanism. The 9, 10-dicyanoanthracene-sensitized photooxygenation of cis- and trans-ä, ä′-dimethylstilbenes occurs exclusively via the singlet oxygen pathway in carbon tetrachloride; in acetonitrile, however, singlet oxygen and electron transfer photooxygenation reactions compete with one another. Addition of tetra-n-butyl ammonium bromide and increasing oxygen concentrations favor the formation of the singlet oxygen product, whereas addition of anisole, increasing substrate concentrations and decreasing oxygen concentrations favor the electron transfer photooxygenation products. In carbon tetrachloride, exciplexes of the sensitizer and the dimethylstilbenes are formed which give rise to cidrrans-isomerization of the substrates. In acetonitrile, neither exciplex formation nor cisltrans-isomerization are observed. A mechanism is proposed which allows us to calculate product distributions of the competing singlet oxygen/electron transfer photooxygenation reactions and thus to determine the efficiencies with which encounters between the singlet excited sensitizer and the substrates finally result in electron transfer photooxygenation products. Using (I) these efficiencies, (2) the β-value obtained from singlet oxygen photooxygenation sensitized by rose bengal, and (3) the appropriate k-values determined from fluorescence quenching of 9, 10-dicyanoanthracene in MeCN by oxygen and the stilbene, allows the calculation of the quantum yield of oxygen consumption by this stilbene. The quantum yield thus calculated is strictly proportional to the rate of oxygen consumption experimentally obtained; this result is considered as convincing evidence for the mechanism proposed.