THE INTRINSIC FLUORESCENCE CHARACTERISTICS OF THE MYELIN BASIC PROTEIN

—The encephalitogenic basic protein has been isolated from the myelin sheath of ox brain white matter and the purity and amino acid composition have been verified. The intrinsic fluorescence characteristics of the purified basic protein have been determined and the results interpreted in terms of current ideas on the structure of the protein. Fluorescence data obtained from the basic protein in aqueous solution indicate that the tyrosine and tryptophan residues are largely exposed to the solvent and that resonance energy transfer from tyrosine to tryptophan is very inefficient. Denaturing conditions in 8 m -urea have little effect on the fluorescence properties of the protein. The ionic detergent, sodium dodecyl sulphate, interacts with the basic protein and alters the fluorescence properties in a manner which indicates that the tryptophan residue is in the hydrocarbon chain region of the detergent while the local positive charge around the tyrosine residues is neutralized by the negatively charged sulphate head-groups. The fluorescence results suggest that the basic protein can be used as a natural, non-perturbing probe which will report on its environment after it has reacted with other membrane components.     

Charge transfer between tryptophan and tyrosine in casein: a pulse radiolysis study

Charge transfer from tyrosine to tryptophan radicals in bovine milk casein, as observed using pulse radiolysis technique, is reported. The reactions of casein with hydroxyl, azide, Br(2)(*-) and CCl(3)O(2)(*) radicals have also been studied. Radical transformation was found to take place at a rate of 1.5 x10(4) s(-1). The effect of pH, oxidising radical and the proximity of tyrosine and tryptophan on this radical transformation, as well as repair of the casein radical by ascorbate, have also been studied.     

Reactivities of tyrosine, histidine, tryptophan, and methionine in radical pair formation in flavin triplet induced protein nuclear magnetic polarization

In order to check the validity of several basic assumptions of protein photochemically induced nuclear polarization (protein photo-CIDNP), we have investigated the quenching processes of the dye triplets by the side chains of tyrosine, histidine, and tryptophan in a variety of molecular systems and environments. The quenching (H atom or electron transfer) is the generating process of the triplet electron-spin-correlated radical pair, the evolution of which gives rise to nuclear polarization. At pH 7 the quenching of 10-(carboxyethyl)flavin triplets by tyrosine and tryptophan is almost diffusion controlled. Quenching by histidine is slower. We have also investigated the slow quenching (by electron transfer) by the side chains of methionine and could show that quenching by cysteine S derivatives is negligible. Quenching by tyrosine and histidine peptides and by the tyrosines of the pancreatic trypsin inhibitor protein is slightly slower than by free side chains. Quenching is strongly viscosity controlled, to be expected of a process requiring bimolecular contact. Reactivity trends at high viscosities resemble those observed in fluid aqueous solutions. Activation energies of quenching by tyrosine, tryptophan, and histidine are similar. No difference could be detected in the mechanism of quenching by these side chains. No fast static quenching was observed that could compete with the diffusional process.     

Radiation damage to histone H2A by the primary aqueous radicals

Histone H2A has been examined for radiation-induced changes in structure and in amino acid composition. The effects of the individual radical intermediates--the hydroxyl radical, solvated electron, and superoxide radical--have been determined by irradiating in dilute aqueous solution under controlled environmental conditions. Amino acid analysis of irradiated histone H2A shows a selective attack on a few residues; only the aromatic residues, phenylalanine and tyrosine, and the heterocyclic residue, histidine, are significantly decreased. A significant increase in aspartic acid is also observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the hydroxyl radical is the effective radical for promoting changes in protein structure.     

Viscosity B-coefficients and activation parameters for viscous flow of a solution of heptanedioic acid in aqueous sucrose solution

Viscosity and density data for the system of heptanedioic acid dissolved in aqueous sucrose solution at temperature range from 288.15 to 313.15 K have been measured. The viscosity B-coefficients for heptanedioic acid in aqueous sucrose solution has been calculated. The effect of temperature and sucrose concentration on the B-coefficients is discussed. On the basis of the Feakins equation, the activation parameters (Deltamu3++, DeltaH3++, DeltaS3++, DeltaG12(0)++, DeltaH12(0)++ and DeltaS12(0)++) for viscous flow of the solution have been evaluated, together with the Gibbs energy of transfer for the solute from the ground state solvent to the hypothetical viscous transition state solvent (DeltaG3++(1-1')). The effect of sucrose concentration and temperature on the activation parameters has been discussed.     

