Recognition of various biomolecules by the environment-sensitive spectral responses of hypocrellin B (original) (raw)
Related papers
Biophysical Chemistry, 1996
Evidence is presented that a compartmentalised protein exists in its native state only within a particular size of aqueous cavity. This behaviour is shown to exist in AOT reverse micelles using fluorescence quenching and circular dichroism (CD) studies of human serum albumin (HSA). In particular. far ultraviolet CD measurements show that a reduction in quencher accessibility to the fluorophore is consistent with the protein being nearest to its native conformation at a waterpool size of around X0 i diameter. We also show that the biexponential fluorescence decay of N-acetylk-tryptophanamide (NATA) in AOT reverse micelles arises from the probe being located in two distinct sites within the interfacial region. The more viscous of these two sites is located on the waterpool side of the interface and the other is located on the oil bide of the interface. Kcm em/.,; Human serum albumin (HSA): N-acetyl-btryptophanamide (NATA): Fluorescence quenching: Circular dichrolsm: Rcver\e micelIe\: .Acrowl OT (AOT) Corresponding author. Tel.: -34-141-552-4400 ext. 3377: fax. ~44-1-11-552-2891. 0301.462~/96/$15.(K) 0 1996 Elsevier Science B.V. All rights reserved P/I SO30 I -1622(96)000 16-6
Glycobiology, 2013
Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below 150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20-150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between 150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases.
Spectroscopic studies of the interaction between hypocrellin B and human serum albumin
Bioorganic & Medicinal Chemistry, 2006
Previous work has proved that hypocrellin B (HB) binds to human serum albumin (HSA) at a specific site instead of distributed randomly on the surface of a protein. In the current work, further investigation by using bilirubin as a site I marker indicates that HB can compete for the same site with bilirubin, suggesting that the HB binding site is located at sub-domain IIA (site I) of HSA. Moreover, bound to HSA, the HB fluorescence was found to be pH sensitive in physiological range (pH 6.0-8.0). The increasing of binding constant of HB to HSA in the pH range 6-8 also indicates that the N M B transition modulates the microenvironment changes of the binding site and influences considerably the binding between HB and HSA. Furthermore, picosecond time-resolved fluorescence spectra of HB-HSA complex in PBS indicate an additional short-lived component compared to that for HB in benzene, which may be assigned to the process of electron transfer from Trp-214 to HB.
Analytical Biochemistry, 2003
Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c 0. Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3 Â 10 6 M À1 for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.
Degree of exposure of membrane proteins determined by fluorescence quenching
Biochemistry, 1977
Quaternary salts of 4-picoline are shown to act as efficient quenchers of tryptophan fluorescence in membrane proteins. Fluorescence quenching determinations of sarcoplasmic reticulum membranes from rabbit muscle and of human erythrocyte membranes of different cholesterol to phospholipid mole ratios (C/PL) were carried out with quaternary picolinium salts in phosphate-buffered saline (PBS) and in 2,2,2-trifluorethanol (TFE)-water 2:1 (v/v), where the membrane is presumably completely disintegrated. In both solvent systems, the tryptophan quenching characteristics were typical of heterogenous systems and were analyzed as such. The ratio of the fraction of fluorescence intensity available for quenching with N-methylpicolinium perchlorate in PBS and in 2:1 TFE-water, (formula: see text), was taken as an index for the bulk degree of exposure of the membrane proteins to the aqueous surrounding. This value was found to increase with C/PL which is in line with the notion that increase in lipid microviscosity results in increase of exposure of membrane proteins. Analogous experiments were performed with N-hexyl- and N-benzylpicolinium, which can quench tryptophyl residues in both the aqueous phase and the hydrocarbon-water interface, and with N-hexadecylpicolinium which is dissolved in the membrane lipid layer and acts mostly as a static quencher of tryptophan at the hydrocarbon-water interface. With these quenchers the complementary indices (formula: see text) and (formula: see text), which represent the fraction of the protein mass located in the hydrocarbon-water interface and in the hydrocarbon layer, respectively, could be semiquantitatively resolved.
