Comparison of binding interactions of lomefloxacin to serum albumin and serum transferrin by resonance light scattering and fluorescence quenching methods

The interaction between lomefloxacin (LMF) and two drug carrier proteins, human serum albumin (HSA) and serum transferrin (TF), were studied and compared by fluorescence quenching, resonance light scattering (RLS), and circular dichroism (CD) spectroscopic along with molecular modeling. Fluorescence data show that LMF has a stronger quenching effect on HSA than on TF. The binding constant and the number of binding sites were calculated as 6.00 x 10(5) M(-1) and 0.77 for HSA, and 4.66 x 10(5) M(-1) and 1.02, for TF, respectively. Also, these binding parameters were calculated by RLS data, as a novel approach and were compared to that obtained from fluorescence. The micro-environment changes of Trp residues were evident in both proteins. The quantitative analysis of the secondary structure in both proteins further confirmed the drug-induced conformational changes. The distance (r) between donors (HSA and TF) and acceptor (LMF) were obtained by fluorescence resonance energy transfer (FRET) theory and found to be 1.83 nm and 1.71 nm for HSA and TF respectively. Moreover, molecular modeling studies suggested the sub-domain IB in HSA and N-lobe in TF as the candidate place for the formation of the binding site of LMF on these proteins.     

Quasielastic light scattering,electrophoresis, and fluorescence studies of lysozyme-poly(2-acrylamido-methylpropylsulfate) complexes

Complexation between lysozyme and sodium poly(2-acrylamido-2-methylpropanesulfonate) (PAMPS) was studied by quasielastic light scattering, electrophoretic light scattering, fluorescence, and turbidimetry in electrolyte solution. These techniques show that complexation occurs at pH 9.6 in an ionic strength buffer of 0.25M NaCl + 25 mM Na₂B₄O₇. At constant lysozyme concentration (C_pro). The structure of the complex depends on the polymer concentration. At low polymer concentration (relative to C_pro), an intrapolymer complex is formed. This intrapolymer complex aggregates to an interpolymer species upon increase in polymer concentration. Complex formation was also studied by fluorescence using pyrene-labeled PAMPS (Py-PAMPS). Energy transfer from singlet-excited tryptophan residues in lysozyme to the pyrene label occurs when the complex is formed. Fluorescence and turbidity data indicate that lysozyme interacts with Py-PAMPS preferentially at pyrene sites, which leads to static quenching of tryptophan fluorescence via energy transfer to the pyrene label. © 1995 John Wiley & Sons, Inc.     

Comparison of fluorescence and resonance light scattering for highly sensitive microarray detection of bacterial pathogens

Microarrays have emerged as potential tools for bacterial detection and identification. Given their high parallelism, they might represent a breakthrough in current diagnostic methods, provided they can be coupled to simplified labeling protocols and detected with adequate sensitivities. We describe here a technique to directly label total bacterial RNA, thus avoiding the multiple steps and possible biases associated with enzymatic amplification (e.g. PCR). We have then compared the performances of one white-light source and two laser-based fluorescence scanners for detection reliability and sensitivity. Our study reveals that nanoparticle-labeled bacterial RNA generates reproducible resonance light scattering signals that are at least 50 times more intense than state-of-the-art confocal-based fluorescence signals.     

Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

Comparison of methods to assess water column primary production in wetlands

We compared a number of techniques to measure water column autotroph production in a shallow, hypereutrophic wetland: diurnal oxygen changes; light and dark bottle incubations; chlorophyll a concentrations; daily changes in pH; and algal volume. Productivity from diurnal oxygen changes calculated at 0.25, 0.5, 1, 2, 3, and 4 h intervals give similar estimates, but not 12 h intervals (dawn-dusk-dawn). Net productivity in bottles was slightly lower than that indicated by diurnal oxygen changes, and gross productivity in bottles was much lower than diurnal changes. Changes in pH correlated well with gross and net productivity measurements, as well as algal volume. Chlorophyll a is correlated with diurnal and bottle net productivity measurements and pH changes, but not algal volume. Since daily pH flux and oxygen changes provide a better overall assessment of ecosystem processes than standing crop or bottle incubations, they could be useful measurements for ecological engineers interested in assessing the ecosystem function.     

