A circular dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions.

A Circular Dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions fully confirms the occurence of a very weak Cotton effect around 435 nm in the Circular Dichroic spectrum of the reduced coenzymes and therefore the very faint transition (λ_max = 435 nm; ?_max ～ 1 M^?1 cm^?1) from which the Cotton effect originates.     

Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H₂O₂ at pH 8.0 emit chemiluminescence with quantum yield $\text{Ф ? 10}{}^{\mathrm{?9}}$ and absolute maximum intensity $\text{I}{}_{\mathrm{<mtext>max</mtext>}}\text{? 10}{}^5\text{hv/}\text{s}$ per cm³ (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s^?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H₂O₂, a weak fluorescence in the region 400–620 nm with λ_max = 465–510 nm (λ_exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λ_exc = 280 nm, λ_em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ_max = 530 nm (λ_exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ is not involved in either peroxidation or chemiluminescence. (6) The haem Fe³⁺ group and H₂O₂ appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.     

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

Protocatechuate 3,4-dioxygenase. Resonance Raman studies of the oxygenated intermediate.

Resonance Raman spectra of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have been investigated during the reaction of the enzyme with substrate and oxygen. It is found that the spectrum of the turned-over enzyme is indistinguishable from that of the resting enzyme in the absence of substrate, and is characterized by resonance-enhanced tyrosinate ring vibrational modes at 1263 and 1174 cm^?1. In the ternary ESO₂ complex, however, the tyrosinate vibrational modes are shifted to 1252 and 1165 cm^?1, respectively. There is no evidence for any dioxygen vibrations in the spectra of ESO₂ complexes prepared with ¹⁶O₂, ¹⁸O₂, and ¹⁶O¹⁸O in the region between 1300 and 200 cm^?1. The results of this resonance Raman study are interpreted to indicate that molecular oxygen is attached only to the substrate (but not iron) in the stable intermediate, and that the concomitant rearrangement at C4 of the substrate induces a substantial change in geometry of the tyrosine residues associated with the iron complex. Furthermore, the optical spectrum of the ESO₂ complex (λ_max = 520 nm) is dominated by tyrosinate → Fe(III) charge transfer and contains little or no peroxide → Fe(III) charge transfer. These results invalidate the previously advanced analogy in spectral properties between this enzyme and the respiratory protein, oxyhemerythrin.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

Oxygen binding to hemocyanin: a resonance Raman spectroscopic study

Oxygenation of hemocyanin gives rise to resonance Raman peaks at 742 and 282 cm^?1. The 742 cm^?1 peak which is in resonance with the 575 nm charge transfer band shifts to 704 cm^?1 when ¹⁸O₂ is substituted for ¹⁶O₂. Our results establish that the bound oxygen is in the form of peroxide (O₂^2?). The 282 cm^?1 peak which is in resonance with the 340 nm optical transition is insensitive to isotopic substitution, suggesting that the 282 cm^?1 peak corresponds to a vibration involving the magnetically-coupled Cu(II)··Cu(II) centers.     

Photoredox reactivity of copper(II)-3,5-diisopropylsalicylate induced by ligand-to-metal charge transfer excitation of the copper-phenolate chromophore

The electronic spectrum of Cu^II(dps)₂ in CH₃CN with dps=3,5-diisopropylsalicylate shows a ligand field absorption at λ_max=711 nm (ε=140 M⁻¹ cm⁻¹), and a phenolate to Cu(II) ligand-to-metal charge transfer (LMCT) band at λ_max=428 nm (ε=950). LMCT excitation of Cu^II(dps)₂ leads to the reduction of Cu(II) to Cu(I). Copper(II) disappears with φ=2.8×10⁻³ at λ_irr=436 nm.     

