Independent Classes Of Binding Sites Research Articles

Multi-spectroscopic studies on the interaction between traditional Chinese herb, helicid with pepsin.

Study on the binding properties of helicid by pepsin systematically using multi-spectroscopic techniques and molecular docking method, and these interactions comprise biological recognition at molecular level and backbone of biological significance in medicine concerned with the uses, effects, and modes of action of drugs. We investigated the mechanism of interaction between helicid and pepsin by using various spectroscopic techniques viz., fluorescence spectra, UV-Vis absorption spectra, circular dichroism (CD), 3D spectra, synchronous fluorescence spectra and molecular docking methods. The quenching mechanism associated with the helicid-pepsin interaction was determined by performing fluorescence measurements at different temperatures. From the experimental results show that helicid quenched the fluorescence intensity of pepsin via a combination of static and dynamic quenching process. The binding constants (Ka) at three temperatures (288, 298, and 308K) were 7.940 × 107, 2.082 × 105 and 3.199 × 105 L mol-1, respectively, and the number of binding sites (n) were 1.44, 1.14, and 1.18, respectively. The n value is close to unity, which means that there is only one independent class of binding site on pepsin for helicid. Thermodynamic parameters at 298K were calculated as follows: ΔHo (- 83.85kJ mol-1), ΔGo (- 33.279kJ mol-1), and ΔSo (- 169.72J K-1 mol-1). Based on thermodynamic analysis, the interaction of helicid with pepsin is driven by enthalpy, and Van der Waals' forces and hydrogen bonds are the main forces between helicid and pepsin. A molecular docking study further confirmed the binding mode obtained by the experimental studies. The conformational changes in the structure of pepsin was confirmed by 3D fluorescence spectra and circular dichroism.

Protein Surface Structural Recognition in Inactive Areas: A New Immobilization Strategy for Acetylcholinesterase.

This work reported a new method of design for the immobilization of acetylcholinesterase (AChE) based on its molecular structure to improve its sensitivity and stability. The immobilization binding site on the surface of AChE was determined using MOLCAD's multi-channel functionality. Then, 11 molecules ((+)-catechin, (-)-epicatechin, (-)-gallocatechin, hesperetin, naringenin, quercetin, taxifolin, (-)-epicatechin gallate, flupirtine, atropine, and hyoscyamine) were selected from the ZINC database (about 50 000 molecules) as candidate affinity ligands for AChE. The fluorescence results showed that the binding constant Kb between AChE and the ligands ranged from 0.01344 × 104 to 4.689 × 104 M-1 and there was one independent class of binding site for the ligands on AChE. The AChE-ligand binding free energy ranged from -12.14 to -26.65 kJ mol-1. Naringenin, hesperetin, and quercetin were the three most potent immobilized affinity ligands. In addition, it was confirmed that the binding between the immobilized ligands only occurred at a single site, located in an inactive area on the surface of AChE, and did not affect the enzymatic activity as shown through a competition experiment and enzyme assay. This method based on protein surface structural recognition with high sensitivity and stability can be used as a generic approach for design of the enzyme immobilization and biosensor development.

Prochlorperazine interaction with melanin and melanocytes.

Prochlorperazine is a phenothiazine-class antipsychotic drug usually used to treat nausea, vomiting and schizophrenia. Phenothiazine derivatives have been known to cause serious side effects, like extrapyramidal symptoms, but also skin disorders which mechanism has not been fully established. The aim of this study was to examine the interaction between prochlorperazine and melanin as well as to estimate the effect of prochlorperazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. We have demonstrated that prochlorperazine forms stable complexes with melanin, characterized by two classes of independent binding sites with the association constants K1∼106 M-1 and K2∼102 M-1. It has been shown that prochlorperazine induces concentration-dependent loss in cell viability. The value of EC50 was calculated to be 18.49 μM. Prochlorperazine in a concentration of 0.001 μM stimulated melanogenesis, while in concentrations 1.0 and 10.0 μM melanization process was inhibited. Furthermore, the drug in concentrations of 0.1, 1.0 and 10.0 μM caused changes in cellular antioxidant defense system, what indicated the induction of oxidative stress. The observed changes in cell viability, melanization and antioxidant defense system in normal human melanocytes after prochlorperazine treatment may explain a potential role of melanin, oxidative stress and melanocytes in mechanisms of undesirable side effects after accumulation of this drug in pigmented tissues.

