Equilibrium-dialysis studies of the interaction between cholic acid and 100000g-supernatant preparations from the rat liver

1. The binding of cholic acid to 100000g supernatants from rat livers was investigated by equilibrium dialysis and gel-exculsion chromatography. 2. Supernatants were found to contain at least two classes of binding site for cholic acid. 3. These recptor molecules are probably proteins since incubation with proteolytic enzymes resulted in complete loss of cholic acid binding. 4. Supernatants were added to columns of Sephadex G-75, and two groups of fractions were shown to bind cholic acid. One of these contained low-affinity binding sites and the other contained both low- and high-affinity binding sites. 5. Feeding cholestyramine had no effect on cholic acid binding. 6. Increased cholic acid binding occurred after injection of phenobarbitone. There was an increase in the amount of the low-affinity component but no change in the high-affinity component. 7. The dissociation constants of the binding of cholic acid suggest that the binding proteins may be involved in bile acid transport.     

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS)

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.     

Glutamate receptor binding in juvenile and adult Rana pipiens CNS

Autoradiographic methods were used to map NMDA- and quisqualate-sensitive glutamate binding sites in the brain of mature and juvenile Rana pipiens frogs. NMDA- and quisqualate-sensitive sites were consistently co-localized in the CNS. The highest glutamate binding occurred in the telencephalon, hypothalamus, and cerebellum. Glutamate binding sites were also specifically localized in visual pathways, including the superficial neuropil of the optic tectum, consistent with glutamate being the retinal ganglion cell neurotransmitter. The distribution of glutamate binding sites in the brain of juvenile postmetamorphic frogs was similar to that in adults. In general, Quis binding increased about twofold in adults compared to juveniles, whereas NMDA binding did not show a comparable developmental increase. To test whether glutamate binding sites are located on retinal axon terminals or on tectal cell dendrites in the optic tectum, juvenile postmetamorphic frogs were enucleated unilaterally, and receptor binding was performed following 1, 3, 7, and 14 days survival. The denervated tectal neuropil showed a delayed decrease in NMDA- and quisqualate-sensitive binding, consistent with the receptors being located on postsynaptic tectal cell dendrites.     

Hyperalgesia and temporal summation of pain after heat injury in man

Proton NMR spectra of maltodextrin binding protein from Escherichia coli were used to monitor conformational changes that accompany ligand binding. Chemical shift changes associated with the binding of different maltodextrins to maltodextrin binding protein were studied using one-dimensional difference spectra. Line-shape analysis of an isolated upfield methyl resonance was used to measure the kinetics of maltose binding at several temperatures. Maltose and linear maltodextrins caused similar changes to the upfield protein spectrum with no detectable differences between alpha and beta sugar anomers. Binding of a cyclic ligand, beta-cyclodextrin, caused smaller chemical shift changes than binding of linear maltodextrins. Two maltodextrin derivatives were also studied. Both maltohexaitol and maltohexanoic acid gave one-dimensional difference spectra that were intermediate between those of linear maltodextrins and beta-cyclodextrin. The methyl resonances at -1 and -0.35 ppm were assigned to leucine 160 on the basis of homonuclear COSY and TOCSY experiments and theoretical chemical shift calculations using the X-ray crystal structure of maltodextrin binding protein.     

Glycosphingolipid binding specificities of Neisseria meningitidis and Haemophilus influenzae: detection, isolation, and characterization of a binding-active glycosphingolipid from human oropharyngeal epithelium