Ultraviolet resonance Raman spectra of insulin and alpha-lactalbumin with 218- and 200-nm laser excitation

Ultraviolet resonance Raman (RR) spectra, with 200- and 218-nm excitation from a H2-shifted quadrupled Nd:YAG laser, are reported for insulin and alpha-lactalbumin in dilute aqueous solution, at pH values known to produce differences in the exposure of the aromatic residues to solvent. At 200 nm, the spectra are dominated by tyrosine bands, whose intensity is lowered somewhat in protein conformations in which tyrosine is exposed to solvent. The expected shift in the relative intensities of the components of the approximately 850-cm-1 tyrosine doublet is difficult to discern because the higher energy component shows much greater resonance enhancement and the lower energy component appears as a weak shoulder. The peptide vibrations, amides I, II, and III, are also enhanced at 200 nm. The infrared active amide II mode is particularly prominent, although it is not observed in Raman spectra with visible excitation. In addition, the amide I band is quite broad in the 200-nm RR spectra, and the peak frequency is lower than that seen in visible excitation Raman spectra and is close to the infrared frequency. It appears that 200-nm excitation produces resonance enhancement of the infrared-active components of both amide I and amide II. Excitation at 218 nm enhances tryptophan modes strongly. The 876-cm-1 band, assigned to a deformation mode of the five-membered ring, shows a measurable upshift upon exposure of tryptophan to solvent, attributable to N-H hydrogen bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments

The one-electron reduction potentials of the radical cations of five dietary carotenoids (β-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.     

Static and dynamic quenching of protein fluorescence. I. Bovine serum albumin.

The fluorescence parameters, lifetime, relative quantum yield, maximum and mean wavelength, half-width, and polarization, of bovine serum albumin (BSA) were measured at 15°C in aqueous solutions containing varying concentrations of different chemical perturbants, glycerol, Cu²⁺ ions, guanidine hydrochloride, and urea. By considering a quenching mechanism as being either dynamic or static, depending upon whether the quenching is or is not accompanied by a change in the fluorescence lifetime, we were able to correlate the changes produced in the various fluorescence parameters by the different chemical perturbants with changes in macromolecular structure as the concentration of perturbant was gradually increased. The addition of glycerol and of Cu²⁺ ions indicated that in aqueous BSA both tryptophan residues are below the surface of the macromolecule, out of contact with solvent water, and, as a consequence, they are statically quenched. “Ultra-Pure” guanidine hydrochloride at 2.4 M or more caused a drastic conformation change, which resulted in the emergence of a visible tyrosine peak at 304 nm in the BSA fluorescence spectrum when either 260- or 270-nm excitation was employed. With the same excitation, the enhancement of BSA tyrosine fluorescence by 6–8 M ultra-pure urea produced only a shoulder near 304 nm in the BSA fluorescence spectrum. We have introduced the use of a new relative quantum yield for protein fluorescence, q′, referenced to the quantum yield of unquenched free tryptophan, which eliminates the quenching action of water from the reference.     

Intrinsic fluorescence of a hydrophobic myelin protein and some complexes with phospholipids

The fluorescence characteristics of lipophilin, a proteolipid apoprotein from human myelin, were determined in aqueous and lipid environments. In all cases the tryptophan residues were located in buried hydrophobic sites of uniform, but limited, accessibility to the permeant quenching agent acrylamide; only in the helicogenic solvent 2-chloroethanol were the protein fluorophores exposed to the medium. Quantum yields were dependent on the state of aggregation of the protein in aqueous solution and increased considerably on treatment with lysolecithin micelles, or when the protein was combined with phosphatidylcholine by codialysis from 2-chloroethanol into water. Fluorescence titrations indicated that lipophilin bound to lysolecithin with an association constant greater than 10(6) L/mol. Radiationless singlet excitation energy transfer from tyrosine to tryptophan residues was found to decrease markedly when the protein was combined with lipids. When the protein was introduced into dimyristoylphosphatidylcholine vesicles, the tryptophan fluorescence did not detect any solid-liquid phase change. These results were consistent with strong hydrophobic interactions between lipophilin and phospholipids, which lead to conformational adjustments in the protein, and to establishment of an immobilized layer of boundary lipid in bilayer systems.     

