Optimal design of experiments for the estimation of precise hyperbolic kinetic and binding parameters.
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Enzymes↗
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Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.
8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using ultraviolet/visible spectroscopy, and the data were consistent with the kinetic data (K = 4.2 microm). Among the other compounds tested, two potential transition state analogs, 4-carboxybutyl(1-amino-1-carboxyethyl)phosphonate (4) and 2-amino-3-hydroxy-2-methylnonadioic acid (5) were found to be competitive inhibitors with respect to l-alanine with Ki of 68 microm and 80 microm, respectively.
Acyl Coenzyme A↗
Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.
Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.
Abrin↗
Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system.
The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.
Blotting, Northern↗
Kinetics and mechanism of CO binding to cytochromes P-450LM2 and P-450LM4. Effect of phospholipid, nonionic detergent, and substrate binding.
The kinetics of carbon monoxide binding to purified, soluble cytochrome P-450LM2 from liver microsomes of phenobarbital-induced rabbits and P-450LM4 from liver microsomes of 5,6-benzoflavone-induced rabbits was determined in the presence of catalytically essential phospholipid, d-benzphetamine or nonionic detergent (0.1% Tween 80, 0.1% Triton N-101, or 0.3% n-octylglucoside). The kinetics under these conditions was consistent with a two-step binding mechanism consisting of bimolecular association followed by a unimolecular rearrangement step. However, when d-benzphetamine was added to P-450LM2 in the presence of sonicated dilauroylglyceryl-3-phosphorylcholine (0.15 mM) or any of the above detergents, CO binding followed simple second order kinetics with a bimolecular rate constant of approximately 3 x 10 M-1 s-1 at 20 degrees C. P-450LM4-CO binding kinetics was not affected by concurrent addition of phospholipid and d-benzphetamine. These results imply that simultaneous binding of amphiphile and substrate induces a conformation of reduced P-450LM2 which is different from that found when either substance is bound alone.
Animals↗
A conserved Val to Ile switch near the heme pocket of animal and bacterial nitric-oxide synthases helps determine their distinct catalytic profiles.
Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.
Amino Acid Substitution↗
The kinetics of binding of oxygen and carbon monoxide to Gastrophilus haemoglobin.
The dimeric haemoglobin in the tracheal cells of the Gastrophilus larva was extracted and purified, and the spectral properties of its oxy- and carbon monoxide adducts are recorded. In dilute solutions the kinetic parameters of binding of oxygen and carbon monoxide were determined. In solutions between 0.1 and 50mum for oxygen k(on) is 1x10(7)m(-1).s(-1) and k(off) is 1s(-1); for carbon monoxide l(on) is 6.5x10(5)m(-1).s(-1) and l(off) is 0.14s(-1). These values are in agreement with previous equilibrium results on oxygen binding and carbon monoxide/oxygen partition. These results are discussed and compared with the known values for other monomeric protohaem proteins.
Animals↗
Species differences in PAF receptor binding in the lungs between hamster and guinea pig.
Platelet-activating factor (PAF) receptor in normal Golden Syrian hamster lung was characterized using radioligand binding studies and compared with guinea pig lung PAF receptor. [3H]WEB2086, a potent and specific PAF antagonist, was used as a radioligand for equilibrium binding, kinetic studies, competitive binding in receptor preparation (0-110000 g fraction of lung homogenate) from hamster and guinea pig lungs. Binding of [3H]WEB 2086 to the receptor preparation was saturable, reversible and specific in both hamster and guinea pig lungs. Scatchard plot analysis of equilibrium binding data indicates a single binding site in hamster lung with the equilibrium dissociation constant (KD) of 66.1 +/- 36.7 nM (n = 4) and maximal binding (Bmax) of 135.4 +/- 63.1 fmol/mg, but two binding sites in guinea pig lung with a high affinity site (KD = 1.7 +/- 0.6 nM; Bmax = 48.6 +/- 2.6 fmol/mg) and a low affinity site (KD = 83.8 +/- 32 nM; Bmax = 480.8 +/- 158 fmol/mg). The heterogeneity of [3H]WEB2086 binding to guinea pig lung but not to hamster lung was also confirmed by dissociation kinetic studies, in which biphasic dissociation kinetic was shown in guinea pig and monophasic kinetic in hamster lung. Although the specific [3H]WEB 2086 binding to lungs of both species was displaced by PAF-C18 and antagonists L659989 and CL184005 in a dose-dependent manner and not by lyso-PAF (a biologically inactive form of PAF), the potencies of the competitive inhibition were significantly different between the two species. The relative potencies ranked WEB2086 approximately L659989 > PAF > CL184005 in hamster lung, whereas in guinea pig lung the potencies ranked PAF > WEB2086 approximately L659989 approximately CL184005. The present study demonstrates for the first time the existence of PAF receptor in the hamster lung and its binding characteristics different from guinea pig lung suggest the possible existence of different PAF receptor subtypes in hamster lung.
