Spectral studies of the interactions of Escherichia coli alkaline phosphatase with 4-(4-aminophenylazo)-phenylarsonic acid.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is reversibly inhibited by a variety of phenylarsonic acids, including some N-haloacetylated derivatives. The inhibition is of the competitive type, and Ki values are reported. The action on the enzyme of one of the arsonate inhibitors, the azo dye, 4-(4-aminophenylazo)-phenylarsonic acid was studied in detail, using spectrophotometric and kinetic methods. The azo dye binds more strongly to E. coli alkaline phosphatase than do the other arsonates. Spectrophotometric titration indicates the presence of a single, strong dye-binding site on the enzyme dimer molecule in the concentration range covered. In 0.1 M Tris - HCl buffer pH 8.0, 25 degrees C K diss for the dye - enzyme complex is 1.50 - 10(-5) M as determined by spectrophotometric titration. This value is in good agreement with the Ki = 1.30 - 10(-5) M obtained from kinetic measurements. The dye can be displaced from alkaline phosphatase by phosphate and competitive inhibitor 2-aminoethyl phosphonate. These results indicate that the dye binds with its arsonic acid group to the anion binding site of the active site of the enzyme. The binding of the dye to the native enzyme is associated with a red shift in the visible spectrum of the dye. It seems that the aromatic portion of the dye interacts with a hydrophobic region close to the anion binding site. The spectrum of the dye is not changed in the presence of the apoenzyme. When zinc is added to an apoenzyme-dye solution, the spectral changes of the dye depend on both the ratio of zinc per apoenzyme and the pH. The presence of Mg2+ had no effect on the observed phenomenon.     

Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

Interactions of urocanic acid with bovine serum albumin and the influence of pH on binding affinities: a docking simulation study

In the present study, the interactions of urocanic acid (UA) with bovine serum albumins (BSA) at pH 5.0 and 7.4 were investigated by means of docking simulations. The binding modes of trans- and cis-UA to BSA at pH 5.0 and 7.4 were analysed. In addition, the theoretically predicted binding abilities of zwitterion and anion of UA with BSA are in good agreement with the experimental results. Through comparison with the binding patterns, we revealed that the stronger interactions of UA anion with BSA relative to the zwitterion primarily result from: (1) the increased number of hydrogen bonds between UA anion and BSA; (2) the attractive electrostatic interaction between the deprotoned carboxyl group in UA anion and Arg433 in comparison with the repulsion between the imidazole moiety in zwitterion and the same residue in BSA. This provides a rational explanation for the experimental finding that the binding of UA to BSA at pH 7.4 is much stronger than at pH 5.0.     

Spectrophotometric and kinetic studies on the binding of the bioflavonoid quercetin to bovine serum albumin

This work investigates the binding of the bioflavonoid, quercetin, to bovine serum albumin (BSA) by spectrophotometric techniques involving both the conventional and stopped-flow methods. Both the neutral and negatively-charged forms of quercetin bound to BSA with a red shift in the maximal absorption. At high pH values, quercetin was rapidly degraded in an oxygen-dependent process, but this decomposition was substantially slower when the flavonoid was bound to BSA. At pH 7.4, the difference spectrum of quercetin with and without BSA was maximal at 425 nm; this wavelength can be conveniently used to monitor the extent and speed of binding. Spectrophotometric studies with a range of equimolar mixtures of quercetin and BSA at pH 7.4 suggest the binding was maximal when the concentration was 10 microM. It is postulated that the binding site of BSA for quercetin was less available at higher protein concentrations, perhaps because of conformational change or self-association. The rate of spectrophotometric change when quercetin bound to BSA was fairly slow; the process was not quite complete within 45 seconds and was biphasic. When a pre-mixed equimolar mixture of BSA and quercetin was diluted with an equal volume of the buffer, there was a surprising further increase in absorbance at 425 nm (rather than the fall anticipated if the binary complex were to dissociate). It is concluded that, upon dilution, the effective concentration of BSA's binding site increased, providing more scope for quercetin to bind.     