Fluorescence studies of endothelin-1

Fluorescence measurements and singlet singlet energy transfer experiments on endothelin-1 provide information on the conformation of this peptide in dilute aqueous solution. The tyrosine fluorescence quantum yield in the absence of transfer (in [Phe²¹]endothelin-1) is relatively large (Φ_tyr = 0.39), indicating the side-chain is oriented away from fluorescence quenching groups such as the two disulfide bonds of the peptide. The fluorescence emission maximum (λ = 351 nm) and quantum yield (Φ_trp = 0.099) of tryptophan in endothelin-I suggests that this residue is fully accessible to the solvent and that the indole ring is not located near the fluorescence quenching histidinium moiety or the disulfide bonds.

Singlet-singlet fluorescence energy transfer measurements of the Tyr¹³/Trp²¹ intramolecular distance by both donor fluorescence quenching and relative enhancement of acceptor fluorescence yield a distance of about 12.8 ± 0.6 Å. Molecular modeling of a fully extended C-terminal hexapeptide indicates a Tyr¹³/Trp²¹ distance of about 25 Å. Thus, the C-terminal residues must bend back towards the bicyclic portion of the molecule.     

Vesicles from peptidic side-chain polymers synthesized by atom transfer radical polymerization

Block copolymers can adopt a wide range of morphologies in dilute aqueous solution. There is a significant amount of interest in the use of block copolymer vesicles for a number of applications. We show that a series of oligo(valine) and oligo(phenylalanine) peptides coupled to a methacrylic group can be prepared by conventional peptide coupling techniques. These can be successfully polymerized by atom transfer radical polymerization (ATRP) in hexafluoroisopropanol (HFIP) giving access to poly(ethylene oxide)- b-poly(side-chain peptides). Many of these polymers self-assemble to form vesicles using an organic to aqueous solvent exchange. One example with a divaline hydrophobic block gives a mixture of toroids and vesicles. Circular dichroism demonstrates that secondary structuring is observed in the hydrophobic region of the vesicle walls for the valine side-chain containing polymers.     

Solubility and property of chitin in NaOH/urea aqueous solution

NaOH/urea aqueous solution has been used as a solvent for chitin for the first time. Effects of this solvent composition and temperature on the solubility and stability of chitin solution were studied with an optical microscope, from which 8 wt% NaOH/4 wt% urea concentrations were deduced as suitable and −20 °C as the appropriate temperature. The original and regenerated chitin were characterized by viscosity, elemental analysis, FI-IR and X-RD analysis, and the effect of solvent composition and temperature on chitin structure was investigated. It was inferred that 8 wt% NaOH/4 wt% urea solvent under low temperature adventitiously has little effect on chitin structure and the urea is of benefit to the stability of chitin solution. In addition, the rheological properties suggested that chitin aqueous solution in high concentration is a pseudoplastic fluid and that chitin aqueous solution in low concentrations is a Newtonian fluid. This chitin aqueous solution is sensitive to temperature and will transform it to a gel when temperature increases.     

On the contemporaneous, reversible interaction of different ligands with serum albumins in dilute aqueous solutions: Fluorescein and phenylbutazone

The binding affinity of fluorescein and of phenylbutazone to human serum albumin (HSA) and to bovine serum albumin (BSA), respectively, as well as of the two drugs together to each protein in dilute aqueous solution has been studied by means of gel permeation chromatography, circular dichroism, U.V. absorption and fluorescence spectroscopy. Identity of and/or interdependence between primary binding sites for the two ligands considered on HSA and BSA are evidenced and correlated with a simple theoretical approach to mixed drugs binding.     