The glycosphingolipid binding specificities of Haemophilus influenzae and Neisseria meningitidis were investigated as to the binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby, similar binding profiles, for the binding of the two bacteria to lactosylceramide, isoglobotriaosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and sialylneolactohexaosylceramide, were obtained. On a closer view the binding preferences of the bacteria could be differentiated into three groups. The first specificity is recognition of lactosylceramide. The second specificity is binding to gangliotriaosylceramide and gangliotetraosylceramide, since conversion of the acetamido group of the N-acetylgalactosamine of gangliotriaosylceramide and gangliotetraosylceramide to an amine prevented the binding of the bacteria, and thus the binding to these two glycosphingolipids represents a separate specificity from lactosylceramide recognition. Preincubation of H. influenzae with neolactotetraose inhibited the binding to neolactotetraosylceramide, while the binding to lactosylceramide, gangliotetraosylceramide, or lactotetraosylceramide was unaffected. Thus, the third binding specificity is represented by neolactotetraosylceramide, and involves recognition of other neolacto series glycosphingolipids with linear N-acetyllactosamine chains, such as sialyl-neolactohexaosylceramide. The relevance of the detected binding specificities for adhesion to target cells was addressed as to the binding of the bacteria to glycosphingolipids from human granulocytes, epithelial cells of human nasopharyngeal tonsils and human plexus choroideus. Binding-active neolactotetraosylceramide was thereby detected in human granulocytes and the oropharyngeal epithelium.     

Immunolocalization of a novel annexin-like protein encoded by a stress and abscisic acid responsive gene in alfalfa

Rainbow trout (Oncorhynchus mykiss, 1-5 g) were exposed to approximately 7.5 microM Co in soft water for 2-3 hr at pH approximately 6.5. The water contained either complexing ligands such as nitrilotriacetic acid and natural dissolved organic matter (DOM) or competing cations such as Ca, Na, or H. After exposure, gills were excised and analyzed for total bound Co using a graphite furnace atomic absorption spectrophotometer. A Langmuir isotherm was used to calculate the conditional equilibrium binding constant (K) for Co binding to trout gills plus the concentrations of gill Co binding sites. The calculated binding constant for Co to trout gills was log KCo-gillCo = 5.1, with 88 nmol Co binding sites per g of wet gill tissue. Conditional equilibrium binding constants were also calculated for Ca, Na, and H binding to the gill Co sites and for Co binding to DOM. The experimentally determined binding constants were entered into an aquatic equilibrium chemistry program, MINEQL+, to predict Co binding by fish gills. Predicted and observed results indicate that Co would not accumulate on or in gills of trout held in a series of natural and 1:1 diluted natural waters supplemented with approximately 8.7 microM Co. Model analysis of the reasons for Co being kept off gills of trout held in natural waters indicated that Ca competition and DOM complexation were the most important factors in preventing Co binding by trout gills.     

In vitro binding of MX2 (KRN8602) and epirubicin to human plasma protein

This study compares the human plasma protein binding characteristics of MX2 and epirubicin. The binding characteristics were determined by equilibrium dialysis at various concentrations of the drugs. The binding dissociation constant (Kd), binding capacity (Bmax) and partitioning constant (Kp) were obtained by Scatchard analysis of the free and bound drugs in the dialysis compartments. Our results have demonstrated that plasma protein binds epirubicin or MX2 in an unsaturable appearance over the concentration up to 150 mumol/l. At the same concentrations, plasma protein binds more epirubicin than MX2. The nature of the interaction may consist of two classes of specific binding, and a partitioning. The binding dissociation constants were 18 and 17.5 mumol/l for the higher binding class (Kd1) and 315.8 and 316.9 mumol/l for the lower binding class (Kd2), respectively, for epirubicin and MX2. The respective maximum binding capacities (Bmax) of plasma protein for epirubicin and MX2 were significantly different, 0.045 and 0.029 mumol/g protein for the higher binding class (Bmax1), and 0.39 and 0.29 mumol/g protein for the lower binding class (Bmax2). The partitioning constants (Kp) were 21.5 x 10(-5) and 20 x 10(-5) litres/g protein for epirubicin and MX2, respectively. The results suggest that plasma protein binds epirubicin or MX2 with a similar affinity, but has less binding sites for MX2. One contributing mechanism to the difference in activity noted between epirubicin and MX2 may be changes in free drug fractions.     

