Number Of ANS Binding Sites Research Articles

Insights into Hydrophobicity and the Chaperone-like Function of αA- and αB-crystallins: AN ISOTHERMAL TITRATION CALORIMETRIC STUDY

Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.

Determination of the inner surface potential of the erythrocyte membrane

The time dependent uptake of 1-anilino-8-naphthalene sulphonate (ANS) by bovine erythrocytes was studied. Experimental results were compared with theoretical predictions in order to determine the inner erythrocyte membrane surface potential. The value of the inner membrane surface potential under physiological conditions was estimated as −35 mV. In addition, the number of ANS binding sites on the outer surface of the single bovine erythrocyte membrane was determined as 2.2 × 10 6.

Interactions of triply phosphorylated human β-casein: Fluorescence spectroscopy and light-scattering studies of conformation and self-association

Structural changes of triply phosphorylated human β-casein, caused by shifts in temperature between 5 and 40 °C, were studied using intrinsic and extrinsic fluorescence, fluorescence polarization, turbidity, and light scattering measurements. Intrinsic fluorescence declined between 5 and 20 °C then rose between 25 and 40 °C, indicative of a shift of the tryptophan fluor toward a more nonpolar environment. The fluorescence of the extrinsic probe, 8-anilino-1-naphthalene-sulfonic acid (ANS), increased only slightly between 5 and 25 °C, and then more sharply between 25 and 40 °C, suggesting a change in conformation leading to a change in either the dissociation constant, K d , or the number of ANS binding sites, N. The presence of Ca 2+ ions did not significantly alter the pattern of changes of intrinsic and extrinsic fluorescence with changing temperature. For ANS binding, values of K d and N were calculated by two different procedures, each based upon different assumptions. The results point to increased exposure of hydrophobic surfaces with increased temperature, strongly supportive of conformational changes. Although more opportunity for hydrophobic interaction leads to increased protein-protein association, turbidity and light-scattering also suggest ion bridge formation between protein molecules. A comparison of the primary sequences of β-caseins from six species reveal residues that are common in all species examined and thus are pivotal in protein folding and conformation, intermolecular hydrophobic interactions and ion bridge formation with Ca 2+ and inorganic phosphate.

Estimation of the hydrophobicity modifications in meat proteins upon thermal treatment

A fluorescent probe method using 8-anilino-l-naphthalene sulfonic acid (ANS) and all-trans-retinol (RET) has been used to study the effects of heat treatments on the hydrophobicity of meat proteins. The number of ANS binding sites per unit protein increased from 0.75 for unheated samples to 2.12 for meat proteins heated at 100° C for 30 min. The number of RET binding sites increased from 0.13 to 0.46 with the same heat treatment. This is discussed in terms of % increase in aromatic (182.7%) and aliphatic (253.5%) hydrophobicity for meat proteins heated at 100° C for 30 min. On the other hand, the assay was able to discriminate between samples of meat proteins receiving heat treatments with 100° C intervals (between 40 and 100° C).

A study of carbachol-atropine interaction on intestinal smooth muscle vesicles, using a fluorescent probe.

Plasma membrane vesicles were prepared from guinea pig ileum longitudinal muscle. The vesicles were characterized by electron microscopy and analysis of lipid and protein content. They were shown to be free of gross contamination from actomyosin, sarcoplasmic reticulum, and mitochondria. 8-Anilino-1-naphthalene sulphonic acid (ANS) binding characteristics were similar to those found in other membranes. Both carbachol and atropine increased the fluorescence of ANS bound to this membrane, the maximum increase for atropine being greater than that for carbachol. Since neither drug effected the apparent affinity constant for the ANS-membrane interaction. It may be assumed that the increased fluorescence was due to an increase in the number of ANS binding sites. The carbachol-dependent increase in ANS fluorescence was blocked noncompetitively by atropine but not by tubocurarine or diphenhydramine. These latter two antagonists also increased ANS fluorescence but at much higher concentrations than either carbachol or atropine. Neither atropine nor carbachol increased ANS fluorescence on either erythrocyte ghosts or liposomes (prepared from a lipid extract of the muscle membrane).

The interaction of calcium and procaine with hepatocyte and hepatoma tissue culture cell plasma membranes studied by fluorescence spectroscopy

The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca 2+ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; K D = 120, μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca 2+ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in K D (40–50 μ m). Procaine was also shown to completely displace Ca 2+ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; K D = 74 μM) from the hepatocyte system. The addition of Ca +2 resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K D values of 15 μ m and 50 μ m. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca 2+. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.

Effects of a series of alcohols on the binding of a fluorescent dye to erythrocyte membranes

1. The effects of a series of aliphatic alcohols (methanol to octanol) on membrane proteins of erythrocytes were studied by monitoring the flueorescence of a dye (1-anilino-8-naphthalenesulfonic acid (ANS)) that adsorbs to erythrocyte ghost membranes. Low concentrations of all the alcohols reduced the ANS fluorescence of the membrane-ANS suspensions; lent to those which protect against hypotonic hemolysis on intact erythrocytes; higher concentrations markedly increased the fluorescence. Ethanol and methanol decreased ANS fluorescence at all concentrations. 2. Lytic concentrations of saponin did not increase ANS fluorescence and did not modify the membrane action of the alcohols. 3. None of these effects were observed in liposomes prepared from lipid extracts of the erythrocyte membrane. 4. Since the apparent dissociation constant for the ANS-membrane interaction was unchanged in the presence of the alcohols, it was assumed that the fluorescence changes anesthetic concentration of the alcohols alter the conformation of membrane proteins, as indicated by the decreased number of ANS binding sites.

Interaction of the fluorescent probe, 1-anilino-8-napthalene sulfonate, with the sulfate transport system of Ehrlich ascites tumor cells.

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculate the apparent dissociation constant of ANS-membrane complex (Kd = 6.53 times 10(-5) M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight). Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 times 10(-3) M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 times 10(-6) M) and (3) the transport system has a higher affinity for ANS than for sulfate. These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.

A study of the interaction of human spermatozoa membrane with ATP and cyclic-AMP

Summary Human spermatozoa suspensions produce a specific fluorescence spectrum which is significantly quenched when adenine nucleotides (AMP, ATP and cAMP) are added to the medium. These same nucleotides modify the pattern of interaction of the human spermatozoa membrane with a fluorescent probe (ANS). ATP induces a decrease in the polarity of the membrane, increases the number of ANS binding sites, and the quantum yield per site, and induces a blue shift of the membrane tryptophan intrinsic fluorescence. Cyclic-AMP is 2 × as active as ATP, but induces an increase in the polarity of the membrane. AMP is considerably less effective than the other two nucleotides.