William H. Sawyer

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua, New Guinea

SummaryMelanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.

Properties and the locations of a set of fluorescent probes sensitive to the fluidity gradient of the lipid bilayer.

The synthesis and properties of a set of four fluorescent probes (n-(9-anthroyloxy) fatty acids, n = 2, 6, 9, 12) sensitive to the fluidity gradient of the lipid bilayer are described. Fluorescent quenching experiments show that the probes locate at a graded series of depths in the bilayer. A fifth probe, methyl-9-anthroate, locates near the bilayer centre. As an example of their application, the probes are used to study the phase transitions of dipalmitoyl phosphatidyl-choline. Changes in the rotational relaxation times of the probes across the transitions are more pronounced at the centre of the bilayer than at the surface.

Spectral properties of the prototropic forms of fluorescein in aqueous solution

The commonly used fluorescent probe, fluorescein, can exist in seven prototropic forms. We have used global analysis procedures to reanalyze the absorption data of Diehl and Horchak-Morris (Talanta34, 739–741, 1987) in terms of five alternative ionization models. We identify the forms of fluorescein present in aqueous solution and the pKa of each ionisation transition. The pKa values of the neutral xanthene, carboxylic acid, and cationic xanthene groups are 6.3, 3.1–3.4, and 3.1–3.4, respectively, and the pKa value of lactonization is 2.4. As a consequence, the neutral form of fluorescein is a mixture of the lactone (70%), zwitterionic (15%), and quinoid (15%) forms. A knowledge of the forms present in solution permits the characterization of their spectral properties. It is shown that the quinoid and monoanion forms have similar absorption spectra, while the zwitterion spectrum is similar to that of the cation but blue-shifted by 3 nm. The emission spectra of the monoanion and quinoid forms are also identified and shown to be similar but not identical. A model for the excited-state reactions of fluorescein is presented.

The molecular weight and stability of concanavalin A

Abstract The stability of concanavalin A is investigated in a wide range of environments by optical rotation, sedimentation and turbidity measurements. In the range of pH 4.5–5.6 ( I = 0.1), a single species of molecular weight 53 000 exists in solution. Below pH 4.5 this molecular weight is maintained, but a time-dependent change in protein conformation occurs. Above pH 5.6 the protein dimerizes, the extent of the reaction increasing with increasing pH. At pH 7 and above ( I = 0.1), the dimerization is accompanied by a time-dependent development of turbidity, which is essentially eliminated at pH 7 by either an increase of ionic strength (to values equal to or greater than 0.3) or by the addition of glucose. Nevertheless, even at I = 0.3 (pH 7) comparable proportions of monomer and dimer coexist as a non-equilibrium mixture. The results are discussed in relation to previous conflicting reports on the molecular weight of the protein and in terms of environments suitable for carbohydrate binding studies.

[12] Fluorescence anisotropy applied to biomolecular interactions

Publisher Summary This chapter focuses on the applications of polarization or anisotropy data. The appeal of fluorescence spectroscopy in the study of biomolecular systems lies in the characteristic time scale of the emission process, the sensitivity of the technique, and its ability to accommodate rapid and facile changes in the solvent milieu under conditions corresponding to thermodynamic equilibrium. On excitation of a fluorescent solution and observation of the emission at right angles both to the direction of propagation of the exciting light and to the direction of the electric vector, the polarization of the emission is defined in this chapter. The optical densities of the solution may be unavoidably high (even allowing for smaller cuvette or front-face geometries), and the requisite inner filter corrections may lower confidence in the binding data. Polarization/anisotropy measurements—such as lifetime measurements—are intensive quantities and as such are not subject to first-order inner filter corrections.

The ring-infected erythrocyte surface antigen of Plasmodium falciparum associates with spectrin in the erythrocyte membrane

The malaria parasite Plasmodium falciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P. falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton.

Spectral Properties of Fluorescein in Solvent‐Water Mixtures: Applications as a Probe of Hydrogen Bonding Environments in Biological Systems

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 Martin (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 α and β 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 water–solvent 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.

Stoichiometry, Kinetic and Binding Analysis of the Interaction between Epidermal Growth Factor (EGF) and the Extracellular Domain of the EGF Receptor

Abstract The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF: 2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF: sEGFR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2–20 nM and the other with a KD of 400–550 nM.

Depth-dependent fluorescent quenching of a tryptophan residue located at defined positions on a rigid 21-peptide helix in liposomes

Five lipophilic 21-peptide analogs of the potential-dependent pore-former, alamethicin, were synthesized bearing tryptophan residues at the position 1, 6, 11, 16 and 21 on a long, conformationally rigid, alpha-helix. The alpha-helical conformation was induced and stabilized using the sequential oligomers (Ala-Aib-Ala-Aib-Ala)n as analyzed by CD and NMR. The partitioning of the N-t-butoxycarbonyl 21-peptide methyl esters and the N-terminally deprotected alpha-helices was followed by fluorescence enhancement in phospholipid bilayer vesicles. Quenching experiments were performed by titrating with n-doxyl stearic acids bearing the nitroxide label at positions 5, 7, 10, 12 and 16. This well-defined system revealed that the N- and C-terminal tryptophan residues become situated in the hydrophilic region. Tryptophan at position 11 was found in the lipophilic core, whereas the tryptophan at positions 6 and 16 were localized at intermediate depths of the lipid membrane. Therefore, the helices span the lipid bilayer with their long axis normal to the membrane surface.

Thermodenaturation of bovine β-lactoglobulin: Kinetics and the introduction of β-structure

The thermal denaturation of the A and B variants of bovine β-lactoglobulin has been examined using optical rotatory dispersion and sedimentation velocity techniques. An equation is derived which describes the time dependence of optical rotation in terms of parameters relevant to a system involving two consecutive first order reactions. Analysis of simulated and experimental curves according to the procedure of Guggenheim (Phil. Mag., 2 (1926) 538) suggests that two consecutive first order unfolding reactions dominate the early stages of the denaturation. Optical rotatory dispersion and circular dichroism analysis in the far ultraviolet region of the spectrum shows that there is a significant increase in the proportion of β-structure in the molecule on thermodenaturation. In addition, thermodenaturation causes an aggregation of the protein, the extent of which depends on both the time and temperature of heat treatment.