Binding of meso-tetra (4-N-methylpyridyl) porphine to DNA.

The porphyrin photosensitizer, meso-Tetra (4-N-methyl-pyridyl) porphine tetraperchlorate is shown to unwind supercoiled ColEI DNA at a somewhat lower concentration than ethidium bromide. In contrast to this, the Fe(III) chelate of T4MPyP cannot unwind supercoiled DNA. It is concluded that these results corroborate our previous findings that, despite its large bulk, T4MPyP is fully capable of intercalating in DNA.     

Photosensitization of wild and mutant strains of Escherichia coli by meso-tetra (N-methyl-4-pyridyl)porphine

Wild type Escherichia coli cells as well as some mutant strains lacking specific DNA repair systems are efficiently killed upon visible light-irradiation after 5 min-incubation with meso-tetra(4N-methyl-pyridyl)porphine (T4MPyP). The presence of oxygen is necessary for cell photoinactivation. The porphyrin appears to exert its phototoxic activity largely by impairing some enzymic and transport functions at the level of both the outer and cytoplasmic membrane. Thus, SDS-PAGE electrophoresis shows a gradual attenuation of some transport protein bands as the irradiation proceeds, while a complete loss of lactate and NADH dehydrogenase activities is caused by 15 min-exposure to light. On the other hand, DNA does not represent a critical target of T4MPyP photosensitization as suggested by the closely similar photosensitivity of the wild E. coli and E. coli strains defective for two different DNA repair mechanisms, as well as by the lack of any detectable alteration of the pUC19 plasmids extracted from photosensitized E. coli TG1 cells.     

Bilirubin and haeme binding to human serum albumin studied by spectroscopy methods

Cobinding of bilirubin and of haeme to human serum albumin was investigated by means of difference absorption spectroscopy and fluorescence spectroscopy. Two specific sites for bilirubin and two for haeme binding occur on the albumin molecule. The primary binding site for bilirubin (Ka = 2.5 microM-1) is different from the primary heame binding site (Ka = 50 microM-1; Beaven et al., Eur J. Biochem. 41, 539-546, 1974), the former, however, might be identical with the secondary center for haeme binding. Similarly, the primary haeme binding center might be identical with the secondary bilirubin binding site.     

Cytochemical study of the fluorescence reaction of chromatin by the porphyrin derivative meso-tetra (4-N-methylpyridyl) porphine

Application of meso-tetra (4-N-methylpyridyl) porphine (TMPP) on smears of chicken blood revealed an intense red fluorescence on the nuclei under blue-violet exciting light, which seemed to be dependent on the DNA component of chromatin. The possibility that an intercalative binding mechanism based on hydrophobic interaction occurs between TMPP molecules and paired bases of DNA is briefly discussed.     

Photodynamic effect of lanthanide derivatives of meso-tetra(N-methyl-4-pyridyl)porphine against Staphylococcus aureus

Photodynamic therapy (PDT), used for cancer treatment, is also an alternative method for eradication of drug-resistant bacteria. This method utilizes a nontoxic light-activated dye, called a photosensitizer, and visible light to produce reactive oxygen species that lead to bacterial cell death. The purpose of this study was to investigate the bactericidal effect of PDT using lanthanide derivatives of meso-tetra(N-methyl-4-pyridyl)porphine against Staphylococcus aureus strains. The new photosensitizers appeared to be photodynamically ineffective. No enhancement of antistaphylococcal activity of TMPyP was observed after the conjugation of the porphyrin with lanthanide ions. Additionally, a significant difference in the susceptibility of two bacterial strains to unmodified TMPyP was observed.     

Characterization of the self-assembly of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in aqueous solutions

The aggregation of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in phosphate solutions was investigated as a function of pH, concentration, time, ionic strength, and solution preparation (either from dilution of a freshly prepared 2 mM stock or by direct preparation of μM solution concentrations) using a combination of complementary analytical techniques. UV-vis and fluorescence spectroscopy indicated the formation of staggered, side-by-side (J-type) assemblies. Their size and self-associative behavior were determined using analytical ultracentrifugation and small-angle X-ray scattering. Our results indicate that in neutral and basic solutions of H(2)TPPS(4-), porphyrin dimers and trimers are formed at micromolar concentrations and in the absence of NaCl to screen any ionic interactions. At these low concentrations and pH 4, the protonated H(4)TPPS(2-) species self-assembles, leading to the formation of particularly stable aggregates bearing 25 ± 3 macrocycles. At higher concentrations, these structures further organize or reorganize into tubular, rod-like shapes of various lengths, which were imaged by cryogenic and freeze-fracture transmission electron microscopy. Micron-scale fibrillar aggregates were obtained even at micromolar concentrations at pH 4 when prepared from dilution of a 2 mM stock solution, upon addition of NaCl, or both.     