Animals↗
Adenosinetriphosphate sulfurylase from Penicillium chrysogenum: steady-state kinetics of the forward and reverse reactions, alternative substrate kinetics, and equilibrium binding studies.
The kinetics of the forward ATP sulfurylase-catalyzed reaction were examined using a new assay based on 32PPi released from [gamma-32P]MgATP in the presence of inorganic sulfate. Replots yielded Vmaxf = 6.6 units mg protein-1, KmA = 0.13 mM, Kia = 0.33 mM, and KmB = 0.55 mM, where A = MgATP and B = SO2-4. Thiosulfate, a dead-end inhibitor of the reaction, was competitive with sulfate and noncompetitive with respect to MgATP. The ratio kcat/KmA was determined for several alternative inorganic substrates, B, where A = MgATP and B = SO2-4, SeO2-4, MoO2-4, WO2-4, or CrO2-4. For SO2-4 and SeO2-4, the ratio was 5-6.5 X 10(4) M-1 S-1; for the others, the ratio was 5.8-7.3 X 10(5) M-1 S-1. The results support a random addition of MgATP and inorganic substrate. The kinetics of the reverse reaction were examined using a new assay based on 35SO2-4 release from [35S]APS (adenosine 5'-phosphosulfate) in the presence of MgPPi. Reciprocal plots were linear, intersecting below the horizontal axis. Replots yielded Vmaxr = 50 units mg protein-1, KmQ = 0.3 microM, Kiq = 0.04 microM, and KmP = 4 microM, where Q = APS and P = PPi (total of all species). MgATP and SO2-4 were both competitive with APS and noncompetitive with respect to MgPPi. Taken together with earlier results suggesting that APS is competitive with both MgATP and SO2-4 and that MgPPi is noncompetitive with respect to both substrates, the qualitative results point to a random A-B, ordered P-Q kinetic mechanism. The Scatchard plot for [35S]APS binding was curved, indicating either negative cooperativity or more than a single class of sites. [gamma-32P]MgATP displayed half-site saturation in the presence of saturating FSO-3.
Adenosine Triphosphate↗
Kinetic and equilibrium studies of the ligand binding reactions of eight electrophoretic components of sperm whale ferrimyoglobin.
The reactions of eight electrophoretic components of sperm whale ferrimyoglobin with fluoride, azide, and cyanide have been studied. There do not appear to be significant differences in rate constants or equilibrium constants among the various components. We conclude that at pH 7.0 in 0.05 M potassium phosphate these ligand binding kinetics and equilibria are insensitive to the net charge on the protein. The variation of the azide equilibrium constant with ionic strength from mu 0.01 M to 0.11 M is not in accord with the predictions of the Debye-Hückel theory. On the other hand, azide association kinetic and equilibrium constants are respectively six- and threefold greater for beef ferrimyoglobin than for the isoelectric whale ferrimyoglobin (band V). An examination of the data for whale, horse, and beef myoglobins reveals that large differences in azide (but not CO) association rate constants are associated with amino acid substitutions at residues 45 and 99 in the heme cavity.
Animals↗
Estradiol and progesterone alter ethanol-induced effects on mu-opioid receptors in specific brain regions of ovariectomized rats.
This study was conducted to evaluate the effects of estradiol and progesterone on ethanol-induced alterations of mu-opioid receptor binding kinetics in specific brain regions. Female ovariectomized rats were injected with ethanol (3 g/kg, i.p.), estradiol (50 microg/kg, s.c.) and/or progesterone (5 mg/kg, s.c.), or ethanol plus estradiol and/or progesterone daily for 7 days. Control animals received saline and olive oil. Brains were immediately removed and the cortex, hippocampus, hypothalamus, and midbrain were dissected and assayed for mu-opioid receptor binding kinetics. In the hypothalamus, ethanol alone and in combination with estradiol and/or progesterone significantly decreased Bmax. Ethanol alone also decreased Bmax in the midbrain and cortex. When administered with estradiol only, ethanol increased Bmax and Kd in the hippocampus. The administration of estradiol alone and progesterone alone decreased Bmax in the hypothalamus, while not affecting Bmax in any of the other brain regions. However, when estradiol and progesterone were combined, Bmax, as well as Kd, increased in the cortex. Progesterone alone and in combination with estradiol also increased Kd in the midbrain. In addition, Kd significantly increased following administration of ethanol in combination with either of the hormones, or both, in the midbrain and cortex. These results clearly indicate that the female hormones modulate the effects of ethanol on binding kinetics of mu-opioid receptors in specific brain regions. The present findings may in part explain sex differences in alcohol effects.