Molecular modeling and multi-spectroscopic approaches to study the interaction between antibacterial drug and human immunoglobulin G.

Mechanistic and conformational studies on the interaction of sulfamethoxazole (SMX) with human immunoglobulin G (HIgG) were performed by molecular modeling and multi-spectroscopic methods. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HIgG. The binding parameters and thermodynamic parameters at different temperatures had been calculated according to the Stern-Volmer, Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the fluorescence intensity of HIgG was quenched by the gradual addition of SMX. The binding constants of SMX with HIgG decreased with the increase of temperature, which meant that the quenching mechanism was a static quenching. Meanwhile, the results also confirmed that there was one independent class of binding site on HIgG for SMX during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH(0) and entropy ΔS(0), had been calculated to be -14.69 kJ·mol(-1) and 22.99 J·mol(-1) ·K(-1), respectively, which suggested that the electrostatic and hydrophobic interactions were the predominant intermolecular forces in stabilizing the SMX-HIgG complex. Furthermore, experimental results obtained from three-dimensional fluorescence spectroscopy, UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy confirmed that the conformational structure of HIgG was altered in the presence of SMX.

Spectroscopic investigation of interaction between bovine gamma globulin and gold nanoparticles

The interaction of Citrate-capped gold nanoparticles (AuNPs) with Bovine gamma globulin (BGG) was studied using Fourier transform infrared (FTIR), UV-Visible and Fluorescence spectroscopy. FTIR has confirmed the conjugation of AuNPs and BGG. Fluorescence quenching of tryptophan has confirmed the strong interaction between BGG and AuNPs. UV-Visible and Fluorescence spectroscopy have investigated the extent of interaction by determining the binding constants. Binding constants evaluated from UV-Visible and Fluorescence data are in good agreement with each other. An independent class of binding site on BGG for AuNPs has been predicted, where AuNPs interact with a highly solvent accessible tryptophan residue.

Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi-spectroscopic approach in vitro.

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ-HIgG complex. Fluorescence spectroscopy, ultraviolet-visible (UV-vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH(0) and entropy ΔS(0), had been calculated to be -19.12 kJ · mol(-1) and 20.22 J · mol(-1) · K(-1), respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ - HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three-dimensional fluorescence spectroscopy, UV-vis absorption spectroscopy and CD spectroscopy.

Interactions of PCBs with human serum albumin: In vitro spectroscopic study

Following absorption, polychlorinated biphenyls (PCBs) bind to albumin and are transported via blood into the target tissues. PCBs then accumulate in tissues and induce a variety of harmful chronic and developmental effects. The aim of the present study is to determine binding parameters, such as binding constant, quenching constant, and number of binding sites for three PCB congeners (PCB118, PCB126 and PCB153) in complex with human serum albumin (HSA). The binding parameters for the complexes of HSA–PCB118, HSA–PCB126, and HSA–PCB153 excited at 280nm were compared with those excited at 295nm. Quenching (static and dynamic) of HSA fluorescence was analyzed based on the Stern–Volmer method. Binding (Ka) constants were calculated according to the Scatchard method and analysis of non-linear regression was based on a two-component model with the Lavenberg–Marquardt algorithm. For all analyzed complexes, a single independent class of binding site for PCB congeners was found in HSA subdomain IIA. Tyrosine residues appear to play the most prominent role in binding of PCB126 to HSA, while tryptophan-214 played a dominant role in interactions of PCB153 with HSA. Among studied PCB congeners, PCB118 formed the most stable complexes with HSA. These results illustrate the importance of studies targeting the binding of PCBs to serum albumin as part of the strategy to understand and protect against toxicity of these environmental toxicants.

Investigation of interaction of antibacterial drug sulfamethoxazole with human serum albumin by molecular modeling and multi-spectroscopic method

Interaction of sulfamethoxazole (SMX) with human serum albumin (HSA) was investigated by molecular modeling and multi-spectroscopic methods under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HSA. The binding parameters and the thermodynamic parameters at different temperatures for the reaction had been calculated according to the Stern–Volmer, Hill, Scatchard and the Van’t Hoff equations, respectively. One independent class of binding site existed during the interaction between HSA and SMX. The binding constants decreased with the increasing temperatures, which meant that the quenching mechanism was a static quenching. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH0 and entropy ΔS0, had been calculated to be −16.40kJmol−1 and 32.33Jmol−1K−1, respectively, which suggested that the binding process was exothermic, enthalpy driven and spontaneous. SMX bound to HSA was mainly based on electrostatic interaction, but hydrophobic interactions and hydrogen bonds could not be excluded from the binding. The conformational changes of HSA in the presence of SMX were confirmed by the three-dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and circular dichroism (CD) spectroscopy. CD data suggested that the protein conformation was altered with the reduction of α-helices from 55.37% to 41.97% at molar ratio of SMX/HSA of 4:1.