Neutron and light scattering studies of light-harvesting photosynthetic antenna complexes

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been employed in studying the structural information of various biological systems, particularly in systems without high-resolution structural information available. In this report, we briefly present some principles and biological applications of neutron scattering and DLS, compare the differences in information that can be obtained with small-angle X-ray scattering (SAXS), and then report recent studies of SANS and DLS, together with other biophysical approaches, for light-harvesting antenna complexes and reaction centers of purple and green phototrophic bacteria.     

Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates

Fiber size analysis, water retention value, and Simons’ stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons’ stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered.     

Quasielastic light scattering study of amyloid beta-protein fibril formation

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid beta-protein.     

Rapid light curves: A new fluorescence method to assess the state of the photosynthetic apparatus

Photosynthetic electron transport rates (ETR), calculated from chlorophyll fluorescence parameters, were compared in long term light and dark adapted as well as photoinhibited Pisum sativum leaves using a novel chlorophyll fluorescence method and a new instrument: rapid light curves (RLC) generated with the MINI-PAM. RLCs are plots of ETRs versus actinic irradiances applied for 10 s. Large changes in maximum electron transport rates (ETR_max) were observed when leaves were shifted from dark to moderate light, or from dark to photoinhibitory light and vice versa. Maximum ETRs were very low following long term dark adaptation, but increased to maximum levels within 8 to 15 minutes of illumination. It took more than 3 hours, however, to return irradiance-exposed leaves to the fully dark adapted state. Quenching analysis of RLCs revealed large q_E development in long-term dark adapted leaves accounting for the low ETRs. Leaves photoinhibited for 3 hours had similarly reduced ETRs. In these leaves, however, q_I was largely responsible for this reduction. Actinic irradiance exposures and saturating flashes affected leaves with different irradiance histories differently.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Identifying protein complexes based on density and modularity in protein-protein interaction network

Background

Identifying protein complexes is crucial to understanding principles of cellular organization and functional mechanisms. As many evidences have indicated that the subgraphs with high density or with high modularity in PPI network usually correspond to protein complexes, protein complexes detection methods based on PPI network focused on subgraph's density or its modularity in PPI network. However, dense subgraphs may have low modularity and subgraph with high modularity may have low density, which results that protein complexes may be subgraphs with low modularity or with low density in the PPI network. As the density-based methods are difficult to mine protein complexes with low density, and the modularity-based methods are difficult to mine protein complexes with low modularity, both two methods have limitation for identifying protein complexes with various density and modularity.

Results

To identify protein complexes with various density and modularity, including those have low density but high modularity and those have low modularity but high density, we define a novel subgraph's fitness, f _ρ , as f _ρ = (density) ^ρ *(modularity)^1-ρ, and propose a novel algorithm, named LF_PIN, to identify protein complexes by expanding seed edges to subgraphs with the local maximum fitness value. Experimental results of LF-PIN in S.cerevisiae show that compared with the results of fitness equal to density (ρ = 1) or equal to modularity (ρ = 0), the LF-PIN identifies known protein complexes more effectively when the fitness value is decided by both density and modularity (0<ρ<1). Compared with the results of seven competing protein complex detection methods (CMC, Core-Attachment, CPM, DPClus, HC-PIN, MCL, and NFC) in S.cerevisiae and E.coli, LF-PIN outperforms other seven methods in terms of matching with known complexes and functional enrichment. Moreover, LF-PIN has better performance in identifying protein complexes with low density or with low modularity.

Conclusions

By considering both the density and the modularity, LF-PIN outperforms other protein complexes detection methods that only consider density or modularity, especially in identifying known protein complexes with low density or low modularity.

     

Comparison of chlorophyll fluorescence with several other techniques used to assess chilling sensitivity in plants

