共查询到20条相似文献,搜索用时 24 毫秒
1.
G Blauer 《Archives of biochemistry and biophysics》1986,251(1):315-322
Light absorption and circular dichroism (CD) were recorded at 26 to 27 degrees C in dilute aqueous solutions (10(-4) M) of ferriprotoporphyrin IX (FP) in the presence of (+)-quinidine. In contrast to the appearance of relatively small and positive CD bands between pH 7 and 10, two bands of opposite sign, having unusually large molar ellipticities of the order of 10(6) deg X cm2 X dmol-1 FP were observed in the Soret region near 400 nm at pH 11.0-11.5. This unique complex A was formed only slowly over periods of hours or days at 26 degrees C. By lowering the pH of Complex A below 10, under certain conditions, an "enantiometric" mirror-image CD spectrum, with all bands having opposite sign to Complex A, was obtained in the range of 650 to 300 nm (Complex B), while the light-absorption spectra of Complexes A and B were similar. The formation of Complex A was inhibited at mole fractions of FP greater than 0.5. Also, this complex was not measurably formed at low salt concentrations. Ultracentrifugation measurements of the complex solutions indicated the presence of very high aggregates. Possible interpretations of the optical properties observed are based on interactions between FP molecules, which are assumed to be arrayed chirally within aggregates of FP with quinidine. A comparison between quinine and quinidine complexes of FP is presented. 相似文献
2.
Close coorelation of atomic absorption measurements for Ca(II) contents indicates that from pH 5.8-7.4 a twentyfold excess of EGTA1 removes but one of two Ca(II) from carp parvalbumin. Thus binding of the two Ca(II) appears to be noncooperative. The maximum in emission intensity observed at a nonintegral 1.4-1.7 equivs of added Tb(III) is shown to be due to quenching by excess Tb(III). The emission intensity at the maximum increased 40% upon dialysis to remove Tb(III) not bound in the CD or EF sites. Atomic absorption results show that both Ca(CD) and Ca(EF) of native parvalbumin are easily replaced by Tb(III). Emission of Tb(EF) is not quenched by Tb(CD), but by solution Tb(III) bound at a third site, perhaps the single water molecule bound to Tb(EF). Labeling of the single sulfhydryl group with a trifluoroacetonyl gorup yields a protein with ultraviolet circular dichroism, emission, and circularly polarized emission spectra closely similar to those of native parvalbumin. 相似文献
3.
Sequence-specific assignment of the 1H NMR spectrum of the 36 amino acid polypeptide porcine neuropeptide Y (pNPY) at pH 3.1 is reported. It was achieved by use of standard two-dimensional techniques and by a combination of the sequential and main-chain-directed assignment strategies. The secondary structure was derived from inspection of the nuclear Overhauser spectra, slow hydrogen-deuterium exchange effects, chemical shifts of main-chain HA resonances, and coupling constants. These studies indicate that the C-terminal segment (residues 11-36) folds into an amphiphilic alpha-helix; the N-terminal segment, containing three prolines in both cis and trans conformations, assumes no regular structure. CD studies of pNPY at pH 3.1 and 7.4 show an increase in ordered structure at neutral pH. The difference spectrum, however, is typical of an alpha-helix and suggests a stabilization of residues 11-36, possibly via Maxfield-Scheraga pair interactions involving side-chain residues. This is supported by a comparison of the one-dimensional 1H NMR spectra of pNPY at pH 3.1 and 7.4, where no remarkable differences are observed. 相似文献
4.
5.
The effects of phenylalanine, NaCl and pH on the conformation of chorismate mutase/prephenate dehydratase have been investigated, using measurements of far and near-ultraviolet circular dichroic spectra and ultraviolet difference spectra. At pH 8.2 in 20 mM Tris-Cl buffer the enzyme was found to contain 10-20% helix and 40-50% beta-structure. There was little or no change in these values on the addition of 1 mM phenylalanine (the allosteric effector) or 0.4 M NaCl or by decreasing the pH to 7.4. Both phenylalanine and NaCl caused significant changes in the conformation of the enzyme. The most prominent of these was the movement of a tryptophan residue into a more hydrophobic environment. There was also a slight perturbation of this tryptophan when the pH was decreased to 7.4. The conformational changes can explain sigmoidal kinetic behaviour observed previously [Gething et al. (1976) Eur. J. Biochem. 71, 317-325]. 相似文献
6.