The solubility of fibrinogen in dilute salt solutions

Highly purified human fibrinogen was dialyzed versus eleven different sodium salts at ionic strengths of 0.005–0.3 and pH values of 4.5–8.0. After equilibration and centrifugation of the protein solutions, fibrinogen solubilities were determined spectrophotometrically and were analyzed as functions of pH, ionic strength, and specific anion. Bell-shaped curves are obtained when fibrinogen solubility is plotted as a function of pH. The solubility exhibits a minimum at a given pH and rises at acid and alkaline values. As the ionic strength is increased, the solubility curves shift toward more acid pH values. At constant pH values between 6 and 7, fibrinogen solubility increases with an increase in ionic strength. At constant pH values below pH 6, a decrease in solubility occurs as the ionic strength is increased. The isoionic pH of a saturated aqueous fibrinogen solution has been determined to be 6.25, meaning that fibrinogen in pure water behaves as a weak acid with a mean net charge of ?0.9. At pH values acid to 6.25, the anions solubilize fibrinogen in the following order of increasing efficacy: thiocyanate, perchlorate, sulfate, citrate, bromide, nitrate, phosphate, chloride, acetate, fluoride, and formate. This order is reversed at pH values alkaline to 6.25. Anion binding parameters calculated from the solubility data indicate that those anions which most effectively solubilize fibrinogen at alkaline pH and precipitate it at acid pH have the highest apparent binding affinities for the protein. Anions with less pronounced solubility effects have lower binding affinities.     

Equilibrium and kinetic studies of the binding of tri- and tetra-anionic ligands to bovine serum albumin.

The binding of tri- and tetra-anionic azo dyes (Amaranth, Ponceau 4R, and Ponceau 6R) to bovine serum albumin (BSA) at pH = 7.0 and 25 degrees C has been studied by equilibrium dialysis, spectrophotometry, and by stopped-flow and temperature-jump methods. Equilibrium dialysis revealed that BSA has one primary binding site and about two secondary sites for each dye. The values of the binding constant for the primary site show that the stability of the complex at the primary site progressively increases with an increase in the number and the density of anionic charges on ligand. Kinetic data have been found to be consistent with a scheme in which a rapid bimolecular binding is followed by two isomerizations of the complex (in the case of Amaranth) or by one isomerization (in the cases of Ponceau 4R and Ponceau 6R). Equilibrium and rate constants for each step of the scheme were determined. From the results it was found that the increment of the number and the density of anionic charges on ligand accelerates the forward process of the final isomerization step but retards the backward one of it, resulting in the enhancement of the stability of the complex at the primary site. On the basis of these results and the structure of the ligands, the detailed binding mechanism has been discussed in the light of the electrostatic interaction between the ligands and the binding site on BSA.     

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.     

Protein purification by polyelectrolyte coacervation: influence of protein charge anisotropy on selectivity

The effect of polyelectrolyte binding affinity on selective coacervation of proteins with the cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC), was investigated for bovine serum albumin/β-lactoglobulin (BSA/BLG) and for the isoforms BLG-A/BLG-B. High-sensitivity turbidimetric titrations were used to define conditions of complex formation and coacervation (pH(c) and pH(?), respectively) as a function of ionic strength. The resultant phase boundaries, essential for the choice of conditions for selective coacervation for the chosen protein pairs, are nonmonotonic with respect to ionic strength, for both pH(c) and pH(?). These results are explained in the context of short-range attraction/long-range repulsion governing initial protein binding "on the wrong side of pI" and also subsequent phase separation due to charge neutralization. The stronger binding of BLG despite its higher isoelectric point, inferred from lower pH(c), is shown to result from the negative "charge patch" on BLG, absent for BSA, as visualized via computer modeling (DelPhi). The higher affinity of BLG versus BSA was also confirmed by isothermal titration calorimetry (ITC). The relative values of pH(?) for the two proteins show complex salt dependence so that the choice of ionic strength determines the order of coacervation, whereas the choice of pH controls the yield of the target protein. Coacervation at I = 100 mM, pH 7, of BLG from a 1:1 (w/w) mixture with BSA was shown by SEC to provide 90% purity of BLG with a 20-fold increase in concentration. Ultrafiltration was shown to remove effectively the polymer from the target protein. The relationship between protein charge anisotropy and binding affinity and between binding affinity and selective coacervation, inferred from the results for BLG/BSA, was tested using the isoforms of BLG. Substitution of glycine in BLG-B by aspartate in BLG-A lowers pH(c) by 0.2, as anticipated on the basis of DelPhi modeling. The stronger binding of BLG-A, confirmed by ITC, led to a difference in pH(?) that was sufficient to provide enrichment by a factor of 2 for BLG-A in the coacervate formed from "native BLG".     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Interactions between m‐phenylenediamine and bovine serum albumin measured by spectroscopy

This study explored interactions between m‐phenylenediamine (MPD) and bovine serum albumin (BSA) by spectrophotometry. The Stern‐Volmer equation and UV‐vis spectra examination at different temperatures and pH were used to explore different quenching mechanisms. Under simulated physiological conditions, the binding distance between MPD and BSA was 5.18 nm with a ratio of 1:1. The quenching effect of MPD on BSA intrinsic fluorescence depended strongly on pH, and maximum quenching was observed at alkaline pH. Moreover, the thermodynamic parameters of the MPD‐BSA system showed that the predominant acting force between MPD and BSA was a hydrophobic force. The impact of MPD on the conformation of BSA and the effects of co‐ions on binding interactions were also examined. Copyright © 2012 John Wiley & Sons, Ltd.     

