Kunio Takeda

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.

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of α-helix, β-structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of α-helix decreased above 30°C and those of β-structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.

Secondary Structural Change of Bovine Serum Albumin in Thermal Denaturation up to 130 °C and Protective Effect of Sodium Dodecyl Sulfate on the Change

The secondary structure of bovine serum albumin (BSA) was first examined in the thermal denaturation up to 130 degrees C. The helicity (66%) of the protein decreased with rise of temperature. Half of the original helicity was lost at 80 degrees C, but the helicity of 16% was still maintained even at 130 degrees C. When the BSA solution was cooled down to 25 degrees C after heating at temperatures above 50 degrees C, the helicity was not completely recovered. The higher the thermal denaturation temperature was, the lower was the recovered helicity. On the other hand, upon the addition of sodium dodecyl sulfate (SDS), the secondary structure of BSA was partially protected against the thermal denaturation above 50 degrees C where the structural change became irreversible. A particular protective effect was observed below 85 degrees C upon the coexistence of SDS of extremely low concentrations. For example, the helicity was 34% at 80 degrees C in the absence of SDS, but it was maintained at 58% at the same temperature upon the coexistence of 0.75 mM SDS. Upon cooling down from 80 to 25 degrees C, the helicity of BSA was recovered to 62% in the presence of 0.75 mM SDS. Such a protective effect of SDS was not observed above 95 degrees C. In the interaction with the surfactant, this protein structure appeared likely to have a critical temperature between 90 and 100 degrees C in addition to the critical temperature in the vicinity of 50 degrees C. This protective effect of SDS, characterized by the specific amphiphilic nature of this anionic surfactant, is considered to be attained by building cross-linking bridges between particular nonpolar residues and particular positively charged residues in the protein molecule.

Protective effect of small amounts of sodium dodecyl sulfate on the helical structure of bovine serum albumin in thermal denaturation

In the presence of sodium dodecyl sulfate (SDS), the secondary structure of bovine serum albumin (BSA) was almost protected against thermal denaturation above 50 degrees C, where the structural change became irreversible. Beyond 30 degrees C, the helicity (66%) of the protein sharply decreased with rise of temperature. In response to this, the proportions of beta-structure and random coil increased. The helicity and the beta-structural proportion were 44% and 13% at 65 degrees C, respectively. The protective effect was observed upon the coexistence of SDS of extremely low concentrations: the molar ratio of [SDS]/[BSA] of 15 was enough to induce the maximal protective effect on the helical structure of the protein. The maximal protected helicity was 58% at 65 degrees C, increasing to 64% upon cooling down to 25 degrees C. This protective effect became greater with an increase of chain length of alkyl sulfate ion. On the other hand, a cationic surfactant did not protect the BSA structure at all against the thermal denaturation. This protective effect was characterized by the specific amphiphilic nature of anionic surfactant. Such an anionic surfactant is considered to protect the protein structure by building bridges between particular nonpolar residues and particular positively charged residues located on different loops of the protein.

Stepwise formation of complexes between sodium dodecyl sulfate and bovine serum albumin detected by measurements of electric conductivity, binding isotherm, and circular dichroism

Abstract Bovine serum albumin in aqueous solution of low salt concentration was titrated conductometrically with sodium dodecyl sulfate (SDS). Plots of conductance versus concentration of SDS revealed four breaks. Difference absorption, circular dichroism, and binding curves were measured in parallel to make clear the nature of the breaks. Three transitional points were commonly observed in the plots of these parameters versus concentration of SDS, suggesting stepwise nature of the denaturation of bovine serum albumin by SDS. These points were found to correspond to the three of the four breaks observed in the conductometry.

Secondary structures of bovine serum albumin in anionic and cationic surfactant solutions

Abstract Secondary structure changes of bovine serum albumin were examined in solutions of sodium decyl sulfate (SDeS), sodium dodecyl sulfate (SDS), decyltrimethylammonium bromide (DeTAB), dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB), and hexadecyltrimethylammonium bromide (HTAB). The relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed circular dichroism (CD) spectrum of reference spectra of the corresponding structures to the experimentally obtained spectrum of the protein. The helical proportion of the protein was 66% at neutral pH. This helical proportion of the protein decreased to 52% in SDeS, to 50% in SDS, to 51% in DeTAB, to 49% in DTAB, and to 47% in TTAB and HTAB. All of these surfactants caused changes in the helical proportion from 66 to approximately 50%. However, the shorter the hydrocarbon chain a surfactant had, the higher the concentration necessary to cause the same decrease in the helical proportion. The results also indicate that the longer the hydrocarbon chain of the surfactant the greater the decrease in the helical proportion. In this study the proportion of β-structure increased slightly in every surfactant solution except DeTAB. The well-known complexes AD n (A = BSA, D = SDS, n = 40–55) and AD 2 n are formed when the helical proportion of BSA decreases to 62 and 54%, respectively. The authors speculated, on the basis of Browns model, which segments of the helix in BSA unfold in surfactant solutions.

Secondary structure changes of disulfide bridge-cleaved bovine serum albumin in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

Abstract Secondary structure of bovine serum albumin, disulfide bridges of which were reduced and blocked by iodoacetamide, was examined in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate. The profile of structural changes in the reduced albumin was compared with that in the unreduced protein with the intact disulfide bridges. The relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed circular dichroism (CD) spectrum of reference spectra of the corresponding structures to the experimentally obtained spectrum of the protein. The helical proportion was 25% for the reduced albumina dn the proportion increased up to 50% in a dodecyl sulfate solution. On the other hand, the helical proportion is 66% for the unreduced protein and it decreases down to 50% by the addition of the identical surfactant [K. Takeda et al., J. Colloid Interface Sci. 117, 120 (1987)]. The helical proportions of both the reduced and the unreduced proteins decreased in the presence of urea and guanidine. the unreduced albumin resisted the unfolding of the helix below 4 M urea and a 1.5 M guanidine. In contrast, the helical proportion in the reduced protein began to decrease even in lower concentrations of the two denaturants.

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.

Circular dichroic study of conformational changes in ovalbumin

By simulation of the circular dichroic spectra (Greenfield and Fasman (1969)) and using reference spectra of Chen et al. (1974), native ovalbumin was estimated to contain 33% α-helix, 5% β-structure, and 62% random coil. Ovalbumin resisted conformational changes in solutions of urea and of SDS. However, guanidine induced transition, starting at about 2 M and completing at about 4.5 M. At concentrations exceeding 4.5 M guanidine, ovalbumin existed as 6–7% α-helical, 12–13% β-structure, and 80–81% random coil. Ovalbumin after denaturation in 6 M guanidine or in 8 M urea (incubated at 4°C for 24 hr) did not recover the native conformation but acquired a new conformation in each case, with a somewhat destabilized helical structure.