Interaction between the plant derived polyphenolic type curcumin molecule having anticarcinogenic, antiinflammatory, and antioxidant activities, and human serum albumin was studied at different pH values by circular dichroism (CD) and electronic absorption spectroscopy. The weak, induced CD spectrum of curcumin-HSA complex measured at pH 7.4 in the visible spectral region shows striking changes upon alkalization; CD spectra collected between pH 7.7 and 9.3 exhibit characteristic, oppositely signed CD band pair according to the visible absorption band of HSA-bound curcumin. At 0.3 curcumin/HSA molar ratio, typical molar CD values are Delta epsilon (496.6nm)+40M(-1)cm(-1) and Delta epsilon (426.8nm)-40M(-1)cm(-1), respectively (pH 9.0, t=37 degrees C). The induced optical activity is attributed to a bent, right-handed chiral conformation of the HSA-bound curcumin molecule within which intramolecular exciton coupling occurs between the electric dipole transition moments of the dissymmetrically juxtaposed feruloyl chromophores. Deprotonation of phenolic OH group(s) of curcumin seems to be the reason leading to the conformational alteration of HSA-bound curcumin. 相似文献
7.
Kamiya M Kumaki Y Nitta K Matsumoto T Hikichi K Matsushima N 《Biochimica et biophysica acta》2005,1722(1):69-76
Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu(2+), circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein's sequences including 1, N(1)Y(2)G(3)H(4)G(5)G(6)G(7)N(8)Y(9)G(10)N(11), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu(2+) binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO(4) caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu(2+) binding unit. 相似文献
8.
The kinetics and spectra of the reactions of clozapine with compounds I and II of myeloperoxidase were investigated using both single- and sequential-mixing stopped-flow techniques, steady-state kinetics, and spectrophotometric measurements. The results show conclusively that both compounds I and II are reduced in one-electron reactions with clozapine. At pH 7.0 the rate constant for compound I reacting with clozapine is (1.5 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (4.8 +/- 0.1) x 10(4) M(-1) s(-1). The physiological pH of 7.4 was found to be optimal for the oxidation of clozapine by compound I. The rate constant for compound I reacting with ascorbate is (1.1 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (1.1 +/- 0.2) x 10(4) M(-1) s(-1), both obtained at pH 7.0. Experiments with both clozapine and ascorbate present showed that ascorbate acts both as a competitive inhibitor and free radical scavenger. 相似文献
9.
A Kumon M Kuba N Murakami S Yasuda T Takashima S Matsumura Y Suezaki 《European journal of biochemistry》1986,160(3):499-506
Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0-0.30 M) at various pH (7.4-8.6) were distinguished from each other by their permeability through a Sepharose 4B column. The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and, upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates. Light meromyosin, which corresponded to the C-terminal two-thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected. Truncated light meromyosin which had lost a small segment from either the C-terminal or N-terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH. It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH-dependent manner by the subfragment 2 segment, the N-terminal third of the rod. 相似文献
10.
