Effect of temperature and guanidine hydrochloride on ferrocytochrome<Emphasis Type="Italic"> c</Emphasis> at neutral pH

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G _D at 25 °C, is 59±5 kJ mol^–1 (DSC) and 65±6 kJ mol^–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Mechanistic aspects of chiral discrimination by surface‐immobilized α1‐acid glycoprotein

The immobilization of the globular protein α‐1‐acid glycoprotein (AGP) onto silica gel led to the commercial availability of an AGP column, which has a high enantioselectivity. The enantioselectivity of AGP columns has been demonstrated in numerous applications. Due to potential AGP structural changes occurring upon its immobilization, the interaction between particular pairs of enantiomers and the stationary phase is very difficult to assess. Therefore, in this paper we report a mechanistic study that probes the nature of these types of interactions. As model ligands, we employed two LTD₄ antagonists (L‐708, 738, MK0476, and their enantiomers) which have a rigid backbone consisting of a conjugated aromatic region and a side chain which is terminated with a carboxylic functional group. The difference between the two compounds is a two‐fluorine versus one‐chlorine substituent in the aromatic region of the molecule. To study the interaction between the two homologues and the AGP stationary phase, several parameters were varied, including pH, ionic strength, organic modifier, and temperature. van't Hoff plots were constructed and found to be nonlinear. They could, however, be divided into two linear regions, one from 0°C to ∼30°C, and another from 39°C to 50°C. The region at lower temperature implied that the separation was entropy‐dominated while the separation at higher temperature was enthalpically driven. The transition from the entropic to the enthalpically driven separation region suggested that bound AGP undergoes a conformational change. Fluorescence spectroscopy performed on the AGP stationary phase found evidence for a limited conformational transition at a similar temperature, consistent with this hypothesis. Chirality 11:224–232, 1999. © 1999 Wiley‐Liss, Inc.     

Quantitative analysis of DNA-porphyrin interactions

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated.     

The influence of temperature on the dynamics of protein precrystallization clusters,studied by photon correlation spectroscopy

Hen-egg white lysozyme was used for studying the influence of temperature on crystallization. The reaction was initiated at variable temperatures, covering the range between 5–50 °C, and was monitored with photon correlation spectroscopy. When aggregation was induced by addition of NaCl, the clusters formed exhibited diffusion limited aggregation behavior and crystals appeared in less than two days. In contrast, (NH₄)₂SO₄ induced aggregation took place mostly in the cross-over regime. In this case, solutions either remained transparent and void of crystals or formed gels within a few weeks. In both cases the kinetics could be dynamically scaled into master curves indicating that the precrystallization formed aggregates are fractals resulting from different collision processes.     

High‐throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.     

Root exudates from sunflower (Helianthus annuus L.) show a strong adsorption ability toward Cd(II)

Abstract

Cd(II) adsorption of root exudates from sunflower (Helianthus annuus L.) seedling was investigated by Cd ion-selective electrode, Fourier Transform Infrared spectroscopy, and fluorescence spectroscopy. Root exudates from Helianthus annuus L. had strong adsorption ability toward Cd(II). The adsorption process was pH-dependent and the maximum adsorption capacity, 150.8 mg g^?1, was observed at pH 7.0. Root exudates had pK _a1 at 4.7 for carboxyl and pK _a2 at 9.2 for phenolic, and amino groups. The aliphatic and aromatic (C?H) groups, amide III group, and the C (=O)?O and sulfonate groups were responsible for Cd(II) adsorption. The excitation emission matrix fluorescence spectroscopy showed protein-like substances participated in Cd adsorption and formed strong complexes, with conditional stability constants of 4.70 and 4.32, which is a little lower than that determined by potentiometric methods, 5.13. The strong Cd complexing ability of root exudates implies that root exudates may significantly affect mobility, toxicity, and phytoavailability of Cd. Cd binding of root exudates may be attributed to its interaction with the proteins, polysaccharides, and phenolic compounds in root exudates.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Lupane,friedelane, oleanane,and ursane triterpenes from the stem of Siphonodon celastrineus Griff

Twenty-one triterpenes consisting of a lupane derivative, two friedelanes, an oleanane derivative, and 17 ursane-type triterpenoids, together with three known triterpenes, three sterols, a fatty acid, a sesquiterpene alkaloid, and a glycerol derivative, were isolated from the stem of Siphonodon celastrineus. Their structures were characterized by various spectroscopic techniques, as well as comparison with literature data. Twenty-seven metabolites of these were evaluated for cytotoxic activity against six human cancer cell lines. The biosynthetic formation of a 1,4-dioxane bridge is also discussed.