Cytotoxic effect of lomefloxacin in culture of human epidermal melanocytes

BackroundLomefloxacin is a potent bactericidal antibiotic. The use of this drug in treatment of various infections is accompanied by serious adverse effects on pigmented tissues. The exact mechanisms of lomefloxacin side effects have not been well established yet. The aim of this study was to characterize the interaction between lomefloxacin and ś, and to examine how this interaction affects the cell viability and melanization in melanocytes. MethodsNormal human epidermal melanocytes and the model DOPA-ś were used. The binding parameters of lomefloxacin-ś complexes as well as the antibiotic effect on cell viability and melanization in pigmented cells were investigated using a spectrophotometric method. ResultsOur results indicate that lomefloxacin forms stable complexes with ś. The analysis of drug binding to ś has shown that at least two classes of independent binding sites are involved in formation of these complexes. The WST-1 assay was used to detect the antibiotic cytotoxic effect. The value of ED50 for lomefloxacin was about 0.75mmol/l. It has been shown that lomefloxacin causes inhibition of tyrosinase activity, and reduces ś content in human skin melanocytes in a dose-dependent manner. ConclusionThe ability of the analyzed fluoroquinolone to form complexes with ś, and the demonstrated inhibitory effect on a melanization process in melanocytes in vitro may explain a potential role of ś biopolymer in the mechanisms of undesirable side effects of lomefloxacin in vivo resulting from its accumulation in pigmented tissues.

Complexation of HSA with different forms of antimony (Sb): An application of fluorescence spectroscopy

Antimony (Sb) pollution has been of a great environmental concern in some areas in China. Sb enters human body via drinking water, inhalation and food chain, unavoidably interacts with human serum albumin (HSA) in blood plasma, and consequently does harm to human health. The harmful effects of Sb on human health depend on the Sb species and their binding ability to HSA. In the present study, binding of three forms of Sb with HSA was investigated by excitation-emission matrix (EEM) spectroscopy. All of antimony potassium tartrate, antimony trichloride and potassium pyroantimonate quenched fluorescence of HSA. Values of conditional stability constant Ka (×105/M) for Sb and HSA systems were 8.13–9.12 for antimony potassium tartrate, 2.51–4.27 for antimony trichloride and 3.63–9.77 for potassium pyroantimonate. The binding constant Kb (×104/M) values of HSA with antimony potassium tartrate, antimony trichloride and potassium pyroantimonate were 0.02–0.07, 3.55–5.01, and 0.07–1.08, respectively. There was one independent class of binding site for antimony trichloride towards HSA. There was more than one Sb binding site and negative cooperativity between multiple binding sites for potassium pyroantimonate and antimony potassium tartrate towards HSA. The binding ability of HSA to complex Sb followed the order: antimony trichloride>potassium pyroantimonate>antimony potassium tartrate.

Interaction between ciprofloxacin and melanin: The effect on proliferation and melanization in melanocytes

There have been described serious adverse events caused by ciprofloxacin in pigmented tissues. It is known that some fluoroquinolones bind well to melanin rich tissues, but the relation between their affinity to melanin and the skin or eye toxicity is not well documented. The aim of this study was to examine whether ciprofloxacin binds to melanin, and how this interaction affects the proliferation and melanization in melanocytes. We have demonstrated that complexes which ciprofloxacin forms with melanin possess at least two classes of independent binding sites. Their association constants are K(1)~10(5) M(-1) and K(2)~10(2) M(-1), respectively. Ciprofloxacin has induced evident concentration-dependent loss in melanocytes viability. The value of ED(50) was found to be ~0.5 mM. It has also been shown that ciprofloxacin reduces melanin content, and decreases tyrosinase activity in human skin melanocytes. The ability of ciprofloxacin to interact with melanin and its inhibitory effect on melanization in melanocytes in vitro may explain a potential role of melanin in the mechanisms of ciprofloxacin toxic effects in vivo.

