Studies of hematoporphyrin and hematoporphyrin derivative equilibria in heterogeneous systems. Porphyrin-liposome binding and porphyrin aqueous dimerization

Two processes of porphyrins in heterogeneous systems containing aqueous and membrane phases have been studied with hematoporphyrin and hematoporphyrin derivative: Dimerization equilibrium in the aqueous phases and porphyrin-membrane binding equilibrium using liposomes as models for biological membranes. The interrelationship of aqueous aggregations and membrane binding was probed and the porphyrin aggregation state in the membrane, at equilibrium, was assessed. Fluorimetric techniques were employed. The dimerization equilibrium constants, at neutral pH and 37°C were found to be 2.8 · 10⁵ M⁻¹ and 1.9 · 10⁶ M⁻¹ for hematoporphyrin and its derivative, respectively. Over a porphyrin concentration range going from monomer-dominant to dimer-dominant systems, we have found that only monomers are bound to the membrane. The respective monomer-liposome binding constants, found to be independent of the initial monomer/dimer distribution in the aqueous phase, were determined to be 1.6 · 10³ M⁻¹ and 4.1 · 10³ M⁻¹ at neutral pH and 37°C for hematoporphyrin and its derivative, respectively. The monomer-liposome interaction was found to perurb the initial monomer/dimer distribution in the aqueous phase, so that the monomers residing at equilibrium in the membrane originate from both monomers and dimers in the aqueous phase.     

Aggregation of tyrocidine in aqueous solutions.

The formation of aggregates of tyrocidine B at 4°C and 20°C in aqueous solutions was studied by means of light scattering and fluorescent techniques. The apparent weight molecular weight of tyrocidine B aggregates was found to be 36,000 at 4°C and 28,800 at 20°C. Fluorescence titration experiments with dansyl-chloride resulted in an aggregational number of 31 (4°) and 28 (20°) indicating that one molecule of dye is bound per monomer of molecular weight 1,200. From a Scatchard plot apparent association constants of 1.22 × 10⁵ M (4°) and 0.95 × 10⁵ M (20°) were calculated. From the angular dependence of scattered intensity the radii of gyration were determined to be 60 Å and 58 Å, respectively.     

Study of kinetic parameters of singlet molecular oxygen in aqueous porphyrin solutions. Effect of detergents and the quencher sodium azide

The kinetic parameters of porphyrin-photosensitized formation and deactivation of singlet molecular oxygen (1O2) and their dependence on the concentration of the 1O2 quencher sodium azide were investigated in air-saturated water, ethanol, and aqueous micellar solutions of detergents using time-resolved measurements of oxygen phosphorescence under pulsed laser excitation. The lifetimes of 1O2 formation and deactivation and the rate constants of 1O2 quenching by sodium azide were determined. It was shown that, with no azide in the solutions, the rise in phosphorescence intensity after the laser flash corresponded to the kinetics of energy transfer from the porphyrin triplet molecules to oxygen, while the decay kinetics corresponded to the kinetics of 1O2 deactivation. In the presence of detergent, a considerable increase in the 1O2 lifetime was observed, which is likely due to the localization of 1O2 molecules mostly in lipophilic micelles and not in the water phase. If relatively high azide concentrations were used, the lifetime of the porphyrin triplet state did not change but the 1O2 lifetime decreased to values similar to those in living cells. In this case, the inversion of the phosphorescence kinetic phases was observed. The rise corresponded to 1O2 deactivation, and the decay, to the energy transfer from triplet porphyrin to oxygen. The data suggest that, in living cells, 1O2 molecules are also located mainly in lipophilic structures and the 1O2 lifetime determines the kinetics of the phosphorescence rise after the laser pulse.     

Thermodynamics of the interaction of sodium-n-dodecyl sulphate with Aspergillus niger catalase in high ionic strength aqueous solutions

The thermodynamic parameters for the interaction between sodium n-dodecyl sulphate (SDS) and Aspergillus niger catalase in aqueous solution at pH 3.2 and 6.4 have been measured by microcalorimetry and equilibrium dialysis over a range of ionic strength from 0.05 to 0.2 at 25 degrees C. Binding isotherms have been interpreted in terms of theoretical models (Hill equation and Wyman binding potential). The Gibbs energies of interaction become increasingly negative with increase in ionic strength and the entropies of interaction become increasingly positive. The ionic strength dependence of the Gibbs energies are much greater than predicted by the Debye-Hückel limiting law indicating a strongly ionic strength dependent hydrophobic contribution to the interactions.     