Arsenazo I and tetramethylmurexide as optical calcium indicators

Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength (K⁰), and molar extinction difference coefficients (Δ?_λ) at the wavelength λ of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1:1 complexes in aqueous solution. For TMX, K⁰ = 1.3 × 10^?3m and Δ?₄₈₀ = 1.5 × 10⁴m^?1 cm^?1; for ArsI, K⁰ = 5.8 × 10^?3m and Δ?₅₆₂ = 1.8 × 10⁴m^?1 cm^?1 at pH 7.0 and T = 293°K. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in 1:1, 1:2, and 2:1 forms.     

Thermodynamics of gelation of sickle cell deoxyhemoglobin.

This paper describes the thermodynamic behavior of gels of deoxyhemoglobin S. The solubility of the protein with respect to assembled hemoglobin fibers has been measured using a sedimentation technique. The solubility in 0.15 m-potassium phosphate buffer (pH 7.15) is found to decrease with increasing temperature, attain a minimum value of 0.16 g cm^?3 at 37 °C, and then increase at higher temperatures. The amount of polymer present at various hemoglobin concentrations and temperatures is presented as part of a phase diagram that may be useful for the calibration of other measurement techniques. The effects of varying pH and urea concentration upon the solubility have also been studied.The heat absorption accompanying gelation has been measured by scanning calorimetry. Using sedimentation data on the amount of polymer formed, molar enthalpy changes are obtained. There is a large negative heat capacity change of ? 197 cal deg. mol^?1 and ΔH = 0 near 37 °C. Calorimetric molar enthalpy changes are found to agree with those calculated from the temperature dependence of the solubility by the van't Hoff equation.Our previous two-phase, two-component thermodynamic model of gelation is extended to include the effects of solution non-ideality. A large contribution to the activity of the hemoglobin in the solution phase results from the geometric effect of excluded volume. Incorporating solution phase non-ideality permits the calculation of standard state thermodynamic quantities for the gelation process at 37 °C: ΔG^O ? ?3 k cal mol^?1, ΔH^O ~ 0, ΔS^O ~ 10 cal deg.^?1 mol^?1. The excluded volume effect is also capable of explaining observations of the minimum gelling concentrations of hemoglobin mixtures containing deoxyhemoglobin S without requiring copolymerization of the non-S hemoglobin.     

Hymenolepis diminuta: Chloride fluxes and membrane potentials associated with sodium-coupled glucose transport

Glucose transport by Hymenolepis diminuta was inhibited when Cl^? in the bathing medium was replaced with acetate (C₂H₃O₂^Post?), but was unaffected when Cl^? was replaced with SCN^?. The relative effectiveness of the anions to inhibit influx of 7.4 mM Cl^? in the presence of 1 mM glucose was SCN^? > Cl^? > C₂H₃O₂^Post?. Glucose stimulated the influxes of 120 mM Cl^? and SCN^?, but had little effect on 120 mM C₂H₃O₂^Post? influx. While the diffusion rates of the anions were C₂H₃O₂^Post? > SCN^? = Cl^?, the preference of the glucose transport system for the anions was SCN^? > Cl^? > C₂H₃O₂^Post?. Efflux of Cl^? was not affected by the rate of glucose influx. Finally, microelectrode recordings of worms anesthetized with 2 mM arecoline revealed a transmembrane potential (TMP) of ?45 ± 3.6 mV (inside negative). Three to four minutes after addition of glucose (5 mM) there was a progressive hyperpolarization of the TMP to ?58 mV. A revised model of the glucose transport system that is consistent with previous observations on this organism is proposed.     

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes,coenzyme analogues,substrates, and inhibitors

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M^?1 cm^?1, 407 nm (3500 M^?1 cm^?1), 505 nm (300 M^?1 cm^?1), 570 nm (?130 M^?1 cm^?1), and 680 nm (?80 M^?1 cm^?1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry.The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH > 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal ion which acts as a Lewis acid in both substrate coordination and catalysis.     

