Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Acrylamide”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,675 records · Page 93Linked to original sources
PubMed2005

Biomechanical properties of high-toughness double network hydrogels.

This study evaluated the wear property of four novel double-network (DN) hydrogels, which was composed of two kinds of hydrophilic polymers, using pin-on-flat wear testing. The gels involve PAMPS-PAAm gel which consists of poly(2-acrylamide-2-metyl-propane sulfonic acid) and polyacrylamide, PAMPS-PDAAAm gel which consists of poly(2-acrylamide-2-metyl-propane sulfonic acid) and poly(N,N'-dimetyl acrylamide), Cellulose/PDMAAm gel which consists of bacterial Cellulose and poly dimetyl-acrylamide, and Cellulose-Gelatin gel which consists of bacterial Cellulose and Gelatin. Ultra-high molecular weight polyethylene (UHMWPE) was used as a control of a clinically available material. Using a reciprocating apparatus, 10(6) cycles of friction between a flat specimen and ceramic pin were repeated in water under a contact pressure of 0.1 MPa. To determine the depth and the roughness of the concave lesion created by wear, a confocal laser microscope was used. As a result, the maximum wear depth of the PAMPS-PDMAAm gel (3.20 microm) was minimal in the five materials, while there was no significant difference compared to UHMWPE. There were significant differences between UHMWPE and one of the other three gels. The PAMPS-PAAm gel (9.50 microm), the Cellulose-PDMAAm gel (7.80 microm), and the Cellulose-Gelatin gel (1302.40 microm). This study demonstrated that the PAMPS-PDMAAm DN gel has an amazing wear property as a hydrogel that is comparable to the UHMWPE. In addition, the PAMPS-PAAm and Cellulose-PDMAAm DN gels are also resistant to wear to greater degrees than conventionally reported hydrogels. On the other hand, this study showed that the Cellulose-Gelatin DN gel was not resistant to wear.

Acrylic Resins↗
PubMed1998

Identification and quantitation of cysteine in proteins separated by gel electrophoresis.

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 37 degrees C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

Alkylation↗
PubMed2003

Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin.

Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.

Fluorescence↗
PubMed1993

Relationship between annexin V tryptophan exposure, calcium, and phospholipid binding.

Annexin V is a Ca(2+)-dependent phospholipid-binding protein that may have one or more membrane-related functions, including inhibition of blood coagulation. The fluorescence of the single tryptophan of annexin V was used to monitor Ca2+ and/or phospholipid binding in terms of emission wavelength, emission intensity, and susceptibility to acrylamide quenching. In the absence of phospholipid, Ca2+ titration showed a strong red shift of the wavelength of maximal emission to approximately 345 nm, where a small increase in intensity occurred and was half maximal at approximately 3 mM Ca2+. The Stern-Volmer quenching constant due to acrylamide was only 5.2 M-1 for annexin V alone, indicating limited aqueous exposure of the tryptophan, but 36 M-1 for a Ca(2+)-bound form, indicating full exposure. Binding to both negatively charged and zwitterionic phospholipids was accompanied by a very large increase in fluorescence emission intensity, a red shift, and low exposure to acrylamide. Calculated concentrations of Ca2+ near the surface of negatively charged vesicles suggested that the exposure of tryptophan by Ca2+ binding to annexin V was sufficient for binding of the protein to all vesicles tested, including those composed of oleic acid and phosphatidylcholine (PC), but not to those composed of pure PC. When binding to PC was monitored, the phenomena associated with phospholipid binding were observed separately, at higher Ca2+ concentration, from the red shift and the high exposure to acrylamide due to Ca2+ binding alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Annexin A5↗
PubMed1993

Nonpolar environment of tryptophans in erythrocyte water channel CHIP28 determined by fluorescence quenching.

CHIP28 is an abundant water-transporting protein in erythrocytes, kidney proximal tubule, and other fluid-transporting tissues. To determine the environment of the four tryptophans in CHIP28, fluorescence spectra and quenching by polar and nonpolar compounds were measured in stripped human erythrocyte membranes containing CHIP28 and in proteoliposomes reconstituted with purified CHIP28; comparative studies were performed in membranes containing MIP26. Functional analysis showed that CHIP28 water permeability was not affected by the polar quenchers iodide and acrylamide nor the nonpolar n-anthroyloxy fatty acids (n-AF). The emission maximum of CHIP28 tryptophan fluorescence was at 324 +/- 2 nm and did not change with the addition of quenchers; the maximum for MIP26 was at 335 +/- 5 nm. There was weak quenching of CHIP28 tryptophan fluorescence by the polar compounds iodide and acrylamide, with Stern-Volmer constants of 0.13 and 0.71 M-1, respectively. HgCl2 inhibited water permeability by > 95% at 50 microM and quenched CHIP28 fluorescence reversibly by up to 70% with a biphasic concentration dependence; quenching by HgCl2 and acrylamide was not additive. The membrane-associated n-AF probes quenched CHIP28 fluorescence by up to 80% with the greatest quenching for n = 2 and 12; addition of HgCl2 or acrylamide after n-AF caused a small, anthroyloxy-position-dependent increase in quenching which was greatest at n = 6. These studies indicate that the tryptophans in CHIP28 are in a nonpolar, membrane-associated environment. Mathematical modeling of the n-AF results suggests that the tryptophans are clustered near the surface and center of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Time-resolved fluorescence investigation of membrane cholesterol heterogeneity and exchange.

