Apparent stability constant
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The partial-filling affinity capillary electrophoresis (pf-ACE) works with a ligand present in a background electrolyte that forms a weak complex with an analyte. In contrast to a more popular mobility-shift affinity capillary electrophoresis, only a short plug of the ligand is introduced into a capillary in the pf-ACE. Both methods can serve for determining apparent stability constants of the formed complexes but this task is hindered in the pf-ACE by the fact that the analyte spends only a part of its migration time in a contact with the ligand. In 1998, Amini and Westerlund published a linearization strategy that allows for extracting an effective mobility of an analyte in the presence of a neutral ligand out of the pf-ACE data. The main purpose of this paper is to show that the original formula is only approximate. We derive a new formula and demonstrate its applicability by means of computer simulations. We further inspect several strategies of data processing in the pf-ACE regarding a risk of an error propagation. This establishes a good practice of determining apparent stability constants of analyte-ligand complexes by means of the pf-ACE.
Affinity capillary electrophoresis (ACE) and pressure-assisted ACE were employed to study the noncovalent molecular interactions of antamanide (AA), cyclic decapeptide from the deadly poisonous fungus Amanita phalloides, with univalent (Li+ , Na+ , K+ , and NH4+ ) and divalent (Mg2+ and Ca2+ ) cations in methanol. The strength of these interactions was quantified by the apparent stability constants of the appropriate AA-cation complexes. The stability constants were calculated using the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of AA on the concentration of the above ions in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pHMeOH 7.8, containing 0-50 mM concentrations of the above ions added in the form of chlorides). Prior to stability constant calculation, the AA effective mobilities measured at actual temperature inside the capillary and at variable ionic strength of the BGEs were corrected to the values corresponding to the reference temperature of 25°C and to the constant ionic strength of 10 mM. From the above ions, sodium cation interacted with AA moderately strong with the stability constant 362 ± 16 L/mol. K+ , Mg2+ , and Ca2+ cations formed with AA weak complexes with stability constants in the range 37-31 L/mol decreasing in the order K+ > Ca2+ > Mg2+ . No interactions were observed between AA and small Li+ and large NH4+ cations.
Capillary affinity electrophoresis (CAE) has been employed to investigate quantitatively the interactions of valinomycin, macrocyclic depsipeptide antibiotic ionophore, with univalent cations, ammonium and alkali metal ions, K(+), Cs(+), Na(+), and Li(+), in methanol. The study involved measuring the change in effective electrophoretic mobility of valinomycin while the cation concentrations in the BGE were increased. The corresponding apparent stability (binding) constants of the valinomycin-univalent cation complexes were obtained from the dependence of valinomycin effective mobility on the cation concentration in BGE using a nonlinear regression analysis. The calculated apparent stability constants of the above-mentioned complexes show the substantially higher selectivity of valinomycin for K(+) and Cs(+) ions over Li(+), Na(+), and NH(4)(+) ions. CAE proved to be a suitable method for the investigation of both weak and strong interactions of valinomycin with small ions.
Affinity capillary electrophoresis (ACE) is typically used for the determination of stability constant, Kst, of weak to moderately strong complexes. Sensitive detection such as mass spectrometry (MS) is required for extension of ACE methodology for estimation of Kst of stronger complexes. Consequently, an efficient interface for hyphenation of CE with MS detection is necessary. For evaluation of interfaces for electrospray ionization mass spectrometric (ESI/MS) detection in ACE conditions, potassium-crown ether complexation was used as model system. The effective mobilities of the crown ether ligands and the Kst of their potassium complexes were measured/determined by ACE-ESI/MS using two lab-made interfaces: (i) a sheathless porous tip CE-ESI/MS interface and (ii) a nano-sheath liquid flow CE-ESI/MS interface, and, in turn, compared with those obtained by ACE with UV spectrophotometric detection. Apparent stability constant of potassium-crown ether complexes in 60/40 (v/v) methanol/water mixed solvent, pH* 5.5, was about 1300 L/mol for dibenzo-18-crown-6, 1600 L/mol for benzo-18-crown-6 and 5200 L/mol for 18-crown-6 ligands, respectively. It was observed that electrophoretic mobilities from CE-MS experiments differ from reference values determined by UV detection by ∼7% depending on the CE-MS interface used. Good agreement of CE-MS and CE-UV data was achieved for nano-sheath liquid flow interface, in which the spray potential and the CE separation potential can be effectively decoupled. As for sheathless porous tip interface, a correction procedure involving a mobility marker has been proposed. It provides typically only ca. 1% difference of effective mobilities and Kst values obtained from CE-MS data as compared to those received by the reference ACE-UV method.
Affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations have been employed for the investigation of noncovalent interactions between hexaarylbenzene-based receptor (R) and ammonium cation NH(4)(+). Firstly, by means of ACE, the binding constant of the NH(4)R(+) complex in methanol was estimated from the dependence of the effective electrophoretic mobility of the receptor R (in advance corrected by our earlier developed procedure to a reference temperature of 25°C) on the concentration of ammonium ion in the background electrolyte using non-linear regression analysis. The logarithmic form of the apparent binding (stability) constant of NH(4)R(+) complex in the methanolic background electrolyte (25 mM Tris, 50 mM chloroacetate, pH(MeOH) 7.8) was evaluated as log K(NH(4)R) = 4.03 ± 0.15. Secondly, the structural characteristics of NH(4)R(+) complex were determined by DFT calculations.
- MeSH
- benzenové deriváty chemie MeSH
- chromatografie afinitní metody MeSH
- elektroforéza kapilární metody MeSH
- kationty chemie MeSH
- kvartérní amoniové sloučeniny chemie MeSH
- methanol MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- teplota MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This article describes the characterization and application of collagenase-based chitosan nanofiber membranes with rat burns. Electrospun chitosan nanofibers were functionalized with clostridial collagenase using carbodiimide chemistry. The immobilized collagenase was characterized by enzyme activity, kinetic constants, and dry storage stability measurements using a Pz-peptide substrate. The apparent kinetic constants KM and Vmax of immobilized collagenase showed a high affinity for the peptide substrate compared to the free enzyme. Drying of chitosan membranes with immobilized collagenase ensured 98 % stability of enzyme activity after rehydration. The effect of collagenase immobilized on chitosan nanofibers on the burn of the rat model was compared with a control treatment with chitosan nanofibers. The healing of the wound with both materials was terminated after 30 days at the same time, although the collagenase wound healed more rapidly during healing. The scar area size after the application of collagenase-containing chitosan nanofiber membranes was 31.6 % smaller than when only chitosan nanofibers were used.
- MeSH
- chitosan terapeutické užití MeSH
- Clostridium histolyticum MeSH
- enzymy imobilizované MeSH
- hojení ran * účinky léků MeSH
- krysa rodu rattus MeSH
- kůže zranění MeSH
- mikrobiální kolagenasa * metabolismus terapeutické užití MeSH
- nanovlákna terapeutické užití MeSH
- pilotní projekty MeSH
- rány a poranění farmakoterapie patologie MeSH
- stabilita enzymů MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Current knowledge of sand fly salivary components has been based solely on Lutzomyia and Phlebotomus species which feed mainly on mammals; their hyaluronidases and apyrases were demonstrated to significantly affect blood meal intake and transmission of vector-borne pathogens. Members of the third sand fly genus Sergentomyia preferentially feed on reptiles but some of them are considered as Leishmania and arboviruses vectors; however, nothing is known about their salivary components that might be relevant for pathogens transmission. Here, marked hyaluronidase and apyrase activities were demonstrated in the saliva of a Sergentomyia schwetzi colony maintained on geckos. Hyaluronidase of S. schwetzi cleaved hyaluronan as the prominent substrate, and was active over a broad pH range from 4.0 to 8.0, with a sharp peak at pH 5.0. SDS PAGE zymography demonstrated the monomeric character of the enzyme, which remained active in reducing conditions. The apparent molecular weight of 43 kDa was substantially lower than in any sand fly species tested so far and may indicate relatively low grade of the glycosylation of the enzyme. The apyrase of S. schwetzi was typical strictly Ca2+ dependent Cimex-family apyrase. It was active over a pH range from 6.5 to 9.0, with a peak of activity at pH 8.5, and had an ATPase/ADPase ratio of 0.9. The apyrase activity increased during the first 3 days post-emergence, then reached a plateau and remained relatively constant until day 8. In comparison with a majority of Phlebotomus and Lutzomyia species tested to date, both the hyaluronidase and apyrase activities of S. schwetzi were relatively low, which may reflect an adaptation of this sand fly to blood feeding on non-mammalian hosts.