Horseradish peroxidase. XXXVII. Compound I formation from reconstituted enzyme lacking free carboxyl groups as heme side chains

Recombination of apo horseradish peroxidase with 2,4 dimethyldeutero hemin and its mono- and dimethyl esters was performed. The number of free carboxyl side chains in these three hemins is 2, 1 and 0 respectively. Despite such a difference, all of these three reconstituted enzymes can react with H₂O₂ to produce compound I. The second order rate constants for compound I formation are 1.3 × 10⁷ M^?1s^?1, 8.5 × 10⁶ M^?1s^?1 and 5.9 × 10⁶ M^?1s^?1. Therefore the propionate side chain of hemin has no direct role in compound I formation.     

Spectral Characteristics of Near-Infrared Surface Plasmon Resonance

Intrinsic properties of surface plasmons (SPs) excited with Kretschmann configuration were analyzed as a function of wavelength, including the propagation length, the penetration depth, the Goos–Hänchen (GH) shift, and the field enhancement. The calculated results indicate that there exists a critical thickness (t _cr) of the gold layer and that the maximum GH shift occurring exactly at the SP resonance wavelength (λ _R) rapidly varies from positive to negative with changing of the gold layer thickness from t?<?t _cr to t?>?t _cr. The maximum field enhancement happens not at λ _R but at a wavelength smaller than λ _R due to the phase retardation between the transmitted and reflected light. Simulations also reveal that a broadband collimated near-infrared beam can simultaneously excite two SPs with different responses to a refractive index (RI) change: the shorter-wavelength SP able to make a small redshift and the longer-wavelength SP capable of yielding a large blueshift. Only the shorter-wavelength SP was experimentally observed and its RI sensitivity was measured to increase from 3,539 nm/RIU at λ _R?=?707.6 nm to 57,143 nm/RIU at λ _R?=?1,398 nm. The SP at λ _R?=?1,013 nm moved to λ _R?=?1,029 nm in response to the saturation adsorption of bovine serum albumin, and the corresponding surface coverage was determined to be Γ?=?1.565 ng/mm² based on a quasilinear dependence of Γ on the resonance wavelength shift (?λ _R) deduced theoretically. Butyrylcholinesterase adsorption from a dilute solution of 10 nM protein in phosphate buffer solution leads to a redshift of ?λ _R?=?10 nm, corresponding to Γ?≈?0.97 ng/mm².     

Measurement of rate constants for quenching singlet oxygen with a Cypridina luciferin analog (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) and sodium azide

The rate constants for [¹O₂] [MCLA] and [¹O₂][NaN₃] were measured by quenching the near-infrared emission (¹Δ_g→³∑_g) in steady state with MCLA and NaN₃, respectively. ¹O₂ was constantly generated by energy transfer to O₂ from Ar laser-excited Rose Bengal. The Stern—Volmer plots yielded the second-order rate constants of 2.94 × 10⁹ M^?1 S^?1 and 3.83 × 10⁸ M^?1 S^?1 for quenching ¹O₂ with MCLA and NaN₃ in water at pH 5.4, respectively. The ¹O₂ + MCLA reaction emitted light with maximum at 465 nm at pD 4.5 identical to the O₂^? + MCLA reaction.     

Exchange rates of pyridine on ferro- and ferriprotoporphyrin (IX) dimethylester in chloroform.

