Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

α-Bungarotoxin binding properties of a central nervous system nicotinic acetylcholine receptor

High-affinity, specific binding of radiolabeled α-bungarotoxin to particulate fractions derived from rat brain shows saturability (B_max ≈ 37fmol/mg, K_D^app = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (K_on = 5 · 10⁶ min^?1 · mol^?1; K_displacement = 1.9 · 10^?4 min^?1, τ_1/2 = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinergic agonists (but not antagonists) induces transformation of α-bungarotoxin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor.     

Cytochalasin B binding by human platelets

Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 10⁴ molecules/cell and dissociation constants (K_D) ranging from 2 × 10^?8 to 10^?6 M. Up to 85% of this saturable binding is displaced by 10^?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with K_D similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with K_D ≌ 4 × 10^?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with K_D ranging from 2.4 × 10^?8 to 1.5 × 10^?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a K_D of 1.5 × 10^?6M, while only 10–15% is CE-sensitive with a K_D of 2.4 ± 10^?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.     

Substrate and metal ion binding to carbamate kinase: NMR and EPR studies

Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn²⁺ as the paramagnetic probe. The enzyme binds Mn²⁺ weakly (K_D = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn²⁺ was observed with K_D = 44 ± 4 μm in the presence of ADP and K_D = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn²⁺-nucleotide and binary enzyme-Mn²⁺ complexes. Mn²⁺ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?_b = 9.3 ± 0.4. When enzyme-Mn²⁺ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn²⁺-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?_t^ADP = 14 ± 1 and ?_t^ATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K₁ = 2.5 ± 0.5 μm and K₁ = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with ¹³C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn²⁺. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase.     

Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K _D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K _D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K _D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K _D in the 10^?8 M range and (ii) a kinetically rapid, low-affinity interaction with K _D in the 10^?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K _D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

A calorimetric study of the binding of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate to thymidylate synthetase.

The thermodynamic parameters, ΔH′, ΔG′, and ΔS′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H⁺?(dUMP-TSase-H⁺). [1] The enthalpy, ΔH₁′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers (I = 0.10 m) show that ΔH₁′ = ?28 kJ mol^?1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of ΔG₁′ = ?30 kJ mol^?1 (K₁ = 1.7 × 10⁵ m^?1). From these parameters, ΔS₁′ was calculated to be +5 J mol^?1 degree^?1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength (I = 0.05 m) reveal a stronger binding, with ΔG₁′ = ?35 kJ mol^?1 (K₁ = 1.2 × 10⁶ m^?1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n_HH⁺?FdUMP₂ ? TSase ? (H⁺)_nH. [2] The enthalpy for this reaction, ΔH₂, was also measured calorimetrically and found to be ?30 kJ mol^?1 with n_H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [Biochemistry15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.     

Specific light uptake rates can enhance astaxanthin productivity in <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

Lumostatic operation was applied for efficient astaxanthin production in autotrophic Haematococcus lacustris cultures using 0.4-L bubble column photobioreactors. The lumostatic operation in this study was performed with three different specific light uptake rates (q _e) based on cell concentration, cell projection area, and fresh weight as one-, two- and three-dimensional characteristics values, respectively. The q _e value from the cell concentration (q _e1D) obtained was 13.5 × 10^?8 μE cell^?1 s^?1, and the maximum astaxanthin concentration was increased to 150 % compared to that of a control with constant light intensity. The other optimum q _e values by cell projection area (q _e2D) and fresh weight (q _e3D) were determined to be 195 μE m^?2 s^?1 and 10.5 μE g^?1 s^?1 for astaxanthin production, respectively. The maximum astaxanthin production from the lumostatic cultures using the parameters controlled by cell projection area (2D) and fresh weight (3D) also increased by 36 and 22 % over that of the controls, respectively. When comparing the optimal q _e values among the three different types, the lumostatic cultures using q _e based on fresh weight showed the highest astaxanthin productivity (22.8 mg L^?1 day^?1), which was a higher level than previously reported. The lumostatic operations reported here demonstrated that more efficient and effective astaxanthin production was obtained by H. lacustris than providing a constant light intensity, regardless of which parameter is used to calculate the specific light uptake rate.     