Thermodynamics of glycophorin A transmembrane helix dimerization in C14 betaine micelles

We have used sedimentation equilibrium analytical ultracentrifugation to measure the free energy change for the glycophorin A transmembrane helix-helix dimerization in C14 betaine micelles. By varying the amount of micellar C14 betaine, we show that the protein association reaction in the micellar C14 phase behaves as an ideal-dilute solution. In this hydrophobic environment, the mole-fraction standard state free energy change for self-association of the SNGpA99 glycophorin A construct is -5.7 (+/-0.3, N=5) kcal mol(-1) at 25 degrees C. Compared with previous results carried out in C(8)E(5) micellar solutions, the free energy of dimerization is 1.3 kcal mol(-1) less favorable in C14 betaine micelles. In contrast, when considered on a per-interface basis, the formation of the glycophorin A transmembrane dimer in C14 betaine micelles may be more favorable than the association of several designed transmembrane peptides.     

Thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes in butanol/aqueous salt biphasic solutions

The thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes wee studied in order to gain further insight into the mechanisms responsible for the sensitivity of the relative solubility of these complexes in aqueous slat and butanol phases to small changes in slat concentration. The dependence of the partition coefficient was measured as a function of temperature at three different salt concentrations. Increasing the temperature was found to favor the form of the complex which was soluble n the aqueous phase. Although the transition could be induced by temperature changes, he transition occurred over a 20°C range in temperature. The transition from the aqueous phase to the butanol phase was strongly exothermic, with ΔH = ?22.3 kcal/mol polymer. The value of ΔS was found to be dependent on the salt concentration, ranging from ?72.7 e.u./mol polymer in 0.05125M NaCl to ?77.1 e.u./mol polymer in 0.05375M MaCl. When placed on a disaccharide basis, the corresponding values are ΔH = ?402 cal/mol and ΔS = ?1.31 to ?1.3 e.u./mol. The sharpness of he transition was found to be due t the similarity in magnitude of ΔH and TΔS, and on the dependence of the later upon the salt concentration.     

Induced CD and interaction of some porphyrin derivatives with α-helical polypeptides in aqueous solutions

Absorption spectra and induced CD have been measured on aqueous solutions of water-soluble porphyrins with α-helical poly(L -glutamic acid) or α-helical poly (L -lysine) at different mixing ratios. For the former, porphyrin is porphine-meso-tetra (4-N-methylpyridinium) (TMpyP), and for the latter, it is porphine-meso-tetra (4-benzenesulfonate) (TPPS) or porphine-meso-tetra(4-benzoate) (TPPC). All the solutions of porphyrin-polypeptide systems show hypochromism in the Soret band and induced CD in the Soret region. The CD is characterized by a positive band at a shorter wavelength and a stronger negative band at a longer wavelength. The hypochromicity and the magnitude of molar ellipticities are much larger for the TPPS– and TPPC–poly (L -lysine) systems than for the TMpyP–poly (L -glutamic acid) system. Porphyrin ions bind to the α-helix electrostatically, and the two components of the Soret transition of porphyrin are subject to dissymmetric perturbation. TMpyP ions bind to the α-helix at isolated sites, while TPPS ions and TPPC ions are in pairs on the α-helix, that is, two ions bind consecutively and dissymmetrically. In the TMpyP–poly (L -glutamic acid) system a single CD band is associated with each of the two components of the Soret transition, and these are of opposite sign. In the TPPS– and TPPC–poly (L -lysine) systems, a pair of positive and negative CD bands is associated with each of the two components, thus giving apparently a single pair of CD bands with a shoulder, owing to partial overlapping.     

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.     

Physicochemical studies of amylose and its derivatives in aqueous solutions: Thermodynamics of the iodine–triiodide complex

Thermodynamic properties of the amylose–iodine–triiodide complex have been studied by spectrophotometry and by calorimetry using previously studied samples of amylose ionic derivatives, carboxymethylamylose and diethylaminoethylamylose. The ratio of triiodide to total molecular iodine ([I₃]_b/[I]_b + [I₂]_b) in the complex is ca. 0.3 over a range of iodide concentration from 10^?5 to 10^?4M, and there is no evidence for an increasing charge at slightly higher iodide concentration. Direct calorimetric experiments have been carried out in different conditions of polymer, iodine, and iodide concentration in order to study the dependence of the heat of the complexation as a function of the above parameters. It is shown that the dependence of the measured ΔH on the iodide concentration simply derives from the rearrangement of the triiodide equilibrium because of the uptake of a fixed ratio of iodine and triiodide molecules in the complex.     