ST/b and DBA/2 mice differ in brain alpha-bungarotoxin binding and alpha 7 nicotinic receptor subunit mRNA levels: a quantitative autoradiographic analysis

Inbred mouse strains vary in sensitivity to a number of behavioral and physiological effects produced by nicotine. Differences in sensitivity to nicotine are correlated with variance in the number of brain nicotinic receptors as measured in regionally dissected brain tissue. The studies reported here used quantitative autoradiography and in-situ hybridization methods to measure regional levels of alpha-bungarotoxin (alpha BTX) binding and alpha 7 mRNA levels. Two inbred mouse strains, ST/b and DBA/2, were compared because these strains differ maximally in sensitivity to nicotine-induced seizures and in alpha BTX binding measured in regional brain homogenates. The binding of alpha BTX was significantly greater in the St/b strain in 42 of 127 brain regions that were analyzed, and a trend towards increased binding was seen in many additional brain regions. The most consistent strain differences were found in hippocampal, thalamic and pontine nuclei. Strain differences in alpha 7 mRNA levels were also detected, but these were not as widespread as were the alpha BTX binding differences. The alpha 7 mRNA levels were significantly correlated with alpha BTX binding in both mouse strains which suggests that the strain differences in binding are related, in part, to the levels of alpha 7 mRNA.     

新型电石成型原料研究

通过分析生石灰的消化曲线和热失重曲线,提出了合理的电石成型工艺.根据电石成型原料对粘结剂的要求,研制出ZJ型复合粘结剂并分析了粘结功效及机理.结合现场的生产要求,通过大量实验与分析,给出了合理的成型加水量、成型压力和养护龄期.     

A comparative study of the reversal by different alpha 2-adrenoceptor antagonists of the central sympatho-inhibitory effect of clonidine

Chronic nicotine treatment often results in tolerance to nicotine as well as increases in brain [3H]-nicotine binding and [125l]-alpha-bungarotoxin (alpha-BTX) binding. Chronic corticosterone (CCS) treatment also produces tolerance to nicotine, but it does not change [3H]-nicotine binding; decreases in alpha-BTX binding are observed, which suggests that tolerance to nicotine may be related to decreases in the number of this nicotinic receptor subtype. In the studies reported here, C57BL/6 mice were implanted subcutaneously with cholesterol or 60% CCS/40% cholesterol-containing pellets and were infused continuously with saline (control) or nicotine for a total of 9 days. Effects of acute nicotine challenge on Y-maze crossing and rearing activities, heart rate, and body temperature were measured. Both chronic nicotine and CCS treatment resulted in tolerance to nicotine for all of the measures, and some evidence for additivity was seen in the animals that were cotreated with CCS and nicotine. Chronic nicotine infusion increased brain nicotine binding and CCS treatment reduced alpha-BTX binding. Decreases in alpha-BTX binding were not detected in the cotreated animals. The latter finding argues that changes in alpha-BTX binding are not reliable predictors of or a cause of tolerance to nicotine.     

Use of affinity capillary electrophoresis for the study of protein and drug interactions

Protein-drug interactions were studied using affinity capillary electrophoresis (ACE). The initial study was performed using a model system, fibronectin-heparin interaction. Two distinct binding constants, 21 and 641 nM, were derived from the Scatchard plots. The results are consistent with reported data obtained using other analytical techniques. The ACE binding assay was applied for studying molecular interactions between kedarcidin chromophore and apoprotein. Conditions with an organic solvent as buffer component were examined to establish a suitable binding assay. It appears that the electrophoretic behavior of the protein shows little distortion in the presence of either dimethyl sulfoxide (up to 10%) or acetonitrile (ACN) (up to 30%). The binding assay was initially conducted in aqueous buffer phase. The saturation concentration of chomophore was found to be around 15 microM. A linear Scatchard plot was derived from binding data with a correlation coefficient of 0.94. The binding constant was determined as Kd = 5.6 microM. The effects of organic solvent content ranging from 0 to 30% ACN on the constant were examined. The binding constants were determined as Kd = 11, 12.5 and 16.2 microM for 5, 10 and 30% ACN, respectively. It appeared that the binding affinity between kedarcidin chromophore and apoprotein is reduced as the organic solvent content in the aqueous phase is increased.     

