CUTANEOUS PHOTOSENSITIZING AND IMMUNOSUPPRESSIVE EFFECTS OF A SERIES OF TUMOR LOCALIZING PORPHYRINS

A series of tumor localizing porphyrins was evaluated with respect to their ability to elicit cutaneous photosensitivity and systemic immunosuppression, two of the most common side effects associated with photodynamic therapy. Using the murine ear swelling response as an indicator, it was found that all the non-metalloporphyrins caused cutaneous photosensitization. Immunosuppressive effects were noted using hematoporphyrin derivative (HPD) and meso-tetra(4-sulfonatophenyl)porphine if sensitization occurred immediately after photoirradiation, but none were evident using Photofrin II (PII) or meso-tetra(4-carboxyphenyl)porphine (TCPP). Subsequent studies indicated that PII and TCPP manifested a delayed type immunosuppression similar to that found following UVB photoirradiation. Manganese (III) meso-tetra(4-sulfonatophenyl)porphine, a prototype magnetic resonance imaging contrast agent, was also evaluated because of its reported demetallation in vivo. It was found to cause neither cutaneous photosensitivity nor immunosuppression.     

STUDIES ON THE RED AND BLUE FORMS OF THE PIGMENT OF BLEPHARISMA*

Abstract— –The intracellular pigment of the ciliate protozoan Blepharisma in the presence of oxygen sensitizes the cells to bright visible light (2700 foot candles (fc)). Illumination of the cells with dim visible light (200 fc) changes the color of the pigment to blue-gray; such cells are no longer sensitive to bright visible light. The pigment which exists in granules can be extruded by cold treatment and is slowly regenerated. The suspension of red cells, the extruded pigment from them and an ethanol extract of the pigment all have very similar absorption spectra. Illumination of the red pigment in ethanol converts it to the blue form of the pigment but only if oxygen is present, indicating a photooxidation. The pigment can be oxidized in darkness to the blue form by ozonation. A suspension of blue cells, the extruded pigment from them and an ethanol extract from them, all have similar absorption spectra. The pigments in red and blue form are very similar spectrophotometrically and in solubility in three species of Blepharisma studies: B. americanum, B. intermedium and B. japonicum. The purified pigment has strong absorption in the far (200–300 nm) ultraviolet (u.v.) and may serve as a screen against damaging U.V. radiation, especially as Blepharisma shows poor photoreactivation.     

THE PHOTOCHEMISTRY OF 14 C-5-BROMO-2''-DEOXYURIDYLYL-(3''→5'')-THYMIDINE DETERMINATION OF QUANTUM YIELDS AS A FUNCTION OF pH

Abstract— Quantum yields for the formation of the major photoproduct of 2-¹⁴C-5-bromo-2'-deoxyuridylyl-(3'→5')-thymidine (BrdUpT) have been determined by irradiation of the 2-¹⁴C-BrU-labeled dinucleotide at pH 2.3, 5.9, 7.05, 8.0 and 10.25 with U.V. light at 280nm. At acidic and neutral pH the quantum yield was 0.0063; the value decreased markedly above pH 8.0 to 0.0025 at pH 10.25. Some evidence of the formation of additional photoproducts at high and low pH was found. Some aspects of the mechanism of the reaction are discussed.
Consideration of p K_a values calculated for singlet and triplet excited states indicates that the decrease in the quantum yield of main photoproduct at high pH is due to dissociation of the excited bromodeoxyuridine moiety. It is suggested that the formation of BrdUpT photoproduct and the debromination of bromouracil-labeled DN A occur via different excited states.     

RUTHENIUM RED INHIBITION OF OXYGEN EVOLUTION AND SPECIFIC RELEASE OF THE EXTRINSIC 16 kDa POLYPEPTIDE IN A PHOTOSYSTEM II PREPARATION

The inhibitory effect of the dye ruthenium red was studied in photosystem II-enriched submembrane fractions. A number of distinct types of interaction were found, which differed in their concentration range and required incubation time. Ruthenium red instantaneously quenches the initial chlorophyll a fluorescence level (F₀) and the maximum fluorescence level (F_m) by enhancing radiationless deactivation in the chlorophyll light harvesting complex. Associated with this quenching of fluorescence is an instantaneous decrease in the quantum yield of oxygen evolution. Ruthenium red also inhibited the light saturated rate of oxygen evolution and the variable fluorescence, monitored 80 µs after a saturating excitation-flash. These inhibitions increased with incubation time and became greater than 50% within 5 min. Although ruthenium red was known to affect Ca²⁺ or Cl^? sites specifically, the inhibitory action was more pronounced than simple Ca²⁺ or Cl^? depletion. Incubation with ruthenium red for 5 min blocks the Z P680⁺ → Z⁺ P680 charge transfer reaction. Upon mixing with the photosystem II preparation, ruthenium red induced specific release of the extrinsic 16 kDa polypeptide associated with water-splitting without release of Mn. It is proposed that the inhibitor produces an ionic imbalance which alters the configuration of the donor side of photosystem II.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