The existence of a practical minimum pH for the dissolution of heat-induced whey gels in alkaline solutions has been studied using beta-lactoglobulin (betaLg) as a model protein. A sharp transition in solubility was observed between pH 11 and 12; this transition shifts to higher pHs for gels formed at higher temperatures and for longer gelling times. The breakdown reactions of heat-induced aggregates in alkali were monitored with size exclusion chromatography. The destruction of large aggregates was faster at higher pH and also showed a transition between pH 11 and 12. Using tryptophan fluorescence and near- and far-UV circular dichroism, this transition was assigned to the base-induced denaturation observed in solutions of aggregates (pK 11.53). It is suggested that the high protein repulsion caused by the large number of charges at pH > 11.5 drives the unfolding of the protein and the disruption of the intermolecular noncovalent bonds. Concentrated urea and GuHCl were found to be less effective than a pH 12 solution in destroying large aggregates. Aggregates formed for a long time (80 degrees C for 24 h) contained a larger number of intermolecular disulfide bonds that hinder the dissolution process. Gels formed at low temperatures (65 degrees C for 60 min), with fewer intermolecular noncovalent bonds, showed a similar solubility-pH profile to that observed for the base-induced denaturation of unheated beta-lactoglobulin (betaLg) (pK 10.63). 相似文献
11.
The pH dependence of the uv/visible and CD spectra of the 1:1 Ni(BSA) complex in aqueous solutions is interpreted in terms of a major square-planar form and an octahedral form. At pH 7.4, the two forms, respectively, account for ca. 70% and 30% of the total Ni(II). The two forms are in rapid equilibrium with each other and so both probably involve Ni(II) binding to the N-terminal region of the albumin protein. The kinetics of the equilibrium reaction of Ni(BSA) with His were studied at 37 degrees C in buffered media of pH 7.4 and 9.3. In line with predictions, the two Ni(BSA) forms show markedly different reactivities, with the square-planar form being the more thermodynamically stable and the less reactive. The octahedral form reacts with an observed zero-order dependence on His concentration while the square-planar form shows both zero-order and first-order dependence, the latter being the more dominant. The significance of the slow equilibrium rate at pH 7.4 to the possible physiological role of Ni-albumin in blood serum is discussed. 相似文献
12.
The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca-actin and Mg-actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this difference was absent in the case of Ca-F-actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg- and Ca-actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca-actin filaments. 相似文献
13.
Tobacco mosaic virus protein aggregates in solution: structural comparison of 20S aggregates with those near conditions for disk crystallization 总被引:2,自引:0,他引:2
Previous X-ray studies (2.8-A resolution) on the crystals of tobacco mosaic virus protein (TMVP) grown from solutions containing high salt have characterized the structure of the protein aggregate as a bilayered cylindrical disk formed by 34 identical subunits [Bloomer, A.C., Champness, J.N., Bricogne, G., Staden, R., & Klug, A. (1978) Nature (London) 276, 362-368]. Under low-salt conditions, 20S aggregates are in equilibrium with 4S species and involved in the efficient nucleation of TMV assembly in vitro [Butler, P.J.G. (1984) J. Gen. Virol. 65, 253-279]. We have investigated by sedimentation velocity and near-UV circular dichroism (CD) measurements the structure of 20S aggregates in low salt (I = 0.1 potassium phosphate at pH 7.0 and 20 degrees C) and the aggregates in high salt [0.2 M (NH4)2SO4 in I = 0.1 tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0 and 20 degrees C, close to the conditions under which TMVP crystallizes as disk aggregates]. At high salt, we observe structures (presumably stacks of disks) having s20,w values around 40, 45, and 50 S, but not the 20S species present in low-salt buffers. The near-UV CD spectrum of 20S aggregates has been obtained for the first time, using computer techniques, from the spectra of the 4S-20S equilibrium mixture and the 4S species. This spectrum of 20S aggregates differs dramatically from that of the stacks of disks examined at both high and low salt (into which the stacks can be returned by dialysis), indicating that the difference is not a solvent effect.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA
The effect of a series of aminooxy analogues of the biogenic polyamines spermidine and spermine on the conformation of calf thymus DNA is studied. These new molecules are isosteric and charge insufficient analogues that are suitable to study the roles of both charge distribution and structural requirements in the molecular physiology of the biogenic polyamines. They are also evidenced as useful tools to inhibit polyamine biosynthesis and cell growth. Circular dichroism (CD) spectra of solutions containing DNA and the aminooxy analogues at different concentrations (100-1000 microM) and different pH values, (5-7.5) are recorded. We use both sonicated and highly polymerized calf thymus DNA. The CD spectra of sonicated DNA showed the formation of Psi-DNA, a highly ordered aggregated structure similar to liquid crystals, in the presence of the aminooxy analogues. Aggregation induced by an aminooxy derivative of spermine is followed by DNA collapse when increasing the polyamine concentration. The features of Psi-DNA are not detected for highly polymerized DNA. Temperature melting measurements support a high degree of structural order of the aggregates. The CD experiments indicate that dications are unable to induce major changes on the macromolecular structure of DNA. In addition, aggregation is only observed when the trimethylene moiety is present between two adjacent positive charges. The observed differences among the CD spectra of DNA solutions with different aminooxy derivatives of spermidine indicate different roles for different amino groups of this biogenic polyamine when interacting with DNA. Our results support the idea that aminooxy analogues can be used as good models in studying the physiological functions of biogenic polyamines. 相似文献
15.