The distribution of tachykinin binding sites in the brain of an electric fish (Apteronotus leptorhynchus)

We mapped the distribution of tachykinin binding sites utilizing quantitative autoradiography of iodinated substance P and eledoisin as prototypic ligands for neurokinin-1 (NK1) and neurokinin-3 (NK3) receptors, respectively. The two ligands produced highly heterogenous and quantitatively different patterns of specific binding, suggesting that they revealed different tachykinin receptor subtypes. Although [125I]substance P and [125I]eledoisin binding were correlated in most brain regions, the binding of substance P was usually denser. [125I]substance P binding and substance P-like immunoreactivity were reasonably correlated in most brain areas, although discrepancies were found in some nuclei. Dense [125I]substance P binding was found in most areas of the subpallium and in parts of the pallium related to the olfactory system, as well as in the glomerular layer of the olfactory bulb. Moderate to dense binding of both ligands was observed in preoptic area, hypothalamus, habenula, parts of the thalamus and preglomerular complex. Especially noteworthy was the presence of [125I] substance P binding in the diencephalic prepacemaker nucleus, a region involved in the control of electroncommuncatory behavior. Substance P-like immunoreactivity is sexually dimorphic in certain diencephalic nuclei, including the prepacemaker nucleus (Weld and Maler, 1992); no obvious difference was seen between [125I]substance P or [125I]eledoisin binding in the brains of male versus female fish. In the mesencephalon striking laminar patterns of binding were seen in the torus semicircularis dorsalis and the optic tectum. Dense binding was also noted in the raphé nuclei, the locus ceruleus and the sensory nucleus of the vagus. Although binding of substance P in the electrosensory lateral line lobe and nucleus preeminentialis was light, it was distributed in a discrete fashion, suggesting a role of substance P in electrosensory processing.     

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.     

Binding of apoB-containing lipoproteins from unfractionated human blood sera to immobilized LDL receptor

Binding of apoB-containing lipoproteins from unfractionated human blood sera to the immobilized bovine receptor of low density lipoproteins (LDL receptor) was studied. Peroxidase-labeled anti-human apoB antibodies were used to evaluate the lipoprotein binding. The equilibrium dissociation constant (Kd) of the interaction between apoB-containing lipoproteins from unfractionated human sera from healthy donors and the immobilized LDL receptor varied from 1 to 20 microg apoB/ml. To describe the binding of lipoproteins to the LDL receptor, a parameter of relative binding affinity (RBA) was used. RBA is inversely related to value of Kd and equal to unity for the standard serum. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera to the immobilized LDL receptor were found to correlate with the RBA values for the binding of isolated VLDL (r = 0.76, p < 0.001) and fail to correlate with the RBA values for the binding of isolated LDL. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera correlated with the RBA values for the binding of apoE-containing lipoproteins from unfractionated sera (r = 0.92, p < 0.001) and with values of triglyceride concentration in the sera (r = 0.93, p < 0.001). The RBA values for the binding of apoB-containing lipoproteins from sera of patients with FDB whose LDL were unable to bind to the LDL receptor did not significantly differ from the RBA values for the normal sera. However, the removal of VLDL from the normal sera significantly decreased the RBA values for the binding of apoB-containing lipoproteins from unfractionated sera. The results indicate that the different binding of apoB-containing lipoproteins to the immobilized LDL receptor mainly depended on the different binding of VLDL and not of LDL.     

Characterization of the fibronectin binding motif for a unique mycobacterial fibronectin attachment protein, FAP