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was investigated as a cholesterol analogue to examine sterol domains in and spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Fluorescence lifetime, acrylamide quenching analyses, and intermembrane exchange kinetics were consistent with the presence of at least two sterol domains in POPC. Fluorescence lifetime was determined by phase and modulation fluorescence spectroscopy and analyzed by nonlinear least-squares as well as continuous distributional analyses. Both methods demonstrated that pure dehydroergosterol in POPC SUV had two lifetime components (C) and fractional intensities (F) near C1 = 0.851 ns (F1 0.96) and C2 = 2.668 ns (F2 0.004). In contrast to component C1, the center of lifetime distribution, fractional intensity, and peak width of dehydroergosterol lifetime component C2 was dependent on the polarity of the medium and vesicle curvature. The sterol domain corresponding to dehydroergosterol component C2 was preferentially quenched by acrylamide. Acrylamide quenching of dehydroergosterol fluorescence demonstrated that the two lifetime components of dehydroergosterol were not due to transbilayer sterol domains with different lifetimes. In a spontaneous exchange assay not requiring separation of donor and acceptor SUV, the lifetime component C2, but not C1, shifted to a shorter lifetime with altered distributional width. The kinetics of these lifetime and distributional width changes best fitted a two-exponential function, with a fast exchange rate constant K1 = 0.0325 min-1, t1/2 = 21.3 min, and a slow rate constant k2 = 0.00275 min-1, t1/2 = 261 min. The fast exchanging pool correlates with the longer lifetime component C2. These kinetics were confirmed both by dehydroergosterol exchange measured with fluorescence intensity and by [3H]cholesterol exchange. In summary, lifetime, distributional width, acrylamide quenching, and classical exchange assay data are consistent with the presence of at least two pools of sterol in POPC SUV.

Cholesterol↗
PubMed1976

Purification and characterization of the histones associated with the macronucleus of Tetrahymena.

Histone fractions have been isolated from the macronucleus of Tetrahymena pyriformis. Five classes of macronuclear histone were purified, using a combination of gel exclusion and ion-exchange chromatography, and were examined with respect to their solubility, electrophoretic, chromatographic, and chemical properties. Tetrahymena H4 is very similar to vertebrate H4, except that it exhibits a larger number of acetylated subfractions. In contrast, the other Tetrahymena histones vary more extensively from their calf thymus counterparts. Tetrahymena H3 resembles calf thymus H3 in its solubility properties and is the only macronuclear histone containing cysteine. However, it differs from vertebrate H3 in composition and has a faster electrophoretic mobility on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gel electrophoresis. Tetrahymena H3 also displays a level of acetylation higher than that reported for its vertebrate homologue. Approximately 45% of macronuclear H2B, which resembles calf thymus H2B in composition and solubility, is present in a (mono)acetylated form, not detected in vertebrate somatic H2B. H1, though similar to its calf thymus homologue in solubility, modification (by phosphorylation), and other properties, differs considerably in its content of basic, acidic, and hydrophobic amino acids. Tetrahymena does not contain a histone strictly homologous to H2A. Although macronuclear histone X resembles H2A in chromatographic and some solubility properties more like H2B than H2A. Fraction X is polymorphic in sodium dodecyl sulfate-acrylamide gels, migrating as two distinct molecular forms. While it is possible that one form is H2A-like and the other more H2B-like, the observation that both forms of X behave identically in solubility fractionation schemes makes this unlikely. Fraction X is both phosphorylated and acetylated which, in addition to two molecular forms, results in a characteristic heterogeneous pattern on urea-acrylamide gels. Characterization of the histone complement of this lower eucaryote should contribute to the understanding of the evolution and biological role of these basic proteins. Moreover, this description represents the most extensive analysis to date of the histones associated with an amitotic, genetically active nucleus. It will serve as a reference to which the histones of the morphologically distinct, mitotically dividing, and genetically inactive micronucleus of this organism can be compared.

Amino Acids↗
PubMed2003

Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid bilayers to determine hydrophobic alpha-helix locations within membranes.

A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission lambda(max) whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.

Amino Acid Sequence↗
PubMed1997

Comparison of DNA bending by Fos-Jun and phased A tracts by multifactorial phasing analysis.

