Categorizing reactivity of bacteriorhodopsin cysteine mutants crosslinking to 4-bromoretinal.

The structure of bacteriorhodopsin (bR) has been probed by a large number of experimental methods. In earlier work distance constraints measured from the 1BRD Brookhaven structure (1, 2) were used to guide site-directed mutagenesis/affinity labeling experiments (3-5). In the present study we report on the use of limited molecular dynamics (MD) investigations of the same bR/affinity label system. We show here that the chiral center introduced when 4-bromo-all-trans retinal is synthesized produces variable impact on potential crosslinking. Our MD analysis suggests the following ranking of binding site mutants in order of reactivity: R118C > S118C >> S121C > R141C >> S141C >>> R121C, R138C, S138C. Chirality appears to have limited effect for the M118C mutants but shows more dramatic impact for the T121C and S141C mutants. These results are in excellent agreement with the experimental observations and offer encouragement that MD can be a useful component of experimental design with considerable predictive power.

Binding studies of four free base porphyrins and six iron (+3) porphyrins with human serum albumin.

The binding stoichiometry and binding equilibria of human serum albumin with water-soluble four free base porphyrins and six chloroiron (+3) porphyrins have been determined in 0.1M phosphate buffer, pH 7.2, by fluorescence quenching and filtration methods. The binding stoichiometry is observed to be 1:1, porphyrin to protein. The dissociation constants, Kd, between the porphyrins and the protein are found to be 1-4 microM. A binding mechanism between the protein and the porphyrins has been presented.

Binding studies of three water-soluble polycationic porphyrins with human serum albumin.

The binding stoichiometry and equilibria of three polycationic water-soluble porphyrins with human serum albumin (HSA) have been determined by fluorescence quenching and filtration methods in 0.1M phosphate buffer, pH 7.2 at 25 degrees. For one porphyrin the binding equilibrium was also measured by measuring the lifetime of tryptophan and also by measuring the polarization of bound porphyrin. Energy transfer between the porphyrin and the tryptophan residue of HSA has been studied.

DNA strand scission by iron complexes of meso-tetra(N-methylpyridyl)porphines.

The DNA strand scission activities of three positional isomers of Fe(III) meso-tetra(N-methylpyridyl)porphine (Fe(III)TnMPyP, where n = 2, 3 or 4) have been investigated using PM2 DNA as a substrate. A significant degree of strand scission activity was noted in the presence of oxygen without the addition of a reducing agent. This activity was probably due to the presence of reducing agents in the agarose gels used to separate the DNA forms, as higher levels were recorded with reducing agents added to the strand scission mixture. The relative order of strand scission activity in the absence of added reducing agents was found to be Fe(III)T2MPyP greater than Fe(III)T4MPyP greater than Fe(III)T3MPyP. Comparative studies were also made with Fe(II)bleomycin. High concentrations of some reducing agents inhibited strand scission. Oxygen was required to produce optimal strand scission activity for all three porphyrins. It was also noted from spectroscopic measurements that the reduced porphyrins were degraded in the presence of oxygen. Studies with a series of potential strand scission inhibitors suggest that hydrogen peroxide and possibly peroxy radicals are intermediates in the reaction mechanism, while diffusible hydroxyl radicals appear to be excluded. However, superoxide radicals cannot be ruled out.

Inability to elicit rapid cytocidal effects on L1210 cells derived from porphyrin-injected mice following in vitro photoirradiation.

The role of the neoplastic cell in both porphyrin localization and the photochemotherapeutic response was investigated with the use of a series of tumor-localizing porphyrins and the L1210 tumor system. In vivo photoirradiation of DBA/2Ha mice bearing L1210 solid tumors and previously given injections of meso-tetra-(4-sulfonatophenyl)-porphine, meso-tetra-(4-carboxyphenyl)-porphine, or hematoporphyrin derivative (Hpd) indicated that all three chemicals elicited a photodynamic response resulting in necrosis of exposed tissue. Isolation of tumor cells from mice given injections of porphyrin with the use of mild mechanical means and physiologic conditions followed by in vitro photoirradiation of the cells under conditions established to optimize rapid cytocidal effects resulted in no appreciable cell death. A similar situation was noted with the use of spleen cells from mice given injections of Hpd, the spleen cells presumably containing substantial amounts of porphyrin. Both fluorescence microscopy and chemical extraction and quantitation of the porphyrins in the cells indicated that the inability to elicit a rapid cytocidal effect upon in vitro photoirradiation resulted from either the absence of or the presence of only very small amounts of porphyrin. These results indicate that in this particular tumor system the neoplastic cell per se plays only a minor role in porphyrin localization and, as a consequence, cannot be readily killed upon photoirradiation, suggesting that rapid cytocidal effects, due solely to porphyrin contained within the cell, probably do not occur among the majority of parenchymal cells during in vivo photoirradiation.