Radiation-induced inactivation of dihydroorotate dehydrogenase in dilute aqueous solution.

The inactivation of dihydroorotate dehydrogenase by gamma irradiation in dilute aqueous solution has been investigated. The activity of the enzyme decreased exponentially as a function of the absorbed dose under aerated and nitrous oxide-saturated conditions. The contributions of the individual radical species derived from water radiolysis were estimated from the inactivation results observed under aerated, argon-saturated, and nitrous oxide-saturated conditions. The hydrogen atom and hydroxyl radical were found to be important in enzyme inactivation. The effect of selected inorganic radical anions such as Br.2-, I.2-, and (SCN).2- on the enzyme activity was also studied, and the results implicate the possible involvement of cysteine and tyrosine residues in the catalytic activity of dihydroorotate dehydrogenase. Changes in the kinetic parameters (Michaelis-Menten constant, Km, and maximal velocity, Vmax) due to irradiation under the conditions investigated suggest that radiation-induced inactivation is due to modification of the substrate binding sites and that of the active site residues in the enzyme. Evidence for the reduction of iron-sulfur centers in the enzyme during the inactivation process has been put forward from the difference spectrum of the irradiated dihydroorotate dehydrogenase. It has also been shown by electrophoretic studies that radiation-induced inactivation was not due to any fragmentation of the protein structure or the formation of any intermolecular crosslinking.     

Radical-Induced Damage to Bovine Serum Albumin: Role of the Cysteine Residue

The reactions of cerium(IV) and the hydroxyl radical [generated from iron(ii)/H₂O₂] with bovine serum albumin (BSA) have been investigated by EPR spin trapping. With the former reagent a protein-derived thiyl radical is selectively generated; this has been characterized via the anisotropic EPR spectra observed on reaction of this radical with the spin trap DMPO. Blocking of the thiol group results in the loss of this species and the detection of a peroxyl radical, believed to be formed by reaction of oxygen with initially-generated, but undetected, carbon-centred radicals from aromatic amino acids. Experiments with a second spin trap (DBNBS) confirm the formation of these carbon-centred species and suggest that damage can be transferred from the thiol group to carbon sites in the protein. A similar transfer pathway can be observed when hydroxyl radicals react with BSA.

Further experiments demonstrate that the reverse process can also occur: when hydroxyl radicals react with BSA, the thiol group appears to act as a radical sink and protects the protein from denaturation and fragmentation through the transfer of damage from a carbon site to the thiol group. Thiol-blocked BSA is shown to be more susceptible to damage than the native protein in both direct EPR experiments and enzyme digestion studies. Oxygen has a similar effect, with more rapid fragmentation detected in its presence than its absence.     

Study of the interaction of artemisinin with bovine serum albumin

The study on the interaction of artemisinin with bovine serum albumin (BSA) has been undertaken at three temperatures, 289, 296 and 303 K and investigated the effect of common ions and UV C (253.7 nm) irradiation on the binding of artemisinin with BSA. The binding mode, the binding constant and the protein structure changes in the presence of artemisinin in aqueous solution at pH 7.40 have been evaluated using fluorescence, UV–vis and Fourier transform infrared (FT-IR) spectroscopy. The quenching constant K_q, K_sv and the association constant K were calculated according to Stern–Volmer equation based on the quenching of the fluorescence of BSA. The thermodynamic parameters, the enthalpy (ΔH) and the entropy change (ΔS) were estimated to be −3.625 kJ mol⁻¹ and 107.419 J mol⁻¹ K⁻¹ using the van’t Hoff equation. The displacement experiment shows that artemisinin can bind to the subdomain IIA. The distance between the tryptophan residues in BSA and artemisinin bound to site I was estimated to be 2.22 nm using Föster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of enough common ions and UV C exposure, indicates that common ions and UV C irradiation have effect on artemisinin binding to BSA.     