Binding Interaction of a Biological Photosensitizer with Serum Albumins: A Biophysical Study
Biomacromolecules, 2007
A photophysical study on the binding interaction of an efficient cancer cell photosensitizer, norharmane (NHM), with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), has been performed using a combination of steady-state and time-resolved fluorescence techniques. The emission profile undergoes a remarkable change upon addition of the proteins to the buffered aqueous solution of the photosensitizer. The polarity-dependent prototropic transformation is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. A marked increase in the fluorescence anisotropy in the proteinous environments indicates that the albumin proteins introduce motional restriction on the drug molecule. Light has been thrown on the denaturing action of urea on the probe-bound protein. The probable binding site of the drug in proteins has also been assessed from the combination of denaturation study, micropolarity measurement, and fluorescence resonance energy transfer (FRET) study. The present study suggests that the stability of serum albumins is enhanced upon binding with the drug.
Preparation and application of biologically active fluorescent hyaluronan oligosaccharides
Glycobiology, 2004
We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2aminobenzoic acid (2AA). Oligosaccharides from 4 to 40 residues in length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is facilitated by 2AA derivatization because it enhances signals in MALDI-TOF MS and improves FACE (fluorophore-assisted carbohydrate electrophoresis) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC)-labeled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatized oligosaccharides, as demonstrated by their ability to compete for polymeric HA binding to the G1domain of human recombinant versican (VG1). An electrophoretic mobility shift assay was used to study VG1 binding to labeled HA 8-, 10-, 20-, 30-, and 40-mers, and although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10 was estimated from densitometry analysis of the free oligosaccharide. Interactions involving HA 20-, 30-, and 40-mers (proposed to be multivalent) could also be studied using this protocol. Oligosaccharides labeled with 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.
Photochemistry and Photobiology, 1998
Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by M a r t i n (Chem. Phys. Lett. 35, 105-111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent-water mixtures. We show that the magnitude of the shift correlates with the OL and fi parameters of Kamlet and Taft (J. Am. Chem. SOC. 98, 377-383; 2886-2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent-water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through watersolvent hydrogen bonding and specific effects due to fluorescein-solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein-antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.
New Fluorescence Probes for Biomolecules
Molecules, 2015
Steady state fluorescence measurements have been used for the investigation of interaction between the bovine serum albumin (BSA) and fluorescence probes: 3-hydroxy-2,4bis[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ6), 3-hydroxy-2,4-bis[(3-methyl-1,3-benzothiazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ7) and 3-hydroxy-2,4-bis[(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)methyl]cyclobut-2-en-1-one (SQ8). The binding constant between bovine serum albumin and squarine dyes has been determined by using both the Benesi-Hildebrand and Stern-Volmer equations. The negative value of free energy change indicates the existence of a spontaneous complexation process of BSA with squarine dyes.
Fluorescence probing of albumin–surfactant interaction
Journal of Colloid and Interface Science, 2005
Protein–surfactant interactions were studied using bovine serum albumin (BSA) and the three surfactants sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and poly(oxyethylene)isooctyl phenyl ether (TX-100). The surfactants used belong to three broad classes, i.e., anionic, cationic, and nonionic. These categories of surfactants were used to elucidate the mechanism of surfactant binding to BSA, at pH 7. The interactions were followed fluorimetrically using both intrinsic tryptophan (Trp) fluorescence and the fluorescence of an external label. The aggregation behavior of the surfactants were studied in the presence of BSA. Steady-state fluorescence studies indicate that all three surfactants bind to BSA in a cooperative manner. This cooperative binding affects the binding of the external label to BSA. All these effects are also manifested in time-resolved fluorescence studies. The effects of surfactants on acrylamide quenching and energy transfer from Trp in BSA to bound dye provided valuable insights into the structural modification of BSA in presence of surfactants. The surfactant-induced conformational change of BSA was also confirmed by circular dichroism studies. However, among the three categories of surfactants, the nonionic surfactant shows the least interaction with BSA.