An assessment of the degree of chromophore photo damage to the heme and globin components in UV-irradiated molecules and electrophoretic fractions of human carboxyhemoglobin

The contribution of hem and globin components of electrophoretic fractions of UV-irradiated human carboxyhemoglobin to photodestruction of the protein was studied. The changes observed are the result of summation of some processes unequal in intensity and direction that take place in microheterogeneous media of photomodified protein. Photosensitivity of hemoproteid in electrophoretic fraction depends on apoprotein condition, whereas the hem photoresistance cannot be the evidence of the photostability of the whole molecule.     

2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin

2,3-Butanedione sensitized the rapid photodestruction of free alpha-amino acids, and the photoinactivation of alpha-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of alpha-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Influence of surface active agents on oxygen absorption to the free interface in a stirred fermentor

Liquid phase coefficients were measured for the absorption of oxygen from air to the free interface in stirred vessels. Coefficients for absorption into soft water were independent of the instantaneous dissolved oxygen concentration. Coefficients for absorption into soft water containing a surface active agent were strongly dependent on the instantaneous dissolved oxygen level. The degree of nonlinearity of the coefficients was a function of the rate of agitation of the liquid. The coefficients were independent of the amount of surface active agent added above a very low level. Absorption coefficients for bubble aeration in the same vessels were independent of dissolved oxygen concentration even when the surface active agent was present.     

Effect of chromium on photosystem 2 in the unicellular green alga, <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

We investigated the effect of chromium (20–40 g m⁻³, 8–72 h) on the photosystem 2 (PS2) activities of Chlorella pyrenoidosa cells. By using chlorophyll fluorescence transients, thermoluminescence, oxygen polarography, and Western blot analysis for D1 protein we found that inhibition of PS2 can be accounted for by the enhanced photodestruction of the reaction centres in the cells cultivated in the presence of Cr(VI) at 25 °C in “white light” (18 W m⁻²). Hence photodestruction of D1 is caused by an enhanced oxidative stress and lipid peroxidation, as indicated by the appearance of a high-temperature thermoluminescence band.     

UV-Induced destruction of light-harvesting complexes from purple bacterium Chromatium minutissimum

We studied UV-induced photodestruction of the native forms of bacteriochlorophyll a (Bchl a) from chromatophores and light harvesting complexes (LHC) of the sulphur photosynthetic bacterium Chromatium minutissimum. Irradiation of chromato- phores with 365-nm light (Soret band) or 280-nm light (absorption region of aromatic amino acids) led to the destruction of all long-wavelength forms of Bchl a. The quantum yields of photodestruction produced by the 280-nm light was higher than that produced by the 365-nm light. For the spectral forms of Bchl a absorbing at 850 nm and 890 nm, the difference was about one order of magnitude, and for the form absorbing at 800 nm the difference was almost two orders of magnitude. Similar UV sensitivity was observed for the Bchl a forms from isolated LHC. As a rule, the quantum yields of photodestruction induced by UV irradiation at 280 nm were about 100-1000 times higher (approximately 10(-3)-10(-4)) than that upon red light irradiation (approximately 10(-6)-10(-7)). We found that irradiation of chromatophores at 280 nm resulted in a crosslink between the core and peripheral LHC.     

Inhibition of respiration and destruction of cytochrome a3 by light in mitochondria and cytochrome oxidase from beef heart

Irradiation of beef-heart mitochondria and of cytochrome oxidase purified from beef-heart mitochondria with blue light inhibited electron transport from substrate (succinate for the mitochondria and reduced cytochrome c for the cytochrome oxidase) to O₂. The irradiation treatment also destroyed cytochrome a₃ as assayed by the absorption band for the reduced cyanide-cytochrome a₃ complex at 587 nm in the low-temperature absorption spectrum. Irradiation under anaerobic conditions was not inhibitory. Cytochrome a₃ was protected against photodestruction if cyanide was present during the irradiation.     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.     

Interaction of tyrosine with dissolved oxygen in solution and spectroscopic evidence for oscillations

Electronic absorption and fluorescence spectra of tyrosine have been studied, using aqueous solutions containing normal and excess amounts of dissolved oxygen, as a function of time. The absorption and fluorescence intensities are found to oscillate with time. Interaction between tyrosine and oxygen is appreciable but it seems that either the formation of a complex between the two molecules is not favoured, or the process of complexation is very slow and the complex weak.     

The mechanism of the riboflavin sensitized photodestruction of mitomycin C.

A detailed in vitro study was made of the riboflavin sensitized photodestruction of mitomycin C. The dependences of the quantum yield in the system were examined on the introduction of various amounts of quenchers, such as halogen ion, paramagnetic ion, 1,4-diazabicyclo[2,2,2]octane, 2,6-di-tert-butyl-p-cresol, and p-hydroquinone. The results are consistent with a mechanism involving oxygen molecule in the excited singlet state as the photochemically reactive species. The rate constant of the reaction between the excited singlet oxygen and mitomycin C was calculated to be 8.9 x 10(9) M-1.S-1.     