A reinvestigation of chlortetracycline fluorescence: effect of pH,metal ions,and environment

The pH dependence of chlortetracycline fluorescence has been reinvestigated. Below pH 7.5, excitation at 400 nm results in an emission band at 530 nm. Above pH 7.5, in addition to the above band, a strong fluorescence is observed at 430 nm on excitation at 345 nm. pK_a values of 3.5 and 7.7 have been determined for the 530- and 430 nm bands, permitting their assignment to the ring A and ring BCD chromophores, respectively. Both bands are shown to be sensitive to divalent metal binding. Excitation energy transfer from the 430-bband to the 530-nm band is observed in metal complexes, with transfer efficiency being greater for the Mg²⁺ chelate than the Ca²⁺ chelate. The effect of solvent and lipid on band intensities and transfer efficiencies is reported. The interaction of chlortetracycline with BSA in the presence of Mg²⁺ leads to the observation of energy transfer from protein tryptophan residues to the 520-nm band of the complex via the intermediacy of the 430-nm band.     

What causes the depolarization of trypsin and trypsinogen fluorescence. Intramolecular mobility or non-radiative energy transfer?

Analysis of protein data bank information about the coordinates of definite atoms of protein macromolecules provides an opportunity to evaluate the efficiency of non-radiative resonance energy transfer within the model of fixed, strictly oriented oscillators. Such evaluations for trypsin and trypsinogen (and also for trypsin complex with a pancreatic inhibitor) show that the efficiency of energy transfer among each pair of tryptophan residues is negligibly small. It is also shown that a fairly effective energy transfer from tyrosine to tryptophan residues is possible. The conclusions have been made that the Tyr-Trp energy transfer is one of the factors determining the shape of the trypsin polarization spectrum, and that upon fluorescence excitation at the long-wavelength edge of the absorption spectrum, the depolarization of trypsin fluorescence in aqueous solution at ambient temperature - compared to model compounds (tryptophan, N-acetyltryptophan, glycyltryptophan, etc.), under the conditions of infinite viscosity - is due to the Brownian rotational motion of the macromolecules as a whole as well as the intramolecular mobility. The differences in the level and character of intramolecular mobility of trypsin and trypsinogen are discussed.     

Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY

Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.     

Protein damage and degradation by oxygen radicals. II. Modification of amino acids

Exposure of proteins to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical (.OH + O2-) causes gross structural modification. Such modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. In the present study, with the representative protein bovine serum albumin (BSA), we report that alterations to primary structure underlie such gross structural modifications. All amino acids in BSA were susceptible to modification by both .OH and .OH + O2- +O2), although tryptophan, tyrosine, histidine, and cysteine were particularly sensitive. At a radical/BSA molar ratio (nmol of radicals/nmol of BSA) of 10, we observed an average 9-10% destruction of amino acids; whereas at a ratio of 100, the average loss was 45%. Decreasing tryptophan fluorescence provided a useful index of amino acid loss and exhibited a clear dose dependence with .OH or with .OH + O2- (+O2). Linear production of the biphenol bityrosine was observed with .OH treatment. In contrast, .OH + O2- (+O2) induced only a limited bityrosine production rate which reached an early plateau. Studies with various chemical scavengers (t-butyl alcohol, isopropyl alcohol, mannitol, urate) and gasses (N2O, N2, O2, air) revealed that .OH is the primary radical responsible for all amino acid modifications, but that O2- and O2 can further transform the products of .OH reactions. Thus, O2-/O2 can potentiate .OH-dependent destruction of many amino acids (e.g. tryptophan) while inhibiting production of bityrosine by reacting with tyrosyl (phenoxyl) radicals. No amino acid loss or bityrosine production occurred with exposure to O2- (+O2) alone. Amino acid modifications caused both by .OH alone and by .OH + O2- (+O2) progressively affected the overall electrical charge of BSA. In a pH range of 3.7-6.2, some 16 new isoelectric focusing bands were induced by .OH, and some eight new bands were induced by .OH + O2- (+O2). The alterations to primary structure observed provide the key to an understanding of the link between oxidative modification and increased proteolytic susceptibility.