Kazuaki Hachiya

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: A comparative study of the two and one tryptophan(s) of bovine and human albumins

The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (λmax) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the λmax of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The λmax of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecyltrimethylammonium bromide, the λmax positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.

Size and mobility of sodium dodecyl sulfate-bovine serum albumin complex as studied by dynamic light scattering and electrophoretic light scattering

Abstract Electrophoretic light scattering and dynamic light scattering methods were applied to measure the electrophoretic mobilities and radii of the complexes of bovine serum albumin (BSA) with sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB). In the phosphate buffer of pH 7.0 and ionic strength 0.014, the mobility of BSA, μBSA, was −1.7 × 10−4 cm2 s−1 V−1. The negative magnitude of μBSA sharply increased at low SDS concentrations below 2 mM. The negative mobility sharply increased again above 5 mM SDS and reached −4.7 × 10−4 cm2 s−1 V−1 at 8 mM. In the DTAB solution, μBSA remained negative below 6 mM. It crossed zero mobility at 6 mM DTAB and became positive beyond this concentration. The mobilities of BSA—SDS and BSA—DTAB complexes attained at high surfactant concentrations were appreciably smaller than those of the corresponding surfactant micelles. On the other hand, the effective hydrodynamic radius of BSA, RBSA, was estimated to be 3.1 nm. The magnitude of RBSA increased up to 6.0 and 5.2 nm with increases of SDS and DTAB concentrations, respectively. The changes in these μBSA and RBSA values occurred in the surfactant concentration ranges where the secondary structure of BSA was disrupted. The secondary structural change of the protein appeared likely to accompany a large-scale tertiary structural change.

Secondary structure change of myoglobin induced by sodium dodecyl sulfate and its kinetic aspects

Abstract Conformational change of horse myoglobin induced by sodium dodecyl sulfate (NaDodSO 4 ) was studied mainly by measurements of circular dichroism (CD) and stopped-flow methods. The relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed CD spectrum of reference spectra of the corresponding structures to the experimentally obtained CD spectrum of the protein. The helical content of the native myoglobin was found to be 82%. In the presence of 0.6 m M NaDodSO 4 , the helical content was decreased to 58% and the β-structure, which did not exist in the absence of the surfactant, was assumed to some extent above 0.3 m M NaDodSO 4 . This conformational change was followed in the Soret band as well as at 288 nm by the stopped-flow spectrophotometry. The corresponding rate constants were determined by measuring absorbance changes with time at these wavelengths. The experiments indicated that the environmental change of the heme group occurs simultaneously with the destruction of the helix. The rate constants showed NaDodSO 4 concentration dependence similar to those of conformational changes in other proteins in the surfactant solution. The binding isotherm of NaDodSO 4 to myoglobin was obtained using high-performance liquid chromatography. The binding sites of this anionic surfactant were estimated to be 0.8 by the Scatchard plot. The conformational change in myoglobin induced by NaDodSO 4 was correlated with its amino acid sequence in which each amino acid residue was considered to be either helical or non-helical.

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellmans reagent, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.

Kinetic study of ion exchange reaction between lysozyme and carboxymethyl Sephadex C-25

The ion-exchange reaction of lysozyme with carboxymethyl Sephadex C-25 was followed by conductivity change as a function of time just after the rapid mixing of the protein solution with the Sephadex suspension. A single relaxation process was observed; the conductivity increased exponentially with time in the 100 s scale. In this process, protons were released from the Sephadex C-25 in the same time scale. The relaxation process slowed down with an increase in the lysozyme concentration, but it quickened upon the addition of HCl. On the other hand, the ζ potential on the Sephadex C-25 surface changed from a negative value to a positive one with an increase in the amount of lysozyme adsorbed on the surface. On the basis of these data, the relaxation process was attributed to the ion-exchange reaction of lysozyme with several protons of carboxymethyl groups of the Sephadex.

Pressure-jump method to adsorption-desorption kinetics

The adsorption of ions on metal oxides and layered compounds has been studied in various fields of soil chemistry, geochemistry, colloid science and catalytic chemistry (Davis et al. 1978; Davis & Leckie 1978; Davis & Leckie 1980; Whittinham & Jacobson 1982). However, the adsorption reaction is so immeasurably fast that detailed examination of the kinetic mechanism could not be made until recently. Now the pressure-jump method was applied to the proton adsorption-desorption at Ti02-HP interface, and the detailed adsorption-desorption mechanism and kinetic parameters were determined (Ashida et al. 1978). This kinetic method was applied also to the adsorption-desorption kinetics ofpotential determining ions (Ashida et al. 1980; Ikeda et al. 1981; Astumian et al. 1981; Ikeda et al. 1982; Sasaki et al. 1982) and divalent metal ions (Hachiya et al. 1979; Hachiya et al. 1984a, b) and the intercalation-deintercalation kinetics ofadsorbing ions and molecules on layered compounds such as montmorillonite, and zirconium phosphate (Sasaki et al. 1982; Ikeda et al. 1983; Ikeda et al. 1984a,b,c; Mikami et al. 1986). These studies at the solidi solution interface were recently reviewed by Yasunaga and Ikeda (1986). As a result, the pressure-jump method has been recognized as a useful technique to investigate the mechanism of adsorption of ions and molecules on particle surfaces. The system of the adsorption-desorption seems to be different from that of an ordinary homogeneous solution. As a result the kinetic data must be treated under an appropriate concept to describe the system. The present review is mainly concerned with the pressurejump method and the fundamental concept in the kinetic treatment of the adsorptiondesorption reaction by this method.

Application of the pressure-jump method to adsorption—desorption kinetics: adsorption—desorption of the lithium ion on the carboxymethyl group of sephadex C-25(Li)

Abstract A single relaxation has been found in a suspension of the lithium-type Sephadex C-25(Li), which has carboxymethyl groups as cation-exchangeable sites, by using the pressure-jump technique with conductivity detection. The relaxation time decreased with increasing Li + concentration. The pH in the Sephadex C-25 (Li) suspension was 6.5–8.6. In this pH range, the carboxymethyl groups were almost dissociated, forming ion pairs with Li + ions. The amount of Li + ion adsorbed on the Sephadex surface decreased with an increase in LiCl concentration. The increase in LiCl concentration also induced a decrease in the pH. The relaxation was attributed to the adsorption-desorption reaction of Li + ions on the carboxymethyl group. The values of the rate constants of the Li + adsorption and desorption were determined to be 1.8±0.2·10 2 mol −1 dm 3 s −1 and 1.5±1.1·10 −1 s −1 at 25°C, respectively.

Kinetic study of the proton association-dissociation of the functional group on carboxymethyl sephadex C-25 by the pressure-jump method with conductivity detection

Abstract A kinetic study of the proton association—dissociation reaction on a Sephadex C-25 surface was made taking into account its surface potentials. The Sephadex C-25 itself had a ζ-potential of −32 mV in 3 m M NaCI solution. In order to explain this, the amount of negatively charged groups which the Sephadex originally had was determined by HCl titration. Then, the sum of the amount determined by this titration (0.82 mmol/g) and the amount determined by the usual NaOH titration (4.3 mmol/g) was used as the total amount of functional groups in calculating the acid dissociation constant, K a . The surface potential of the Sephadex was estimated from the K a . In parallel, the rate of the reaction was measured by a pressure-jump method with conductivity detection. A single relaxation was observed in the time scale of 0.1 s. The relaxation time became fast with an increase in pH. On the basis of the pH dependences of the relaxation time and the above surface potential, the relaxation was attributed to the proton association—dissociation reaction of functional groups (predominantly the carboxymethyl group) of the Sephadex.