Chilling tolerance in eight taxa was estimated from field and controlled environment observations and compared to tolerance estimated using a variety of techniques. The controlled environment observations suggested that the eight taxa could be ranked from chilling tolerant to chilling sensitive in the order: pea ( Pisum sativum L. cv. Greenfeast), Passiflora edulis Sims., Passiflora ligularis Juss. and Passiflora quadrangularis L., pepino ( Solanum muricatum L.) cultivars Comeraya, Suma, Miski, and mungbean [ Vigna radiata (L.) R. Wilcz]; although the relationship between the passionfruit as a group and the pepinos was unclear.
The change in the variable component of chlorophyll fluorescence (F_R) with time near 0°C in darkness was the most reliable method of ranking the plants according to relative chilling tolerance. It was also sufficiently sensitive to discriminate clearly between the closely related pepino cultivars. The Passiflora species and pea were not susceptible to short term reductions in F_R, with or without a 20 min exposure to light. Exposure to light at temperatures near 0°C emphasised the reductions in F_R in the more sensitive species. Pea was the only species capable of recovering a measurable F_R after a 60 min exposure to white light.
Measurement of electrolyte leakage and ethylene evolution from leaf disks after a low temperature treatment could allow discrimination between closely related varieties, but not between genera. Catalase activity was reduced in all taxa in response to low temperature. However, both initial catalase levels and relative response to dark treatment at 20°C enabled the ranking of plants within the closely related subgroups according to susceptibility to chill injury.
No one method clearly distinguished chilling sensitivity over all taxa.     

Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies

Size parameters of model antigen-antibody (Ag-Ab) complexes formed by the interaction of bovine serum albumin (BSA) and pairs of monoclonal anti-BSA antibodies (mAb) were evaluated by quasielastic light scattering, classical light scattering, and electron microscopy (EM). Mean values for the hydrodynamic radius, radius of gyration, and molecular weight were determined by light scattering. Detailed information regarding the molecular weight distribution and the presence of cycles or open chains was obtained with EM. Average molecular weights were calculated from the EM data, and the Porod-Kratky wormlike chain theory was used to model the conformational behavior of the Ag-mAb complexes. Ag-mAb complexes prepared from three different mAb pairs displayed significantly different properties as assessed by each of the techniques employed. Observations and size parameter calculations from EM photomicrographs were consistent with the results from light scattering. The differences observed between the mab pairs would not have been predicted by idealized thermodynamic models. These results suggest that the geometric constraints imposed by the individual epitope environment and/or the relative epitope location are important in determining the average size of complexes and the ratio of linear to cyclic complexes.     

Ribosome formation from subunits studied by stopped-flow and rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is highlighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix. Published: March 19, 2004.     

Comparison of subjective and objective methods to assess flax straw cultivars and fibre quality after dew-retting

Subjective and objective methods were compared for the assessment of the quality of unretted and retted straw, and the fibres obtained after dew-retting of four flax cultivars, Ariane, Evelin, Laura and Viola. Maturity of straw, degree of retting, ease of decortication after retting, divisibility, handle, cleanliness and colour were assessed as subjective factors and of these, degree of retting and handle appear to be useful for assessing quality. The samples were also analysed for physical and chemical differences using a range of objective methods. Straw quality could not be graded using height and maturity, although stem diameter and technical length could indicate quality. Changes in N, lipid and ash content, as a result of retting, were observed. The increase in the proportions of fibre fractions present in retted compared with unretted straw, due to microbial degradation of pectin, could be identified by thermal analysis. Analyses of the fibre samples to determine fineness, strength, cellulose, hemicellulose, lignin and minerals were carried out and significant differences in some of the parameters were identified. Cultivar Laura produced the best fibre. Fibre fineness, ash, lipid, caustic weight loss and fibre fractions as determined by thermal analysis were found to be important objective parameters for quality assessment. The assessment of fibre samples performed subjectively by an expert grader and by selected objective methods gave similar quality rankings.     

Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations

The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm.     

Comparison of X-ray fluorescence and optical fluorescence measured behind scattering layers as a basis for X-ray fluorescence endoscopy.

Recent developments in the technology of capillary-fiber optics suitable for X-rays in the range of approximately 4-10keV point to the possible realization of endoscopes applicable in X-ray fluorescence analysis. A general problem is the determination of scattering and absorption processes with consideration to tissue optics, X-ray scattering and X-ray absorption in a diagnostic partial volume. Therefore comparative investigations were performed in order to answer these questions. Zinc-oxide nanoparticles configured as single particles and ZnO clusters provided the fluorescence source in cell layers. An artificial scattering material was employed, which closely approximated the tissue optical conditions and the X-ray optical application conditions in possible diagnostic situations. As a result imaging of spatially resolved X-ray contrasts was better than adequate optical fluorescence imaging by approximately one magnitude. Hence a very important precondition for realizing X-ray fluorescence endoscopy is fulfilled.