- MeSH
- glykosylace MeSH
- hmyzí proteiny chemie genetika metabolismus MeSH
- hyaluronoglukosaminidasa chemie metabolismus MeSH
- Psychodidae enzymologie genetika MeSH
- slinné proteiny a peptidy chemie genetika metabolismus MeSH
- stabilita enzymů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
ACE and density functional theory were employed to study the noncovalent interactions of cyclic decapeptide glycine-6-antamanide ([Gly6]AA), synthetic derivative of native antamanide (AA) peptide from the deadly poisonous fungus Amanita phalloides, with small cations (Li+, Rb+, Cs+, NH4+, and Ca2+) in methanol. The strength of these interactions was quantified by the apparent stability constants of the appropriate complexes determined by ACE. The stability constants were calculated using the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of [Gly6]AA on the concentration of the above ions in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pHMeOH7.8, containing 0-70 mM concentrations of the above ions added in the form of chlorides). Prior to stability constant calculation, the effective mobilities measured at actual temperature inside the capillary and at variable ionic strength of the BGEs were corrected to the values corresponding to the reference temperature of 25°C and to the constant ionic strength of 10 mM. From the above ions, Rb+and Cs+cations interacted weakly with [Gly6]AA but no interactions of [Gly6]AA with univalent Li+and NH4+ions and divalent Ca2+ion were observed. The apparent stability constants of [Gly6]AA-Rb+and [Gly6]AA-Cs+complexes were found to be equal to 13 ± 4 and 22 ± 3 L/mol, respectively. The structural characteristics of these complexes, such as position of the Rb+and Cs+ions in the cavity of the [Gly6]AA molecule and the interatomic distances within these complexes, were obtained by the density functional theory calculations.
This chapter deals with the application of affinity capillary electrophoresis (ACE) to investigation of noncovalent interactions (complexes) of valinomycin, a macrocyclic dodecadepsipeptide antibiotic ionophore, with ammonium and alkali metal ions (lithium, sodium, potassium, rubidium, and cesium). The strength of these interactions was characterized by the apparent binding (stability, association) constants (K b) of the above valinomycin complexes using the mobility shift assay mode of ACE. The study involved measurements of effective electrophoretic mobility of valinomycin at variable concentrations of ammonium or alkali metal ions in the background electrolyte (BGE). The effective electrophoretic mobilities of valinomycin measured at ambient temperature and variable ionic strength were first corrected to the reference temperature 25 °C and constant ionic strength (10 or 25 mM). Then, from the dependence of the corrected valinomycin effective mobility on the ammonium or alkali metal ion concentration in the BGE, the apparent binding constants of the valinomycin-ammonium or valinomycin-alkali metal ion complexes were determined using a nonlinear regression analysis. Logarithmic form of the binding constants (log K b) were found to be in the range of 1.50-4.63, decreasing in the order Rb(+) > K(+) > Cs(+) > > Na(+) > NH4 (+) ~ Li(+).
The interactions of valinomycin, macrocyclic depsipeptide antibiotic ionophore, with ammonium cation NH4+ have been investigated. Using quantum mechanical density functional theory (DFT) calculations, the most probable structure of the valinomycin-NH4+ complex species was predicted. In this complex, the ammonium cation is bound partly by three strong hydrogen bonds to three ester carbonyl oxygen atoms of valinomycin and partly by somewhat weaker hydrogen bonds to the remaining three ester carbonyl groups of the valinomycin ligand. The strength of the valinomycin-NH4+ complex was evaluated experimentally by capillary affinity electrophoresis. From the dependence of valinomycin effective electrophoretic mobility on the ammonium ion concentration in the background electrolyte, the apparent binding (association, stability) constant (Kb) of the valinomycin-NH4+ complex in methanol was evaluated as log Kb = 1.52 +/- 0.22.
