• YAKHAK HOEJI
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• v.26 no.2
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• pp.85-90
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• 1982

The binding of the 1-anilinonaphthalene-8-sulfonate(ANS) to bovine serum albumin was studied by fluorescence spectroscopy. The effect of pH, ionic strength, and protein concentration on the binding of ANS to protein were compared. The binding between ANS and protein was dependent on pH and ionic strength. It seems that both hydrophobic binding and some electrostatic forces are involved in the binding of ANS to protein. The binding constants for ANS increased with increasing protein concentration. This suggests the possibility of a sharing of one ANS molecule by more than one protein molecule at relatively high protein concentration.

• Journal of Pharmaceutical Investigation
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• v.18 no.3
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• pp.113-123
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• 1988

A series of water-soluble polyparacyclophanes bearing two diphenylmethane or two diphenyl ether skeletons were investigated to develop useful host compounds by using 1-anilinonaphthalene-6-sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as fluorescent hydrophobic substrates in aqueous solution. It was noteworthy that remarkable fluorescent enhancements and blue shifts of ANS and TNS were observed only in the presence of 1,6,20,25-tetraaza[6.1.6.1] paracyclophane (CPM 44) and 1,6,21,27-tetraaza [7.1.7.1] paracyclophane (CPM 55) for diphenylmethane skeleton, and 1,7,21,27-tetraaza-14,34-dioxa [7.1.7.1] paracyclophane (CPE 55) and 1,8,22,29-tetraaza-15,36-dioxa [8.1.8.1] paracyclophane (CPE 66) for diphenyl ether skeleton, comparing with ${\alpha}-\;and\;{\beta}-cyclodextrins$. However, their acyclic analogues such as 4,4'-dimethylaminodiphenylmethane and 4,4'-dimethylaminodiphenyl ether, and paracyclophanes whose cavities were smaller showed only small effects under the same conditions. These facts suggested that hosts and substrates were in an intimate contact which would not occur without larger structures, and thus that guest molecules were strongly incorporated in the hydrophobic cavities of these larger paracyclophanes. The effects of pH on the fluorescent intensity of ANS-CPM 44, ANS-CPM 55, ANS-CPE 55, ANS-CPE 66, TNS-CPM 44, TNS-CPM 55, TNS-CPE 55 and TNS-CPE 66 systems were not significant below pH 2.0, but their fluorescent intensities were markedly reduced with increasing ionic strength.

• Journal of Pharmaceutical Investigation
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• v.21 no.3
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• pp.133-141
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• 1991

The fluorescence characteristics of l-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) made the dyes useful probes for the determination of the polarity at the binding sites of several water-soluble polyparacyclophanes. Polyparacyclophanes used were 1,6,20,25-tetraaza[ 6.1.6.1]paracyclophane (CPM 44), 1,7,21,27 -tetraaza[7.1.7.1]paracyclophane (CPM 55). 1,7,21,27 -tetraaza-14,34-dioxa[7.1.7.1]paracyclophane (CPE 55) and 1,8,22,29-tetraaza-15,36-dioxa[8.1.8.1] paracyclophane (CPE 66). The fluorescence quantum yield, emission maximum, and half bandwidth of ANS or TNS obtained in a variety of solvent systems were plotted as a function of four kinds of empirical solvent polarity scales such as dielectric constant (D), (D-l)/(2D+1). Y and Z values. It was found that the Z-value-emission maximum $(\overline}V_F,\;cm^{-1})$ profile showed the most reliable linearity. ANS and TNS interacted with CPM 44, CPM 55, CPE 55. CPE 66. ${\alpha}-cyclodextrin$ (CyD) and ${\beta}-CyD$ in the aqueous solution, and from the emission maxima the polarities (Z-value) of their binding sites were calculated to be 92.65, 87.50, 93.35, 84.52, 94.36, and 90.48 for ANS, respectively. and 91.07, 89.68, 85.44, 86.74 and 87.6 for TNS except for ${\alpha}-CyD$, respectively.

• Journal of Pharmaceutical Investigation
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• v.19 no.2
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• pp.71-79
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• 1989

Complex formation of water-soluble polyparacyclophanes bearing two diphenylmethane or two diphenyl ether skeletons with l-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated quantitatively to develop useful host compounds comparing with ${\alpha}\;-\;and\;{\beta}-cyc1odextrins$$({\alpha}-\;and\;{\beta}-CyDs$) in aqueous solution. Benesi-Hildebrand type analysis of the fluorescent intensity showed that the dissociation constants (Kd) of paracyclophane-ANS complexes were $1.55\;{\times}\;10^{-4}M$ for 1,6,20,25-tetraaza[6.1.6.1]paracyclophane(CPM 44) and $1.23\;{\times}\;10^{-4}M$ for 1,7,21,27-tetraaza[7.1.7.1]paracyclophane (CPM 55), and those of paracyclophane-TNS complexes were $6.99\;{\times}\;10^{-6}M$ for CPM 44 and $6.23\;{\times}\;10^{-5}M$ for CPM 55, in 1:1 molar ratio. On the other hand, the Kd values of 1,7,21,27-tetraaza-14,34-dioxa[7.1.7.1]paracyclophane (CPE 55)-ANS, 1,8,22,29-tetraaza-15,36-dioxa[8.1.8.1]paracyclophane (CPE 66)-ANS, CPE 55-TNS, CPE 66-TNS complexes were $1.75\;{\times}\;10^{-3}M$, $3.07\;{\times}\;10^{-3}M$, $3.75\;{\times}\;10^{-3}M$ and $2.15\;{\times}\;10^{-3}M$, respectively. On the contrary, the Kd values of ${\alpha}-CyD-ANS$, ${\beta}-CyD-ANS$, ${\alpha}-CyD-TNS$ and ${\beta}-CyD-TNS$ complexes were found to be $3.98\;{\times}\;10^{-2}M$, $1.05\;{\times}\;10^{-2}M$, $1.38\;{\times}\;10^{-2}M$ and $3.52\;{\times}\;10^{-4}M$, respectively. These results mean that the complexation of CPMs with ANS or TNS is by 5.6-1,975 fold stronger than that for ${\alpha}-or\;{\beta}-CyDs$, and the complex formation of CPEs with ANS or TNS is nearly same as or somewhat stronger than that for ${\alpha}-or\;{\beta}-CyDs$. From the Kd values determined at different temperatures, thermodynamic parameters were calculated and the complexation was found to be a spontaneous exothermic reaction. The effects of pH on Kd values of CPM 44-ANS, and CPM 55-ANS complexes were negligible in the range of pH 1.2-1.8. However, the Kd values of these complexes increased significantly with increasing ionic strength.