Nuclear magnetic resonance line-widths data have been used to determine the rate of solvent exchange from the first coordination sphere of ferro-and ferriprotoporphyrin(IX) dimethylester (Fe-PPD) in pyridine/chloroform. The average values of kinetic parameters for pyridine (PY) exchange indicate an SN2 mechanism tor Fe(III)-PPD(ΔH^&;#; = 36 kJ · mol⁻¹ ; ΔS^&;#; = −53 J·mol⁻¹K⁻¹; T_M(298 K) = 0.07 msec) and an SNI mechanism for Fe(II)-PPD (ΔH^&;#; = 67 kJ·mol⁻¹; ΔS^&;#; = 42 J · mol⁻¹K⁻¹; T_M(298 K) = 0.06 msec). Parallel to the accelerated ligand exchange rate at rising temperatures a redistribution of the electrons causing a transition of the metal porphyrin from the low-spin state to the high-spin state is observed. Enthalpy and entropy of the thermodynamic equilibrium between low- and high-spin Fe-PPD have been determined from experimental values of the average magnetic moment. A mean lifetime of low-spin Fe(III)-PPD was estimated from line. widths changes (T_L→H(298 K)≈ 20 msec) and the corresponding activation parameters have been obtained (ΔH^&;#;_L→H(298 K) = 26 kJ · mol⁻¹; ΔS^&;#;_L→H(298K) = −125 J · mol⁻¹K⁻¹).     

Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λ_max?=?390?nm, ??=?32 880 M^?1 cm^?1 at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05?nmol mL^?1 was achieved for peroxidase.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Biophysical characterization of G protein ectodomain of group B human respiratory syncytial virus from E. coli

Human respiratory syncytial virus (hRSV) is an important pathogen of acute respiratory tract infection. The G protein of hRSV is a transmembrane glycoprotein that is a neutralizing antigen and is thus a vaccine candidate. In this study, synthetic codon optimized ectodomain G protein [G(ΔTM)] of BA genotype of group B hRSV was cloned, expressed, and characterized using biophysical techniques. The molar absorption coefficient and mean residue ellipticity at 222 nm ([θ]222) of G (ΔTM) was found to be 7950 M^?1 cm^?1 and ?19701.7 deg cm² dmol^-1 respectively. It was concluded that G(ΔTM) mainly consist of α-helix (74.9%) with some amount of β-sheet (4%). The protein was stable up to 85°C without any transition curve. However, heat-induced denaturation of G(ΔTM) resulted in total loss of β-sheet whereas not much change was observed in the α-helix part of the secondary structure. It was concluded that G(ΔTM) is an α-helical protein and it is highly stable at high temperature, but could be easily denatured using high concentrations of GdmCl/urea or acidic condition. This is the first investigation of cloning, expression, and characterization of G(ΔTM) of BA viruses from India. Structural characterization of G protein will assist in drug designing and vaccine development for hRSV.     

A spectral investigation of the copper(II) complex of the antitumor compound bleomycin

A thorough spectral investigation of the copper(II) complex of the antitumor compound, bleomycin, has been carried out in solution employing optical, difference optical, electron spin resonance, and circular dichroism techniques. The optical spectrum of a pH = 7 solution of the 1:1 complex between copper(II) and bleomycin is characterized by a broad weak band in the visible region (λ_max = 610 nm) that cannot be resolved and intense ultraviolet bands at 317 (? = 2800), 327 (shoulder), 250 (? = 4700), and 257 nm (shoulder). The circular dichroism spectrum in the visible region shows the broad and weak visible absorption band contains at least three components (558, 675, and 880 nm) that are likely to be “d-d” in origin. The electron spin resonance spectrum is characteristic of a tetragonal d⁹ copper(II) system showing no rhombic distoritions at X-band frequencies (g_x = g_y ± 0.002). The spin Hamiltonian parameters for the pH = 7.0 solution corrected for second order effects are A_∥ = 177 × 10^?4 cm^?1, A_⊥ ? 15 × 10^?4 cm^?1, g_∥ = 2.214, g_⊥ = 2.039. Most interesting was the observation of extra hyperfine splitting due to endogenous nitrogen coordination in a 30% glycerol glass (A^N = 12.0 × 10^?4 cm^?1). That pattern is best interpreted as a seven-line sequence associated with three liganded nitrogens. A dramatic change in all spectral properties occurs when the pH of the copper(II)-bleomycin complex is lowered to 2.5. All these data taken together suggest a CuN₃O coordination complex in solution. Details and justifications as well as a discussion of the limitations of the interpretations are presented.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.