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The purpose of the present work is to study the vacuum ultraviolet radiation action on the optical and chromatographic characteristics of human hemoglobin molecules and their components - haem and globin. Using the methods of spectrophotometry and thin layer chromatography (TLC), we have investigated into the structural changes of molecules of human hemoglobin, haem and globin, induced by the influence of vacuum UV light (gamma = 118-134 nm, dose - 1.2 kJ/m2). It has been shown that vacuum ultraviolet radiation induces an infringement of the higher types of the spatial organization ofglobin molecules, thus leading to the changes in the structural state of the albuminous globule.

Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T(m) values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.

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.

Heme oxygenase (HO) breaks down heme to iron, biliverdin, and carbon monoxide, and activity of this enzyme increases in many tissues and cell types after exposure to oxidative stress. There is evidence that increased HO activity is involved in long-term protective mechanisms against oxidative stress. We studied the effect of artificially overexpressed HO activity on the cytotoxicity of oxidative ultraviolet A (UVA) radiation after loading human cells with the HO substrate ferric heme (hemin). In contrast to the reported long-term protection attributed to HO activity, cells overexpressing HO activity were hypersensitive to UVA radiation shortly after heme treatment when compared with control cells. Cells overexpressing HO activity showed an increased rate of heme consumption and a higher level of accumulated free chelatable iron when compared with control cells. The hypersensitivity of cells overexpressing HO to UVA radiation after heme treatment was apparently caused by the increased accumulation of chelatable iron, because the iron chelator desferrioxamine strongly reduced the hypersensitivity. One day after the heme treatment, cells overexpressing HO activity were no longer hypersensitive to UVA radiation. We conclude that increased HO activity can temporarily increase the sensitivity of cells to oxidative stress by releasing iron from heme.

To study porphyrin biosynthesis in human umbilical vein endothelial cells (EC), cultured EC were incubated with delta-aminolevulinic acid (ALA), the precursor of porphyrin synthesis, and porphyrins generated were measured spectrofluorometrically. EC accumulated porphyrins in an incubation time-dependent and ALA dose-dependent fashion. Porphyrin accumulation was enhanced by CaMg EDTA, a ferrochelatase inhibitor, suggesting the participation of ferrochelatase. The effect of porphyrin and irradiation on porphyrin accumulation was evaluated by exposing porphyrin-containing EC to the UVA and blue light, followed by incubation with ALA for 2-48 h. Decreased porphyrin accumulation 2-48 h after the irradiation was noted at all the time-points examined. However, no alteration of ferrochelatase activity was observed, suggesting that the decrease in porphyrin levels was due to suppression of the activity of the other enzymes in the earlier steps of the heme biosynthetic pathway.