Studies were performed to define the fibronectin binding motif of the previously identified Mycobacterium avium fibronectin attachment protein (FAP-A). Using synthetic peptides of a previously identified fibronectin binding region (amino acids 269-292), the minimal binding sequence was determined to be 12 amino acids, 269-280 (FAP-A-(269-280)). Synthetic peptides were prepared in which each amino acid in the 269-280 sequence was substituted with Ala. Assessment of the effect of Ala substitution on fibronectin binding showed that the presence of Ala at amino acids 273-276 (RWFV) completely abrogated fibronectin binding activity. Furthermore, the ability to inhibit the attachment of viable Mycobacterium bovis BCG to fibronectin was abrogated by Ala substitution at the RWFV sites. To validate the function of RWFV, further studies were performed with recombinant FAP-A in which single Ala mutations were generated for the RWFV sites and as controls at amino acids 269 and 280. Mutant FAP-A containing single Ala substitutions at the RWFV sites (amino acids 273, 274, 275, or 276) showed significant abrogation of fibronectin binding function. Recombinant FAP-A with Ala substitutions at either 269 or 280 showed wild type activity. When the four essential amino acids (RWFV) were either substituted en bloc with Ala or were all deleted, complete loss of fibronectin binding function was observed. Control recombinant proteins with en bloc Ala substitutions or deletions at four positions outside the fibronectin binding region (amino acids 255-257) retained functional activity. These data show that the RWFV sequence is necessary for fibronectin binding function of FAP-A. Furthermore, the data suggest that mycobacterial FAP proteins, all of which share the RWFV binding motif, constitute a family of highly homologous proteins that bind fibronectin in a unique manner.     

What do we learn from binding features? Evidence for multilevel feature integration.

Four experiments were conducted to investigate the relationship between the binding of visual features (as measured by their aftereffects on subsequent binding) and the learning of feature-conjunction probabilities. Both binding and learning effects were obtained, but they did not interact. Interestingly, (shape-color) binding effects disappeared with increasing practice, presumably because of the fact that only 1 of the features involved was relevant to the task. However, this instability was only observed for arbitrary, not highly overlearned combinations of simple geometric features and not for real objects (colored pictures of a banana and strawberry), where binding effects were strong and resistant to practice. These findings suggest that learning has no direct impact on the strength or resistance of bindings or on speed with which features are bound; however, learning does affect the amount of attention particular feature dimensions attract, which again can influence which features are considered in binding. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Binding of enterostatin to the human neuroepithelioma cell line SK-N-MC

SK-N-MC cells were found to possess binding sites for enterostatin, a peptide with central effects on appetite and sympathetic activation of brown adipose tissue during high-fat feeding. Scatchard analyses of the binding indicated one high-affinity binding (Kd = 0.5-1.5 nM) and one low-affinity binding (Kd = 15-30 nM) for 3H-enterostatin (APGPR). 125I-YGGAPGPR showed similar binding parameters as for the low affinity binding of 3H-APGPR. Met-enkephalin and beta3-casomorphin1-5 were found to displace the binding of 3H-APGPR to the SK-N-MC cells. Affinity purification of solubilized cells revealed an APGPR-binding protein estimated to 53 kDa which may represent a distinct enterostatin receptor. Cross-linking of 125I-YGGAPGPR to intact cells labeled one major protein with the same molecular size. There was no binding of enterostatin to four other human neuroblastoma/neuroepithelioma cell lines, named IMR-92, LAN#5, NB-1 #14 and SH5-SY.     

Thermodynamic studies on the binding of adenosine diphosphate and calcium to beef cardiac myosin

Thermodynamic quantities for the binding of MgADP, CaADP and Ca2+ to purified beef cardiac myosin have been determined by flow calorimetry at 25 degrees C and by equilibrium dialysis at 4 degrees C in 0.5 M KCl, 20 mM tris-HCl (pH 7.5). About 1.65 +/- 0.15 mol MgADP and 1.9 +/- 0.1 mol CaADP were bound per mol myosin. Free energies of binding of MgADP and CaADP were -6.7 and -5.7 kcal/mol, respectively. Enthalpies for binding of MgADP and CaADP were about -12.5 and -19.0 kcal/mol, respectively. Furthermore, there were 1.8 +/- 0.2 mol high affinity Ca2+ binding sites per mol myosin with an affinity constant of about 10(5) M-1. The enthalpy of Ca2+ binding was about zero. It is concluded that CaADP binds to cardiac myosin with a much greater negative enthalpy than MgADP. Also, the free energy of MgADP binding to cardiac myosin is similar to values reported for skeletal myosin. However, the enthalpy of binding is much less negative than the value obtained for skeletal myosin by Kodama and Woledge (J. Biol. Chem. (1976) 251, 7499--7503). The latter results suggest a subtle difference in the nucleotide binding sites of these myosins.     

Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor

Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.