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [³H]quinuclidinyl benzylate ([³H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K _d), amount (B _max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K _d1 = 0.18 ± 0.08 nM, B _m1 = 221.2 ± 56.7 fmol/mg protein, n ₁ = 2, and for low-affinity receptors, K _d2 = 1.33 ± 0.11 nM, B _m2 = 748.7 ± 53.3 fmol/mg protein, n ₂ = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α₂-adrenoreceptors clonidine, the antagonist of α₂-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.     

Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate

Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{mm}$ Hg and K_eq = 5.4 × 10⁵M^?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K_eq = 5.2 × 10⁵M^?1 with k_on = 1.03 × 10⁵M^?1 · S^? and k_off = 0.198 S^?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.     

Solubilization and partial characterization of the tetrodotoxin binding component from nerve axons

The tetrodotoxin binding component from garfish olfactory nerve membranes has been solubilized using the nonionic detergent Triton X-100. Tetrodotoxin binds to the solubilized component with a dissociation constant K_D = 2.5 × 10^?9$\text{M}$ and under saturating conditions 1.95 × 10^?12 moles of tetrodotoxin are bound per milligram of solubilized protein. Upon solubilization the toxin binding component becomes much less stable towards heat, chemical modification and enzymatic degradation. Sucrose gradient velocity sedimentation yields an S value of 9.2 for the extracted binding component and from gel filtration data the binding component appears to be slightly larger than β-D-galactosidase.     

Specific binding sites for d-α-tocopherol on human erythrocytes

Since vitamin E deficiency is associated with increased susceptibility of erythrocytes to hemolysis, we investigated the presence of tocopherol binding sites in human red blood cells. Erythrocytes were found to have specific binding sites for d-α-[³H]tocopherol with properties of receptors. Kinetic studies of binding demonstrated two binding sites: one with high affinity (equilibrium association constant K_a = 2.6·10⁷ M^?1), low capacity (7600 sites/cell) and the second with low affinity (K_a = 1.24·10⁶ M^?1), high capacity (150000 sites/cell). These sites are at least partly protein in nature.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Zinc ion binding to human brain calcium binding proteins. Calmodulin and S100b protein

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn²⁺ binding sites, with K_D ranging from 8.10^?5M to 3.10^?4M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. K_D = 10^?7 ? 10^?6M. We suggest that S100b protein should no longer be considered only as a “calcium binding protein” but also as a “zinc binding protein”, and that Zn²⁺ ions are involved in the functions of the S100 proteins.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster

The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [³H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [³H]pon A which is saturable, apparently showing two specific components: a very high affinity component (K_D = 0.3 nM) and a high affinity component (K_D = 2 nM). The specific binding of [³H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (k_a) for [³H]pon A was determined to be 3 × 10⁷M^?1min^?1 at 21 °C, while the dissociation rate constant (k_d) for the specifically bound [³H]pon A was found to be 4.4 × 10^?3/min. Together, the kinetic rate constants yield a value of 0.15 nM for the K_D. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell-free extract also contains two high affinity specific binding components (k_D = 0.1 nM and K_D = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G-25, and are susceptible to protease digestion, heat, and treatment with N-ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at ?20°C.     

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN′

The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, k_on, suggested the involvement of the two ionizable groups of pK_a of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (E_A = 39.7 kJ · mol^?1, 9.50 kcal · mol^?1) and insensitivity of k_on to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, K_L, is estimated to be 1.2 × 10^?4m and the rate constant of the second step, k₊₂, to be 770 s^?1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.     

Thermodynamics of mucopolysaccharide–dye binding. II. Binding constant and cooperativity parameters of acridine orange–dermatan sulfate system

In the acridine orange–dermatan sulfate system, free and bound dye can be distinguished from each other spectroscopically. This permits the use of fluorometric methods to study the binding of acridine orange to the acid mucopolysaccharide dermatan sulfate. Experiments were conducted at 24°C in 10^?3 M citrate/phosphate buffer at pH = 7.0. The binding of the dye is highly cooperative, as evidenced by considerable interaction between adjacent bound dye molecules. Analysis of the data indicates that dermatan sulfate binds 2.3 ± 0.3 mol of acridine orange per dermatan sulfate uronic acid residue with a cooperative binding constant, K_q ranging from 4.9 to 6.0 × 10⁵ M^?1 which corresponds to a free energy of 7.74 ? ΔG° ? 7.86. The cooperativity parameter q apparently increases with increasing polymer-to-dye ratio.