Decontamination of aqueous solutions of biological stains.

Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins. Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions. Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16. Reaction times varied from 10 min to 18 hr. Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16. Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium. None of the completely decontaminated solutions were found to be mutagenic.     

Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics.

Dialysis kinetics measurements have been made to study the effect of ionic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals. Using glutaraldehyde cross-linked dimers of lysozyme, we have determined that both monomers and dimers can escape from 25,000 molecular weight cutoff dialysis membranes with velocity constants of 5.1 x 10(-7) and 1.0 x 10(-7) s(-1) for the monomer and dimer species, respectively. The flux from 25K MWCO membranes has been measured for lysozyme in pH 4.0 buffered solutions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concentrations. Assuming that dimerization is the first step in crystallization, a simple monomer to dimer equilibrium was used to model the flux rates. Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respectively. These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions. At high protein concentrations and high supersaturation, the dimerization model does not describe the data well. However, the Li model that uses a pathway of monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer fits the measured flux data remarkably well suggesting the presence of higher order aggregates in crystallizing solutions.     

Riboflavin-photosensitized changes in aqueous solutions of alginate. Rheological studies

Interactions between photoexcited riboflavin (RF), promoted by irradiation in the range of 310-800 nm, and alginate have been studied in air equilibrated aqueous solutions with the aid of rheological methods. Light irradiation of RF causes under aerobic conditions fragmentation of alginate and a decrease in the shear viscosity and other rheological parameters of its solutions. The decrease is most pronounced in concentrated polymer solutions. The photochemical degradation of alginate is inhibited in the presence of the quenchers/scavengers d-mannitol, glutathione, potassium iodide, and sodium azide and in excess oxygen. The addition of thiourea to alginate-RF solutions leads to enhanced degradation of the polymer. Significant shear-thinning effects and deviations from the Cox-Merz rule are observed at higher polymer concentrations.     

Conformation of poly-S-carboxyethyl-L-cysteine in aqueous solutions

Poly-S-carboxyethyl-L -cysteine, a higher side-chain homolog of poly-S-carboxymethyl-L -cysteine, has been prepared from poly-S-carbobenzoxyethyl-L -cysteine with hydrogen bromide in chloroform or acetic acid. The polymer is found to be in the β-conformation of an antiparallel arrangement of polypeptide chains in solid films, both in acid and salt forms, when examined by infrared spectra. Aqueous solutions of t he polymer have been investigated by measurements of rotatory dispersion and circular dichroism as well as by infrared spectra in D₂O. These properties show sharp changes around pH 5.5, as the pH of solution is varied. At higher ionization the polymer is randomly coiled, but at lower ionization it is in the β-conformation. Dependence of the rotatory properties upon polymer concentration as well as on ionic strength has been observed even at the lowest degree of ionization attained, and this has been attributed to the formation of intermolecular β-conformation in solutions. The β-structure is characterized by a negative circular dichroic band at 223 mμ and a positive dichroic band at a wavelength lower than 200 mμ, and furt her by a negative b_o value, ?140°. The pH-induced coil-β transition of the polymer is compared with that of poly-S-carboxymethl-L -cysteine.     

New trends in cryoenzymology: I.-Supercooled aqueous solutions.

A water in oil emulsion technique is proposed to investigate enzyme catalyzed reactions at sub-zero temperatures in the supercooled liquid state to avoid some reversible effects of the usual cosolvents on kinetics. Some results are listed: potentialities and technical problems of the procedure are discussed.     

Thermodynamics of the conversion of aqueous xylose to xylulose

The thermodynamics of the conversion of aqueous xylose to xylulose has been investigated using high-pressure liquid chromatography (HPLC) and microcalorimetry. The reaction was carried out in aqueous phosphate buffer over the pH range 6.8-7.4 using solubilized glucose isomerase with MgSO(4) as a cofactor. The temperature range over which this reaction was investigated was 298.15-342.15 K. A combined analysis of both the HPLC and microcalorimetric data leads to the following results at 298.15 K for the conversion process: DeltaG degrees = 4389 +/- 31 J mol(-1), DeltaH degrees = 16090 +/- 670 J mol(-1), and DeltaC(p) degrees = 40 +/- 23 J mol(-1) K(-1). The temperature dependence of the equilibrium constant for the reaction is expressed as R ln K = -4389/298.15 +16090[(1/298.15)-(1/T)]+40[(298.15/T)-1 + ln(T/298.15)]. Comparisons are made with literature data.