The human erythrocyte anion transport protein, band 3. Characterization of exofacial alkaline titratable groups involved in anion binding/translocation

Chloride self-exchange across the human erythrocyte membrane at alkaline extracellular pH (pHO) and constant neutral intracellular pH (pH(i)) can be described by an exofacial deprotonatable reciprocating anion binding site model. The conversion of the transport system from the neutral to the alkaline state is related to deprotonation of a positively charged ionic strength- and substrate-sensitive group. In the absence of substrate ions ([ClO] = 0) the group has a pK of approximately 9.4 at constant high ionic strength (equivalent to approximately 150 mM KCl) and a pK of approximately 8.7 at approximately zero ionic strength. The alkaline ping-pong system (examined at constant high ionic strength) demonstrates outward recruitment of the binding sites with an asymmetry factor of approximately 0.2, as compared with the inward recruitment of the transport system at neutral pHO with an asymmetry factor of approximately 10. The intrinsic half-saturation constant for chloride binding, with [Cli] = [Clo], increased from approximately 30 mM at neutral to approximately 110 mM at alkaline pHO. The maximal transport rate was a factor of approximately 1.7 higher at alkaline pHO. This increase explains the stimulation of anion transport, the "modifier hump," observed at alkaline pHO. The translocation of anions at alkaline pHO was inhibited by deprotonation of another substrate-sensitive group with an intrinsic pK of approximately 11.3. This group together with the group with a pK of approximately 9.4 appear to form the essential part of the exofacial anion binding site. The effect of extracellular iodide inhibition on chloride transport as a function of pHO could, moreover, be simulated if three extracellular iodide binding constants were included in the model: namely, a competitive intrinsic iodide binding constant of approximately 1 mM in the neutral state, a self-inhibitor binding constant of approximately 120 mM in the neutral state, and a competitive intrinsic binding constant of approximately 38 mM in the alkaline state.     

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function.

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.     

Mechanism of acridine uptake by submitochondrial particles.

Acridines were compared regarding their ability to be taken up by submitochondrial particles under energized conditions. pH dependence of uptake was explored, and it was found that acridines fell into three classes independently of their pK_a value: acridines which are not taken up, acridines taken up at all pH values, and acridines taken up only at alkaline pH. Partition measurements between H₂O and chloroform phase showed a similar pattern, and affinity for the organic phase seemed to parallel uptake. Acridines which are taken up by submitochondrial particles at acidic pH under energized conditions despite a high pK_a value could also be extracted into chloroform at acidic pH, thus implying that the dye's uncharged form has a high affinity for the organic phase. By supplementing the aqueous medium with lipophilic anions, the dye may also be extracted in its charged form. The data support a mechanism for acridine uptake in which diffusion of the uncharged form across the membranes is an obligatory step. Some previously reported inhibitory anion effects on uptake may be explained by ion pair formation, which allows release of the accumulated charged form.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction between lisinopril and bovine serum albumin

To further understand the mode of action and pharmacokinetics of lisinopril, the binding interaction of lisinopril with bovine serum albumin (BSA) under imitated physiological conditions (pH 7.4) was investigated using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and molecular docking methods. The results showed that the fluorescence quenching of BSA near 338 nm resulted from the formation of a lisinopril–BSA complex. The number of binding sites (n) for lisinopril binding on subdomain IIIA (site II) of BSA and the binding constant were ~ 1 and 2.04 × 10⁴ M^–1, respectively, at 310 K. The binding of lisinopril to BSA induced a slight change in the conformation of BSA, which retained its α‐helical structure. However, the binding of lisinopril with BSA was spontaneous and the main interaction forces involved were van der Waal's force and hydrogen bonding interaction as shown by the negative values of ΔG⁰, ΔH⁰ and ΔS⁰ for the binding of lisinopril with BSA. It was concluded from the molecular docking results that the flexibility of lisinopril also played an important role in increasing the stability of the lisinopril–BSA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of Lipoic Acid Induces Conformational Change and Appearance of a New Binding Site in Methylglyoxal Modified Serum Albumin

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.     

Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces.