Binding modes of V(=O)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy

The interactions between water-soluble cationic oxovanadyl[meso-tetrakis(4-N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A-T)2], poly[d(G-C)2], and poly[d(I-C)2] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A-T)2] and poly[d(I-C)2], a positive CD signal at low [VOTMPyP]/[DNA] ratios (R ratios) and strong excitonic CD signals at above R > or = 0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G-C)2] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I-C)2] is similar with that of poly[d(G-C)2], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A-T)2] and poly[d(G-C)2], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420-430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A-T)2], the five-coordinated species favored the poly[d(G-C)2] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base' amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.     

Binding of lead ion to bovine serum albumin studied by ion selective electrode

The binding of Pb2+ to bovine serum albumin (BSA) at neutral pH was studied using lead ion selective electrode. The binding data was treated according to Scatchard Equation. The number of binding classes and the number of binding sites, intrinsic dissociation constants and stepwise binding constants for each class were determined. Two binding classes were found. Four binding sites in the first class and five binding sites in the second class were determined. Binding in the first class was stronger than in the second. Similar binding studies were carried out with heat treated BSA. It was found that not only the number of binding sites but also the strength of binding increases upon heat treatment.     

Tumor localizing agents: the transport of meso-tetra(p-sulfophenyl) porphine by Vero and HEp-2 cells in vitro.

The mechanism of transport of the tumor localizing agent, meso-tetra(p-sulfophenyl)porphine (TPPS4), was investigated in Vero and HEp-2 cells in vitro. Vero cells proved to be basically impermeable to the porphyrin, but a slow transport was observed. The uptake was linear with time and appeared to be carrier mediated, as it was strongly inhibited by cyanide or low temperature and demonstrated saturation kinetics. Transport in HEp-2 cells was more rapid and non-linear, reaching a plateau after about 2 h. Analysis of this uptake over a 20-fold range of porphyrin concentration revealed it to be biphasic. A low affinity, high capacity component appeared to be unsaturable and was unaffected by low temperature or metabolic inhibitors. This system is probably one of a passive diffusion. The high affinity, low capacity phase is probably carrier mediated. The tumor cells appear to be "leaky" to the porphyrin, with respect to the Vero cells. This may explain part of the localizing ability of TPPS4.     

Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of < or =10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G . A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop-loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding.     

Dynamics of human serum albumin studied by acoustic relaxation spectroscopy

Sonic absorption spectra of solutions of human serum albumin (SA) in water and in aqueous phosphate buffer systems have been measured between 0.2 and 2000 MHz at different temperatures (15-35 degrees C), pH values (1.8-12.3), and protein concentrations (1-40 g/L). Several spectra, indicating relaxation processes in the whole frequency range, have been found. The spectra at neutral pH could be fitted well with an analytical function consisting of the asymptotic high frequency absorption and two relaxation contributions, a Debye-type relaxation term with discrete relaxation time and a term with asymmetric continuous distribution of relaxation times. Both relaxation contributions were observed in water and in buffer solutions and increased with protein concentration. The contribution represented by a Debye-type term is practically independent of temperature and was attributed to cooperative conformational changes of the polypeptide chain featuring a relaxation time of about 400 ns. The distribution of the relaxation times corresponding to the second relaxation contribution was characterized by a short time cutoff, between about 0.02 and 0.4 ns depending on temperature, and a long time tail extending to microseconds. Such relaxation behavior was interpreted in terms of solute-solvent interactions reflecting various hydration layers of HSA molecules. At acid and alkaline pH, an additional Debye-type contribution with relaxation time in the range of 30-100 ns exists. It seems to be due to proton transfer reactions of protein side-chain groups. The kinetic and thermodynamic parameters of these processes have been estimated from these first measurements to indicate the potential of acoustic spectra for the investigation of the elementary kinetics of albumin processes.     

Binding modes of V(O)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy

The interactions between water-soluble cationic oxovanadyl[meso-tetrakis(4-N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A–T)₂], poly[d(G–C)₂], and poly[d(I–C)₂] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A–T)₂] and poly[d(I–C)₂], a positive CD signal at low [VOTMPyP]/[DNA] ratios (R ratios) and strong excitonic CD signals at above R ≥ 0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G–C)₂] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I–C)₂] is similar with that of poly[d(G–C)₂], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A–T)₂] and poly[d(G–C)₂], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420–430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A–T)₂], the five-coordinated species favored the poly[d(G–C)₂] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base′ amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.     

Binding of Triton X-100 to bovine serum albumin as studied by surface tension measurements

A previously published computerized drop-weight technique for surface tension measurements, not involving the use of radioactively labelled compounds, has been applied to the study of detergent binding to proteins. The procedure is based on the observation that the protein-surfactant complex is no longer surface-active. As an example, the binding of Triton X-100 to bovine serum albumin has been studied, and the results were found to be in good agreement with those obtained through established but less convenient methods. Our procedure should be useful for measurements of detergent binding to biomembranes.     

Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering

To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein; disordered structures add as the pD decreases. Moreover, beta-aggregates seem to contribute only to oligomers formation, as showed by the good correlation between kinetics of scattering intensity and IR absorption intensity. These results indicate for BSA a general mechanism of aggregation composed by partial unfolding of the tertiary structure and by the decrease of alpha-helix and random coil contents in favor of beta-sheet aggregates. This mechanism strictly depends on pD and gives rise to almost two distinct types of macromolecular aggregates.     

Binding of mercury(II) to poly(dA-dT) studied by proton nuclear magnetic resonance

The binding of Hg(II) to poly(dA-dT) has been examined with proton NMR spectroscopy. Addition of HgCl2 between r (Hg2+/nucleotide) = 0 and 0.25 results in loss of the exchangeable imino N3H resonance of thymine, indicating preferential binding at this site. The nonexchangeable base resonances AH8, AH2, and TH6 shift their intensity downfield in a cooperative manner, indicating complexation which is slow on the NMR time scale and changes in the polymer conformation upon binding. At r = 0.25, the polymer is cross-linked, and an increase in temperature does not result in denaturation of the polymer, as evidenced by the thymine proton resonance chemical shifts. The chemical shifts of the AH2 and T(CH3)5 base resonances allow some general conclusions to be made about the stereochemistry of this complex.     

Species-dependency in chiral-drug recognition of serum albumin studied by chromatographic methods

Stereoselective binding of benzodiazepine and coumarin drugs to serum albumin from human and six mammalian species were studied by chiral chromatographic techniques. The applied methods were affinity chromatography on the albumins immobilized on Sepharose 4B, high-performance liquid chromatography (HPLC) separation on columns based on human serum albumin (HSA) and bovine serum albumin (BSA), and chiral HPLC analysis of ultrafiltrates of solutions containing the racemic drug and the native protein. Substantial differences in preferred configurations and conformations were detected among the species. The binding stereoselectivity of the 2,3-benzodiazepine drug, tofisopam, in human, is opposite to that in all other species. In the binding of 1,4-benzodiazepines, dog albumin is very similar to HSA. Highly preferred binding of (S)-phenprocoumon was found with dog albumin.     

Volume and enthalpy profiles of CO binding to Fe(II) tetrakis-(4-sulfonatophenyl)porphyrin.

The focus of this study is to examine volume and enthalpy profiles of ligand binding associated with CO-Fe(II) tetrakis-(4-sulfonato phenyl)-porphyrin (COFe(II)4SP) in aqueous solution. Temperature dependent photothermal beam deflection was employed to probe the overall enthalpy and volume changes associated with CO-photolysis and recombination. The analysis demonstrates that ligand recombination occurs with a pseudo first order rate constant of (2.5+/-0.2)x10(4) s(-1) (at 25 degrees C) with a corresponding volume decrease of 6+/-1 ml/mol. The activation enthalpy (DeltaH(double dagger)) and volume (DeltaV(double dagger)) change for CO recombination (determined from temperature/pressure dependent transient absorption spectroscopy) are found to be 3.9 kcal/mol and 8.2 ml/mol, respectively. These data are consistent with a mechanism in which photolysis yields a five-coordinate high spin (H(2)O)Fe(II)4SP complex that recombines in a single step to form the low spin (CO)(H(2)O)Fe(II)4SP complex. Base elimination, often associated with CO photolysis from hemes, is not observed in this system. The overall volume changes suggest a transition state with significant high spin character. Furthermore, these results demonstrate the utility of coupling photothermal techniques with variable pressure/temperature transient absorption spectroscopy to probe heme reaction dynamics.     

Translational competition by mRNA species encoding albumin, ferritin, haemopexin and globin

The photoreactive gamma-(p-azidoanilidate) analog of ATP, AzAnATP, was used to affinity-label the chloroplastic and cytoplasmic leucyl-tRNA synthetases of Euglena gracilis. The analog is able to replace the substrate ATP in the tRNA leucylation reaction catalyzed by both enzymes. In the presence of ATP, it is a competitive inhibitor against ATP as well as leucine for the two isoenzymes, as is also shown for the photoinactive gamma-anilidate analog of ATP, AnATP, which does not serve as substrate in the enzyme reaction. During ultraviolet irradiation, the enzymes are irreversibly inactivated by AzAnATP in a concentration-dependent and time-dependent manner indicative of photoaffinity labeling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet-induced inactivation by AzAnATP. Comparative kinetic characterization of the inactivation process reveals differences in the active centers of the two intracellular isoenzymes.     

Binding of 8-bromoguanylic acid to ribonuclease T1 as studied by absorption and circular dichroism spectroscopy

8-Bromoguanosine 2'- and 3'-phosphates have been shown to bind to RNase T1 with the same affinity as the corresponding guanosine phosphates, inducing difference absorption and circular dichroism spectra similar to those induced by the guanosine phosphates. Since the brominated ligands have reduced electron density on N-7 of the guanine ring and syn-fixed conformation due to a bulky, electron-withdrawing Br substituent on C-8, the difference spectra are not attributable to the protonation on N-7 and to the restriction of the ligand to syn-conformation as proposed previously.