Studies of DNA bending by Fos and Jun using different methods have yielded contradictory results. Whereas gel electrophoretic phasing analysis indicates that Fos and Jun bend DNA, results obtained through X-ray crystallography and ligase-catalyzed cyclization suggest that they do not. To test the assumptions underlying phasing analysis and to examine DNA bending by Fos and Jun, a multifactorial phasing analysis approach based on the distinct electrophoretic mobilities of DNA fragments of diverse shapes was developed. In this approach, the spacing between the bends, the length of sequences flanking the bends, and the acrylamide concentration in the gel are varied. Two closely spaced intrinsic bends with long flanking sequences had the same effect on electrophoretic mobility as a single bend corresponding to the sum of the bends when they were arranged in phase, and the difference between the bends when they were arranged out of phase. Based on the phase-dependent electrophoretic mobility variation of fragments containing intrinsic DNA bends of different magnitudes, three criteria for determination whether the phase-dependent mobility variation of protein-DNA complexes is caused by DNA bending were adopted. Complexes formed by the bZIP domains of Fos and Jun fulfilled each of these criteria. First, the electrophoretic mobility variation induced by Fos and Jun was proportional to that caused by an intrinsic bend over a broad range of acrylamide concentrations. Second, the mobility difference between fragments containing in phase and out of phase bends was reduced by an increase in the separation between the bends. The separation between the bends had the same effect on the electrophoretic mobility variation caused by Fos and Jun as well as intrinsic bends on long DNA fragments at low acrylamide concentrations. Third, on short DNA fragments analyzed at high acrylamide concentrations, two intrinsic bends separated by long spacers caused a larger decrease in electrophoretic mobility when they were out of phase than when they were in phase. This reversal of the phase dependence of the electrophoretic mobility variation was also observed for complexes formed by truncated Fos and Jun. Thus, the phase-dependent mobility variation of Fos and Jun complexes is due to DNA bending.

Animals↗
PubMedDate not supplied

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture.

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present. The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems. Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.

Acrylic Resins↗
PubMed1996

Structure-function relationship of xylanase: fluorimetric analysis of the tryptophan environment.

Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177]. The work described here aims at: (i) deducing the structure-function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern-Volmer quenching constants (K(SV)) of the solute quenchers. The analysis of K(SV) and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.

Actinomycetales↗
PubMed1979

Proteins of small subunits of rat liver ribosomes that interact with poly(U). II. Cross-links between poly(U) and ribosomal proteins in 40 S subunits induced by UV irradiation.

(1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.

Animals↗
PubMed2004

Direct test of the critical exponents at the sol-gel transition.

The steady state fluorescence technique was used to study the sol-gel transition for the solution-free radical cross-linking polymerization of acrylamide (AAm), with N,N'-methylenebis (acrylamide) as cross linker in the presence of ammonium persulfate as an initiator. Pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid, trisodium salt) is used as a fluoroprobe for monitoring the polymerization. Pyranine molecules start to bind to acrylamide polymer chains upon the initiation of the polymerization, thus the spectra of the bonded pyranines shift to the shorter wavelengths. Fluorescence spectra from the bonded pyranines allows one to monitor the sol-gel transition, without disturbing the system mechanically, and to test the universality of the sol-gel transition as a function of some kinetic parameters such as polymer concentration, cross-linker concentration, and temperature. Observations around the critical point show that there are three regimes for AAm concentration in which the exponents differ drastically. The gel fraction exponent beta and the weight average degree of polymerization exponent gamma agree best with the static percolation results for higher acrylamide concentrations above 1M, but they cross over from percolation to mean-field (Flory-Stockmayer) values when the AAm concentration is lower than 2M. For very low polymer concentrations, below which the system can not form the gel, the exponents differ considerably from both the percolation and the mean-field values.

Journal Article↗
PubMed1975

Synthesis of two lactogenic proteins by the mouse placenta in vitro.

The culture medium of mouse placental tissues was analyzed on acrylamide gel electrophoresis to localize lactogenic substances. Placental explants from 12- or 14-day-pregnant BALB/cHe mice were organ-cultured for 6 days in leucine-free Way-mouth's medium supplemented with 3H-leucine (10 mu/Ci/ml) and insulin (0.12I.U./ml). The medium was collected every other day and subjected to acrylamide gel electrophoresis. The electrophoretic pattern of radioactive leucine incorporated into proteins was examined on stained 7% gels. Five protein bands were associated with high radioactivity. The location of lactogenic activity on acrylamide gel was when investigated by the technique of organ culture of mouse mammary tissues. Placental explants from 12- to 14-day-pregnant BALB/cHe mice were organ-cultured in Waymouth's medium supplemented with insulin for 2 days. After electrophoresis, proteins were eluted by keeping the segment of acrylamide gel in phosphate buffer, dialyzed and dissolved in tissue culture medium 199 supplemented with insulin and cortisol. Mammary tissues from 8-day-pregnant KA2 mice were cultured for 3 days in the medium containing each eluate. Mammary glands always responded to eluted proteins from two positions of 7% gel, as judged in histological sections. The data suggest the presence of two lactogenic substances in the mouse placenta.