• Archives of Pharmacal Research
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• v.6 no.1
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• pp.55-62
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• 1983

Binding of six cephalosporins (cefotaxime, cefuroxime, cefazoline, cephalothin, cephaloridine, cephacetrile) to bovine serum albumin was studied. Fluorescence probe technique and difference spectrophotometry were employed to evaluate the nature and degree of association of cephalosporin albumin complex. 1-Anilinonaphthalene-8-sulfonate (ANS) was used as the fluorescence probe. 2-(4'-hydroxybenzeneazo) benzoic acid(HBAB) was employed as the UV spectrophotometric probe. Compentitive bindings between cephalosporins and probes were observed. The number of binding sites of bovine serum albumin for each cephalcsporin is 2. Among six cephaloporins, cefotaxime has the highest binding constant followed by cafazoline, cefuroxime, cephalothin, cephaloridine and cephacetrile.

• Archives of Pharmacal Research
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• v.7 no.1
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• pp.11-15
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• 1984

Binding of sic cephalosporins (cefotaxime, cefuroxime, cafazoline, cephalothin, cephaloridine, cephacetrile) to human serum albumin was studied. Fluorescence probe technique and difference spectrophotometry were employed to evaluate the nature and degree of association of cephalosporin-albumin complex. 1-anilinonaphthalene-8-surfonate was used as the fluorescence probe, and 2-(4'-hydroxybenzeneazo)benzoic acid as the UV spectrophotometric probe. Competitive bindings between cephalosporins and probe were observed. For the binding of cephalosporins to human serum albumin, three binding sites were identified by fluorescence probe technique but four binding constants of cephalosporins to human serum albumin measured by fluorescence probe technique are higher than those meausred by UV spectrophotometry.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.10 no.1
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• pp.13-23
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• 1984

The fluorescence intensity rations (F2.F1) of excimer (F2) to monomer (F1) of pyrene were measured as a function of the concentration of sodium dodecyl sulfate (SDS). It was found that there were not gross changes in size and shape of sphere-shape micelles in the first micelle concentration, while at concentrations above the second critical micelle concentration (CMC) the micelles grew in size with increasing concentration. Fluorescence intensities of 8-anilinonaphthalene 1-sulfonate (ANS) were also monitored as a micellar probe with varying concentrations of SDS. Results suggested that a phase transition from sphere-shaped micelles to hemicapped rod-like micelles occurred at the second CMC (17). A general formula for the axial ratio of ellip-soil-shaped micelle in the first micelle concentration was suggested. According to this general formula, the axial ratio of SDS, sodium lauryl ether sulfate and sodium laurate were 1:1, 5:2, and 5:3, respectively. The electrolyte-induced phase transition from spherical to hemicapped rod-like micelles occurred and the size of hemicapped rod-like micelles grew with increasing electrolyte concentrations. The maximum concentrations of solubilzed benzene in sphere-shaped micelles and hemicapped rod-like micelles were measured by differential spectrohpotometry. The hemicapped rod-like micelles in the presence of electrolytes grew in size with increasing amount of benzene solubilized.

• KSBB Journal
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• v.14 no.6
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• pp.651-654
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• 1999

Using recombinant human growth hormone as a model protein, we carried out unfolding by adding a denaturant such as urea, guanidine HCl, or SDS followed by refolding by dilution and dialysis. The objectives were to monitor the structural changes during in vitro refolding process and, based on the results, to develop a quantitative method of refolding progress assessment. The changes in surface hydrophobicity were measured by fluorescence tagging of 1-anilinonaphthalene-8-sulfonate(1,8-ANS) to the hydrophobic portions, and those in the secondary structure were monitored by using far UV-CD(circular dichroism) spectroscopy. Also, we used RP-HPLC to separate and quantify the folded and unfolded proteins to correlate the result with the structure analysis. Our results indicate the surface hydrophobicity are well correlated with the formations of the secondary structure, primarily ${\alpha}$-helices, as well as the disulfide bridges. We expect this monitoring technique can be applied in industrial fields as a means to quantitatively assess the progress of in-vitro refolding of recombinant proteins.