Photogeneration of the superoxide anion oxygen radical and its role in the inactivation of yeast cells by long-wave UV light

Long-wave (320-400 nm) UV-induced oxygen superoxide anion radical (O2-) formation was found in yeast cells. This radical plays an important part in initiation of photodestruction reactions in DNA which serves as a main target of UV irradiation in yeast. The observed cell photoinactivation spectrum at the wavelengths 320-400 nm suggests that NADH can serve as an endogenous sensitizer of O2- formation.     

Use of the glucose oxidase system to measure oxygen transfer rates

This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D -glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, K_m(O₂), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.     

Involvement of active oxygen species in degradation of light-harvesting proteins under light stresses

This paper presents evidence for light-mediated degradation of isolated light-harvesting proteins (Lhc2) and involvement of oxygen free radicals in the process. The time course of light harvesting photodestruction is much slower than that of D1 protein (requiring hours for complete breakdown). By use of mass spectrometry and amino acid sequencing, it has been revealed that the primary cleavages take place in the hydrophilic portion of the NH(2) region where oxygen-containing radicals attack randomly and not at specific sites. Moreover, these chlorophyll binding proteins are completely fragmented. From the effectiveness of scavengers and the preliminary electron paramagnetic resonance measurements reported, it appears that singlet oxygen is involved as a short-lived species, and hydroxyl and alkoxyl radicals act at higher light intensity or over a longer time, whereas hydrogen peroxide and superoxide anions are not observed. Antenna proteins appear more resistance to photodestruction in their monomeric form than in trimeric form, while minor antenna are highly sensitive. However, the organization of both minor and major proteins in the photosystem II supracomplex affords some photoprotection. Interestingly, leaves exposed to strong light contained degraded major antenna, unlike those kept in the dark, which is consistent with studies on the illumination of isolated proteins, supporting the hypothesis that active oxygen species play a role in vivo in the short-term acclimative adaptation of plants.     

The automatic maintenance of low dissolved oxygen using a photobacterial oxygen sensor for the study of microaerobiosis

K avanagh , E. & H ill , S. 1990. The automatic maintenance of low dissolved oxygen using a photobacterial oxygen sensor for the study of microaerobiosis. Journal of Applied Bacteriology 69 , 539–549.
Conventional galvanic and polarographic oxygen electrodes are not sensitive enough to investigate microaerobic phenomena such as nitrogen fixation. Two sensors are available for this application. They are the oxygen-dependent changes in either the absorption spectrum of leghaemoglobin, or the light emission by photo-bacteria. We have coupled the latter to the automatic feedback control of agitation for the maintenance of predetermined low dissolved oxygen concentrations (DOCs) in bacterial suspensions. This method is inexpensive, provides accurate control of DOC, and the photobacterium is easily propagated. We have used this equipment to determine the optimum DOC for microaerobic nitrogenase activity in the facultative anaerobe Klebsiella pneumoniae .     

Singlet oxygen and photodestruction of erythrocyte membranes, sensitized by chlorin e6

By using scavengers of active oxygen forms (AOF) the role of 1O2, O2- and HO in photodestruction of protein and lipid components of erythrocyte membranes and also in photohemolysis of erythrocytes sensitized by chlorine e 6 were studied. It was found that 1O2 plays the dominant role in these processes, but contribution of other AOF, O2- in particular, can not be fully excluded.     

Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers

Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC₅₀ values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction.     

Generation of reactive oxygen species in water under exposure of visible or infrared irradiation at absorption band of molecular oxygen

It is found that in bidistilled water saturated with oxygen hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sourses, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Generation of reactive oxygen species in water under exposure to visible or infrared irradiation at absorption bands of molecular oxygen

It is found that in bidistilled water saturated with oxygen, hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sources, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Evidence for the involvement of singlet oxygen in the photodestruction by chloroaluminum phthalocyanine tetrasulfonate

In recent years, choloroaluminum phthalocyanine tetrasulfonate (A1PCTS) has been shown to be a promising photosensitizer for the photodynamic therapy (PDT) of cancer. Although its mechanism of photodynamic action is not well defined, A1PCTS is going to be under clinical trials of PDT. In this study, in vitro addition of A1PCTS to a suspension of rat epidermal microsomes followed by irradiation with red light (approximately 675 nm) resulted in significant destruction of cytochrome P-450 and associated monooxygenase activities. The photodestructive effect was dependent on both the dose of A1PCTS and the duration of light exposure. Studies using various quenchers of reactive oxygen species showed that only scavengers of singlet oxygen such as histidine, 2,5-dimethylfuran, beta-carotene and sodium azide afforded substantial protection against photodestruction. Our data indicate the direct involvement of singlet oxygen in the A1PCTS-mediated photodestructive process.