Induction of DNA damage by porphyrin photosensitizers.

The photosensitizing effects of hematoporphyrin derivative, meso-tetra(p-sulfonatophenyl)porphine, meso-tetra(p-carboxyphenyl)porphine, and meso-tetra(4-N-methylpyridyl)-porphine on ColE1 supercoiled DNA were studied using agarose gel electrophoresis. Photoinduced single- and double-strand breaks were observed to form under neutral conditions. Singlet oxygen was shown to predominate in the mechanism of the induction of these lesions in the case of hematoporphyrin derivative, meso-tetra(p-sulfonatophenyl)porphine, and meso-tetra(p-carboxyphenyl)porphine and to play a significant role in the case of meso-tetra(4-N-methylpyridyl)porphine. The results suggest the possibility that the risk of photodynamic carcinogenesis may accompany photochemotherapy and fluorescence endoscopic procedures involving porphyrin photosensitizers.

The binding of tumor localizing porphyrins to a fibrin matrix and their effects following photoirradiation.

The binding of two tumor localizing porphyrins, meso-tetra (4-carboxyphenyl) porphine (TCPP) and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) to fibrin clots was determined in vitro. Both TCPP and TPPS4 were found to bind extensively, although weakly, to fibrin. No appreciable binding by Hematoporphyrin IX to the fibrin matrix was detectable. Similar results were obtained whether the clot was non-crosslinked or crosslinked with factor XIII. Photoirradiation of a porphyrin-impregnated non-crosslinked clot rendered it urea insoluble. Electrophoretic analysis indicated that the alpha chain of the fibrinogen molecule was most affected by photoirradiation. This was manifested as a loss of the alpha chain intensity and a concomitant increase of high molecular weight components, suggesting the formation of crosslinks. No substantial differences were noted in susceptibility to plasmin lysis between photoirradiated and non-irradiated clots. Photoirradiated clots were, however, significantly more resistant to lysis induced by the plasminogen activators urokinase and streptokinase, suggesting that inactivation of plasminogen within the clot matrix had occurred during photoirradiation. The relevance of porphyrin binding to fibrin with regard to tumor localization and destruction is also discussed.

Binding of some meso-tetraphenyl porphyrins with apohemoglobin and methemoglobin.

Six tetraphenylporphyrins have been studied for their binding with human apohemoglobin and methemoglobin. Four showed binding with apohemoglobin, in a way hemin binds, to form analogs of recondtituted hemoglobin. Five porphyrins bound with methemoglobin, perhaps, through random ionic interaction.

The effect of tumor localizing porphyrins on the conversion of fibrinogen to fibrin.

The interaction of hematoporphyrin IX and two synthetic porphyrin tumor localizers, meso-tetra (4-carboxyphenyl) porphine (TCPP) and mesotetra (4-sulfonatophenyl) porphine (TPPS4) with fibrinogen was investigated in the presence and absence of light. Both TPPS4 and TCPP were found to bind to fibrinogen in greater than a 1/1 mole ratio in the absence of light. Chromatographic analysis indicated that during illumination TPP4 bound to fibrinogen to a greater extent that did either TCPP or hematoporphyrin. Both TCPP and TPPS4 were found to exhibit a greater inhibitory effect on clotting in the presence of light than did hematoporphyrin. In the absence of light and added Ca2+, both TCPP and TPPS4 were found to stimulate clotting at very specific porphyrin/fibrinogen concentration ratios, with TPPS4 being the more potent stimulator of the two. Hematoporphyrin exhibited little or no effect on clotting times. Fibrinogen, photoirradiated in the presence of the porphyrins was found to inhibit the clotting of unirradiated fibrinogen. A comparison of the stimulatory effects on clotting times by either calcium or TCPP and TPPS4 indicated that the porphyrins were capable of eliciting a greater stimulatory response than did calcium. The magnitude of the stimulatory response caused by the porphyrins alone was substantially reduced in the presence of calcium although the overall stimulatory response was increased but not additive. This suggests some interaction between either the calcium and porphyrin molecules or similarities in the respective stimulatory mechanism(s) involved. A possible correlation between these observations and the porphyrins ability to localize in tumors is also discussed.

Interaction of DNA with a porphyrin ligand: evidence for intercalation.

The porphyrin photosensitizer, meso-Tetra (4-N-methylpyridyl) porphine tetraperchlorate binds to calf thymus DNA by intercalation and by external electrostatic association. This was concluded from the results of measruements involving Scatchard analysis, viscometry, thermal denaturation, and circular dichroism.