Animals↗
Validation of the hemizona assay (HZA) in a monkey model. II. Kinetics of binding and influence of cryopreserved-thawed spermatozoa.
We compared fresh and frozen-thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 x 10(4) motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen-thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.
Animals↗
Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation.
Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.
Adrenal Cortex↗
Spectroscopic analysis of the equilibrium and kinetic DNA binding properties of several actinomycin analogs.
Experiments are described that measure DNA dissociation kinetics and thermal denaturation temperatures for a series of actinomycin analogs containing, in the 3' amino acid position, pipecolic acid, proline or azetidine-2-carboxylic acid. Also included are studies on actinomycin C3. Analysis of the temperature dependence of the slowest rate constant for DNA dissociation shows that both the enthalpy and entropy of ativation increase as the ring size of the 3' amino acid decreases from six to five to four. All compounds increase the DNA melting temperature to the same extent except for the analog containing pipecolic acid, which shows a smaller effect. These results are discussed in terms of a possible role for conformational changes in the actinomycin pentapeptide lactone rings in determining the slow DNA dissociations rates for this class of intercalators. It is suggested that cis-trans isomerization of proline may be important in this regard.
Binding Sites↗
Assembly of human hemoglobin. Studies with Escherichia coli-expressed alpha-globin.
The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native beta-chains. The functional and structural properties of the expressed alpha-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl- and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the alpha1beta1 and alpha1beta2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed alpha-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the alpha-globin refolds and incorporates heme in the absence of the partner beta-chain. Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant alpha-chain assumes alternating conformations in the absence of beta-chain and indicate that the isolated alpha-chain exhibits a higher degree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed alpha-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.
Adult↗
A two-binding-site kinetic model for the ribonuclease-T1-catalysed transesterification of dinucleoside phosphate substrates.
Ribonucleases have been found to have subsites that confer large rate enhancements but do not contribute to substrate binding. In this study, we present a kinetic model that formally explains how subsite binding energy is converted into chemical activation energy. The proposed mechanism takes into account a primary specificity site and a subsite, both of which must be occupied for chemical turnover. An unstable reaction intermediate is formed upon binding of the polymeric substrate monomers at the corresponding subsites. The structure of this reaction intermediate resembles the transition state of the catalysed transphosphorylation reaction. Similar mechanisms may be used by other depolymerizing enzymes including nucleases, glycosidases, and proteases.
Binding Sites↗
Interaction of streptolysin-O with natural and artificial membranes.
1. Kinetic studies on the binding of 125I-streptolysin-O exhibited immediate fixation of activation toxin to natural and artificial membranes. Once fixed to the membrane no release of Streptolysin-O or Streptolysin-O-lipid-complexes has been observed. 2. In contrast to activated toxin (free SH-groups!), oxidized Streptolysin-O was shown to become also fixed to membranes, however, with different binding kinetics. The binding of oxidized amterial was clearly dependent on temperature and time. When the toxin was oxidized twice the amount of labelled material was bound as compared with the hemolytically active Streptolysin-O. This suggests that oxidized Streptolysin-O, too, possesses a "binding site" within the molecule, though free SH-groups were expected to be essential for toxin fixation at the membrane. It has been shown that oxidized (inactive) and reduced (active) Streptolysin-O forms stable "complexes" with liposomes in aqueous which could be separated by chromatography on Sepharose gels. 3. The binding of 125I-toxin to membranes and liposomes was specific since specific antisera against Streptolysin-O inhibited fixation of toxin completely. 4. Hydrolysis of phospholipids and release of lysophosphatides by Streptolysin O esterase (EC 2.1.2.2) has not been observed, thus providing no evidence for an enzymatic concept for membrane damage.
Antigen-Antibody Reactions↗
Kinetics of phloretin binding to phosphatidylcholine vesicle membranes.
The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.
Hydrogen-Ion Concentration↗