Binding of [3H]mianserin to bovine serum albumin, human serum albumin and alpha 1-acid glycoprotein.

Scatchard plots which were curvilinear with negative slopes were obtained when the binding of [3H]mianserin to bovine serum albumin (BSA), human serum albumin (HSA), defatted human serum albumin (D-HSA) and alpha 1-acid glycoprotein (alpha 1-AGP) was studied with equilibrium dialysis with constant protein concentrations and various ligand concentrations. Binding parameters were estimated graphically and with a non-linear least-squares computer program, assuming two classes of independent binding sites. alpha 1-AGP had the highest binding affinity (K) and binding capacity (nK). The binding parameters, n and K were not independent of protein concentration when the BSA concentration was varied. Linear atypical Scatchard plots with positive slopes were obtained when the protein concentration was varied for BSA, HSA and D-HSA, at a fixed ligand concentration.

Negatively cooperative binding of high-density lipoprotein to the HDL receptor SR-BI.

Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 μg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.

Sorption, Intercalation and Cooperativety: the Modes of Interaction of Actinomycin to DNA

The interaction of Actinomycin-D to DNA has been long investigated given it inhibits the synthesis of ribonucleic acid, inhibits the growth of cancer cells and induces apoptosis. So far, thermodynamic and structural studies have demonstrated that Actinomycin-D intercalates to DNA double helix preferentially to G-C pairs. There is also evidence that binding affinity is modulated by nearest base pairs flanking the intercalation site. However, the mechanism of Actinomycin-D interaction to DNA, and thus its energetic, is still ill understood. While some studies show evidence that ActD intercalation to natural DNA occurs via a mechanism consistent with a model of one independent and equivalent sites, other studies show evidence of the existence of two classes of independent binding sites; other yet show that the binding at low saturation is cooperative. In this work we measured the binding of Actinomycin-D to calf thymus DNA by optical titration and dialysis equilibrium under different solution conditions. Thus, we have found the conditions where the different kinds of binding reported in the literature can be reproduced. Through the analysis of the data correlating experimental design and solutions conditions, we were able to characterize the complexity of ActD interactions with DNA. In this work we show experimental evidences that intercalation of ActD at low drug/DNA ratio is cooperative; that the strong binding site is a consequence of cooperative binding; and that ActD not only intercalates to the DNA double helix but it also binds to the helix surface with a affinity which is in the same order of that measured upon intercalation.

Thermodynamic Analysis of Ferrous Ion Binding toEscherichia coliFerritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.

Melanin counter act puromycin-induced inhibition of collagen and DNA biosynthesis in human skin fibroblasts

Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K 1 = 1.84 × 10 6 M −1, and the second class of weak binding sites with the association constant K 2 = 5.26 × 10 3 M −1. The number of total binding sites were n 1 = 0.1260 and n 2 = 0.2861 μmol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC 50 ∼ 2 μM). Melanin at 100 μg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to puromycin - treated cells (2 μM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.

Melanin potentiates gentamicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

One of the recognized side effects of gentamicin is ototoxicity. The mechanism underlying the organ specificity of this side effect of gentamicin has not been fully established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear tissues, we determined whether gentamicin interacts with melanin and how this process affects the biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that gentamicin forms complexes with melanin. The amount of gentamicin bound to melanin increases with increasing of initial drug concentration. The Scatchard plot analysis of drug binding to melanin showed that at least two classes of independent binding sites are implicated in gentamicin–melanin complex formation: one class with an association constant K 1∼4×10 3 M −1, and the second class with an association constant K 2∼3×10 2 M −1. The number of total binding sites ( n 1+ n 2) was calculated as about 1.36 μmol gentamicin per 1 mg melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for gentamicin-induced inhibition of collagen biosynthesis. We found that gentamicin-induced inhibition of prolidase activity (IC 50∼100 μM) and collagen biosynthesis (IC 50∼100 μM). At this concentration of gentamicin, DNA biosynthesis in human skin fibroblasts was inhibited only by about 30%. Melanin at 100 μg/ml produced about 25% inhibition of DNA synthesis and about 30% inhibition of prolidase activity, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to gentamicin-treated cells (100 μM) augmented the inhibitory action of gentamicin on collagen and DNA biosynthesis and partially reversed its inhibitory effect on prolidase activity. A melanin-induced augmentation of the inhibitory effects of gentamicin on collagen and DNA biosynthesis may explain the mechanism for the organ specificity of gentamicin-induced hearing loss in patients administered this drug.

Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

One of the recognized side effects of antineoplastic anthracyclines is poor wound healing, resulting from an impairment of collagen biosynthesis. The most affected tissue is skin. The mechanism underlying the tissue specificity of the side effects of anthracyclines has not been established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanins are abundant constituents of the skin, we determined whether daunorubicin interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. Results indicated that daunorubicin forms complexes with melanin. Scatchard analysis showed that the binding of daunorubicin to melanin was heterogeneous, suggesting the presence of two classes of independent binding sites with K 1=1.83×10 5 M −1 and K 2=5.52×10 3 M −1. The number of strong binding sites was calculated as n 1=0.158 μmol/mg of melanin and the number of weak binding sites as n 2=0.255 μmol/mg of melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracycline-induced inhibition of collagen synthesis. We found that daunorubicin induced inhibition of prolidase activity (IC 50=10 μM), collagen biosynthesis (IC 50=70 μM) and DNA biosynthesis (IC 50=10 μM) in human skin fibroblasts. Melanin (100 μg/ml) by itself produced about 25% inhibition of DNA synthesis and prolidase activity but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to daunorubicin-treated cells (at IC 50 concentration) augmented the inhibitory action of daunorubicin on collagen and DNA biosynthesis without having any effect on prolidase activity. The same effect was achieved when the cells were treated with daunorubicin at one-fourth of the IC 50 given at 0, 6, 12 and 18 h during a 24-h incubation. The data suggest that the melanin-induced augmentation of the inhibitory effects of daunorubicin on collagen and DNA biosynthesis may result from: (i) accumulation of the drug in the extracellular matrix, (ii) gradual dissociation of the complex, and (iii) constant action of the released drug on cell metabolism. The phenomenon may explain the potential mechanism for the organ specificity of daunorubicin-induced poor wound healing in patients administered this drug.

A Structure-Activity Relationship in the Binding of Multicharged Anionic Azo and Pyrene Dyes to Serum Albumin

Abstract The binding of 11 azo and pyrene dyes (which have 1 to 4 sulfonato groups) to bovine serum albumin at pH = 7.0 and 25 °C has been studied by spectrophotometry, equilibrium dialysis, and/or ultrafiltration method. Binding parameters were determined assuming one or two classes of independent binding sites. By combining with data reported before, we examined the effects of hydrophobicity and net charge number of ligands on the binding parameters for a total of 19 dyes. The ratio of the binding ability to the primary sites to the total binding ability, a measure of binding specificity, was found to increase with an increase in the anionic charge number of ligands. The values of binding constant for nonspecific binding of the monoanionic dyes and for the primary binding of the multicharged dyes were found to have a strong correlation to both the hydrophobicity and the anionic charge number of ligands. A multiple regression analysis has derived the equation: where K is a binding constant, P is the partition coefficient in 1-octanol/water system, and m and αi are respectively the number of anionic groups and the degree of dissociation of the i-th group. From this equation, the contribution of electrostatic interactions due to a unit net charge on ligands to the standard free energy change of binding was estimated to be −11.4 kJ mol−1. The role of electrostatic interactions has been discussed in detail.

Micellar Binding of Phenoxide Ions to Cetyltrimethylammonium Chloride (CTAC)

The interaction of phenolic compounds, phenol (PhOH); 2-chlorophenol (2-Cl-PhOH); 2,5-dichlorophenol (2,5-DCPhOH); 2,6-dimethylphenol (2,6-DMPhOH); and 4-bromo-2,6-dimethylphenol (4-Br-2,6-DMPhOH), in their aoionic forms with cetyltrimethylammonium chloride (CTAC) micelles was studied. The concentration of phenoxide ions in the micellar pseudophase was measured from the observed absorbance changes upon the addition of detergent to an alkaline aqueous solution. The luminescence quenching of tris(2,2′bipyridine)ruthenium(II) dichloride was used to measure the concentration of phenoxide ions in the intramicellar aqueous pseudophase for 2,6-DMPhOH and 4-Br-2,6-DMPhOH. To apply this method, two different series of quenching experiments were carried out in which the quencher concentration and the CTAC concentration were either varied or fixed. The results can be interpreted in terms of binding to a single class of independent binding sites. The site-binding constants ( K b), their respective site numbers per micellized detergent ( p), and the critical micelle concentration of the detergent in the presence of the substrates were obtained.