An indicator dye attached to an electrostatically charged macromolecular surface generally has a pK value (pKb') different from that of uncombined dye (pKf'). The question if changes in (pKb' - pKf'), designated as increment incrementpK, records changes in the electrostatic potential at the binding site has been examined in spectrophotometric and binding experiments, using the interaction of Chlorophenol Red and Phenol Red with human serum albumin and cationic micelles as examples. (1) In serum albumin solutions increment incrementpK is decreased by a reduction of pH. The decrease is correlated with the increase in positive charges on the protein molecule, and the response is attenuated by high ionic strength in accordance with electrostatic theory. (2) Opposite changes in binding affinity to serum albumin and increment incrementpK as a function of pH are observed; the binding of basic (bivalent anion) dye is more susceptible to a change in pH than in the acidic (univalent anion) form. (3) Preferential uptake of the basic as compared to the acidic form of dye is observed by binding to cetyltrimethylammonium chloride and cetylpyridinium chloride micelles (mu equals 0.033, [Cl-] equals 0.033 M). An increase in the ionic strength is accompanied by a positive value of increment incrementpK. The results are consonant with the view that the observed increment incrementpK values reflect changes in the electrostatic potential at the binding site with consequently little, if any, effect on the intrinsic pK. The extension of the method to measure changes in the electrostatic potential at binding sites on cell membranes is briefly discussed.     

The isolated cytoplasmic domain of band 3 binds calcium at physiological salt concentration and neutral PH

Calcium is known to be a potent but partial intracellular inhibitor of band 3 anion exchange. Here we test the hypothesis that the cytoplasmic domain of band 3 (CDB3) contains a calcium binding site. Calcium binding to CDB3 was monitored by measuring the formation of the Aresenazo III-calcium complex at various constant CDB3 concentrations. These experiments were performed at physiological salt and neutral pH. The calcium-CDB3 dissociation constant was estimated to be less than or equal to 24 microM. We also found that the Arsenazo III-calcium complex binds to CDB3, while the free dye does not bind. We conclude that CDB3 contains a site which is capable of binding free calcium under physiological conditions. A specific role for this site in inhibition of band 3 anion exchange is suggested, but that role remains to be established.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

Electrostatic selectivity in protein-nanoparticle interactions

The binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to TTMA (a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23-mercapto-N,N,N-trimethyl) was studied by high-resolution turbidimetry (to observe a critical pH for binding), dynamic light scattering (to monitor particle growth), and isothermal titration calorimetry (to measure binding energetics), all as a function of pH and ionic strength. Distinctively higher affinities observed for BLG versus BSA, despite the lower pI of the latter, were explained in terms of their different charge anisotropies, namely, the negative charge patch of BLG. To confirm this effect, we studied two isoforms of BLG that differ in only two amino acids. Significantly stronger binding to BLGA could be attributed to the presence of the additional aspartates in the negative charge domain for the BLG dimer, best portrayed in DelPhi. This selectivity decreases at low ionic strength, at which both isoforms bind well below pI. Selectivity increases with ionic strength for BLG versus BSA, which binds above pI. This result points to the diminished role of long-range repulsions for binding above pI. Dynamic light scattering reveals a tendency for higher-order aggregation for TTMA-BSA at pH above the pI of BSA, due to its ability to bridge nanoparticles. In contrast, soluble BLG-TTMA complexes were stable over a range of pH because the charge anisotropy of this protein at makes it unable to bridge nanoparticles. Finally, isothermal titration calorimetry shows endoenthalpic binding for all proteins: the higher affinity of TTMA for BLGA versus BLGB comes from a difference in the dominant entropy term.     

荧光光谱法研究淫羊藿苷和淫羊藿次苷Ⅰ与牛血清白蛋白的相互作用

在模拟生理条件下应用荧光光谱学方法分别研究了淫羊藿苷和淫羊藿次苷Ⅰ与牛血清白蛋白（BSA）间的结合作用. 根据荧光强度数据,计算出了结合常数KA,结合位点数n和热力学参数（△G, △H 和△S）. 实验结果表明,淫羊藿苷和淫羊藿次苷Ⅰ都能显著猝灭BSA的内源荧光,猝灭机制均为形成基态复合物的单一静态猝灭过程. 不同温度下（17 ℃, 27 ℃, 37 ℃）得到的KA和n值,表明淫羊藿次苷Ⅰ与BSA的结合强于淫羊藿苷. 从得到的热力学参数判断,淫羊藿苷与BSA间的主要作用力是氢键作用和范德华力,而疏水作用和静电引力在淫羊藿次苷Ⅰ与BSA形成复合物过程中起主导作用.而且同步荧光光谱显示,淫羊藿苷和淫羊藿次苷Ⅰ与BSA的结合导致BSA构象发生了变化.