L M Gordon C C Curtain Y C Zhong A Kirkpatrick P W Mobley A J Waring 《Biochimica et biophysica acta》1992,1139(4):257-274
Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis. 相似文献
16.
The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate. 相似文献
17.
Kamiya M Kumaki Y Nitta K Ueno T Watanabe Y Yamada K Matsumoto T Hikichi K Matsushima N 《Biochemical and biophysical research communications》2004,314(3):908-915
Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers. 相似文献
18.
Circular dichroism (CD) spectra in the region of 210-250 nm allow visualization of intrachain phase transition of pH- and thermosensitive polyelectrolytes. Indeed, in 0.001 M citrate and acetate buffers, at pH 4.0-5.5, aqueous solutions of a poly(N-isopropylacrylamide-co-N-methacryloyl-L-leucine) (NIPAAm-MALEU) copolymer containing 90.9 mol% of NIPAAm residues exhibit a well-defined sigmoidal increase in the CD signal at 220 nm with increasing temperature. This phenomenon is suggestive of a highly cooperative transition which occurs at lower temperatures compared to that observed by cloud point measurements. The change in the CD signal is less sharp at higher pH, indicating varying cooperativity with pH. For pH 6.0 and higher, no such phenomena are observed. 相似文献
19.
We have constructed a strain that overproduces ribonuclease I of Escherichia coli and we have purified large quantities of the enzyme. Data from fluorescence, CD, and DSC measurements showed that it was a very stable protein. The conformation energy determined from urea and guanidine hydrochloride denaturation experiments was 11.5 kcal mol(-1) at pH 7.5. Thermal denaturation studies indicated that it had a T(m) of 64 degrees C at pH 4.0. RNase I belongs to the RNase T2/S-RNase group of endoribonucleases, but near the amino terminus it has an unusually long hydrophilic segment. Part of this was removed in the deletion construct, RNase I Delta(26-38). We have obtained crystals of both RNase I and of an enzyme-G2'p5'G complex in the P2(1) space group and oligonucleotide complexes with both wild type and mutant enzymes. The current study lays the groundwork for extensive investigation into the structure, function, and physical properties of this widely distributed group of ribonucleases. 相似文献
20.
The effects of pH on the binding of hematoporphyrin derivative (HpD) to monolayer and bilayer membranes have been studied. Absorption spectra of HpD bound to phosphatidylcholine (PC) liposomes indicate that there is greater binding of HpD to lipid films at acidic, tumoricidal pH conditions than at normal tissue pH. These results were found to correlate with surface pressure measurements of monolayer films formed under similar conditions. Surface potential measurements in conjunction with surface pressure measurements from monolayer films suggest that at low pH (i.e. less than or equal to 6.6) porphyrin intercalates within the lipid film to reach relatively high concentrations, while at higher pH (i.e. greater than or equal to 7.4) the porphyrin preferably adsorbs to the lipid film at the monolayer/water interface. 相似文献