Animals↗
PubMed1984

Analysis of fluorescence quenching of ribosome-bound virginiamycin S.

The two virginiamycin components VM and VS interact synergistically with bacterial ribosomes in vitro and in vivo. Ribosome affinity for virginiamycin S increases about 10-fold upon incubation with virginiamycin M. This effect has been previously traced by spectrofluorimetric measurement based on the enhancement of virginiamycin S fluorescence upon its binding to the 50 S ribosomal subunit. In the present work the action of two virginiamycin S fluorescence quenchers, acrylamide and iodide, has been explored to gather information about the accessibility of ribosome-bound virginiamycin S and the variation of the accessibility level in the presence of virginiamycin M. Both acrylamide (non-ionized quencher) and iodide (ionized quencher) proved powerful quenchers of free virginiamycin S solutions. Since a comparable effect was obtained on 3- hydroxypicolinamide , the latter was indicated as the part of the molecule involved in the fluorescence effect. Fluorescence quenching by either agent was of the dynamic, i.e. collisional, type. Such an inference was based on the fact that these quenchers merely modified the emission spectrum (not the absorption spectrum), the bimolecular rate constant for the quenching process decreased linearly with the viscosity of the medium (static-type quenching is viscosity-independent), and that linear Stern-Volmer plots were obtained. The quenching ability of both agents underwent a sharp decrease in the presence of ribosomes; however, the Stern-Volmer equation was followed only in the case of acrylamide, whereas Lehrer 's relationship had to be applied in the case of iodide. When ribosomes were incubated with virginiamycin M, the fluorescence quenching ability of acrylamide and iodide was significantly reduced. Conclusions are as follows: a) the 3- hydroxypicolinyl residue of virginiamycin S is buried within an open well on the ribosome surface and is likely to be involved in the interaction with the binding site; b) the accessibility to the well is partly controlled by electrostatic forces; c) interaction of ribosomes with virginiamycin M entails a conformational change whereby the access to the well is reduced. These findings provide a molecular explanation for the previously observed increase of the association constant of virginiamycin S to ribosomes incubated with virginiamycin M which was found to be due to the decrease of the dissociation rate constant (the association rate constant remains practically the same).

Anti-Bacterial Agents↗
PubMed1980

Fluorescence quenching as an indicator for the exposure of tryptophyl residues in Streptomyces subtilisin inhibitor.

Fluorescence quenching by acrylamide and trichloroethanol of two equivalent tryptophyl residues in Streptomyces Subtilisin Inhibitor (SSI) was studied in several different conformations of the protein. Fluorescence intensity and polarization degree were simultaneously observed, and the relation between fluorescence intensity and lifetime was examined. The quenching profiles showed deviation from the Stern-Volmer plots. In the denatured form, a nonstoichiometric mechanism which differs from the static and dynamic one was found to be applicable to the interpretation of quenching by acrylamide. Quenching by trichloroethanol proceeds via the nonstoichiometric process for both the native (N) and the denatured (D) form. Each quenching constant obtained from SSI in N- or D-form for acrylamide or trichloroethanol quenching was compared with the others or with that from free tryptophan, as an indicator of the relative exposure of tryptophyl residues. The quenching constant of acrylamide for the urea denatured D-form is larger than that for the acid denatured D-form. The urea denatured state has more exposed tryptophyl residues. Temperature dependence of fluorescence quenching by Cs+ was also studied and a structural fluctuation was found around Trp-86 prior to the massive unfolding of the hydrophobic core containing the residue.

Bacterial Proteins↗
PubMed1997

X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.

Mant (2'(3')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching measurements. Here we present the crystal structure of mant-ADP and beryllium fluoride complexed with Dictyostelium discoideum myosin motor domain (S1dC) at 1.9 A resolution. We complement the structural approach with an investigation of the accessibility of the mant moiety to solvent using acrylamide quenching of fluorescence emission. In contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (Ksv=0.2 M-1), the fluorophore is relatively exposed when bound to Dictyostelium myosin motor domain (Ksv= 1.4 M-1). Differences between the Dictyostelium structure and that of vertebrate skeletal subfragment 1, in the region of the nucleotide binding pocket, are proposed as an explanation for the differences observed in the solvent accessibility of complexed mant-nucleotides. We conclude that protection of the mant group from acrylamide quenching does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes.

Acrylamide↗
PubMed1995

Fluorescence study of Escherichia coli cyclic AMP receptor protein.

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76-83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.

Acrylamide↗