On the Completeness of Sets of Solutions to the Helmholtz Equation

Completeness in L²(D) is established for sets of functions formedfrom solutions to the two-dimensional Helmholtz equation ina domain D. Each function is a linear combination of a solution(found by separation of variables) and its normal derivativeon D, so the sets may be used to solve impedance-type boundaryvalue problems. Sets that contain either regular Bessel functionsor singular Hankel functions are considered. Methods of proofare employed that provide alternatives to the conventional potential-theoreticapproaches. In the majority of cases, the domain of interestis bounded and simply connected. One completeness result fora bounded, doubly-connected domain is proved. In some circumstances,one of the methods leads to a mild but inessential eigenvaluerestriction.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: LIGHT-INDUCED ABSORBANCE CHANGES AND ESR SIGNALS FOR VARIOUS PORPHYRIN-QUINONE AND HYDROQUINONE SYSTEMS IN ALCOHOL SOLVENTS*

Abstract— Light-dark optical difference spectra of degassed ethanol or pyridine solutions of chlorophyll and benzoquinone or hydroquinone at temperatures above — 50°C show only the semiquinone absorbance band. Decay of the signals is second order, with a rate constant in agreement with earlier ESR results. Light-induced optical changes due to chlorophyll can be elicited by lowering the temperature of ethanol solutions of chlorophyll and benzoquinone to a region of high viscosity. Hydroquinone is not effective in producing these optical changes. Similar results are achieved at room temperature by using as solvent a degassed mixture of the alcohols: cyclohexanol, tert-butanol, and ethanol (CBE). Difference spectra show bleaching of the chlorophyll bands and increased absorbance in the intermediate wavelength region (460–580 nm). Decay kinetics are first order, while the rise is complicated (probably biphasic). ESR signals have no hyperfine structure and also decay by first order kinetics, at a rate which is faster than that of the optical changes. The ESR signals reach a steady state more rapidly than the optical signals, without biphasic kinetics. These results demonstrate that at least two species are generated. Addition of acid increases the amount of bleaching in CBE, while small amounts of base decrease it. Larger amounts of base cause chlorophyll bleaching to completely disappear and only the semiquinone anion is observed. Activation energies for the chlorophyll a-benzoquinone photoreaction in CBE are 10–14 kcal/mole. Lower potential quinones give lower activation energies. The rate constant for quenching of the triplet state of chlorophyll a by β-carotene in CBE is 7.5±0.5×10⁸ (M set)^-1. β-carotene also quenches photoproduct formation. The bimolecular rate constant for formation of the photoproduct with benzoquinone was calculated to be 7×10⁸ (Msec)^-1. The redox potential of the quinone affects both the magnitude of the chlorophyll absorbance changes and the rate of decay. The higher the potential, the larger the changes and the slower the decay. Other porphyrin systems show similar photoreactions only if they are chelated with a group II metal, such as Mg²⁺, Cd⁺², or Zn⁺². The results are interpreted in terms of the formation, by a triplet-sensitized one-electron transfer from solvent to quinone, of a chlorophyll-semiquinone complex which is stabilized via coordination with the chelated metal.     

SINGLET AND TRIPLET REACTIVITY IN THE PHOTOREDUCTION OF OXONINE(3,7-DIAMINOPHENOXAZIN-5-IUM CHLORIDE) BY IRON (II)

The oxazine dye, oxonine (3,7-diaminophenoxazin-5-ium chloride), 1, is photoreduced by Fe (II) sulfate in dilute sulfuric acid. The reaction mechanism is analogous to that for the photo-reduction of thiazine dyes by Fe (II), the most important difference being that reduction of oxonine occurs predominantly from its excited singlet state, S1, rather than from the triplet state, T1. The latter is formed with an intersystem crossing (isc) quantum yield of ca 1.7 x 10(-3). The quenching of S1 by Fe (II) has a rate constant kSQ = 2.2 +/- 0.1 x 10(9) M-1 s-1 and affords the one electron reduced product, semioxonine (R), with a limiting quantum yield, phi SR, of 0.26 +/- 0.02. In contrast, quenching of T1, generated by bromide ion quenching of S1 or by diacetyl sensitization, occurs with KTQ approximately 1.2 x 10(6) M-1 s-1, extrapolated to zero ionic strength, and affords R with a limiting probability, phi TR = 1.1 +/- 0.2. Three possible reasons for the lower quantum yield of the more exothermic S1 reduction are discussed. These are energy transfer from S1 to Fe (II), different rates of escape of R from the encounter complex as a consequence of the different states of protonation of R as initially formed from S1 and T1, and spin allowed back electron transfer in an exciplex formed between S1 and Fe (II). Evidence is also presented for a very low probability (ca 1%) induced isc from the encounter of S1 with paramagnetic Fe (II). Rate parameters for other processes important to the overall reduction mechanism such as disproportionation of R to leucooxonine L and oxonine, k(R)DIS = 1.7 +/- 0.2 x 10(9) M-1 s-1, oxidation of R by Fe (III), k(R)OX = 1.5 +/- 0.1 x 10(5) M-1 s-1, and oxidation of L by Fe (III), kLOX = 1.1 +/- 0.1 x 10(3) M-1 s-1, have also been measured. These results are contrasted with those for the closely related thionine/Fe(II) photoredox reaction, the most well understood system for photogalvanic energy conversion.