Yoshiko Moriyama

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.

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.

Circular dichroism studies on helical structure preferences of amino acid residues of proteins caused by sodium dodecyl sulfate

The extent of helical structure of 19 intact proteins and of 15 proteins with no disulfide bridges in the absence and presence of 10 mM sodium dodecyl sulfate (SDS) was determined using the curve-fitting method of circular dichroic spectra. The change in helicity caused by the addition of SDS was examined as a function of each amino acid fraction. An increase in the helicity upon the addition of SDS occurred in most of the proteins with no disulfide bridges (C proteins) and containing more than 0.06 Lys fraction. In most of the intact proteins (B proteins), most of which contained disulfide bridges, helicity in SDS decreased with an increase in Lys fraction. The helicity of the C proteins in SDS also tended to increase with an increase in the Leu and Phe fractions, while it decreased with an increase in the Gly fraction. For the helicity of the B proteins in SDS, there was a tendency to increase with increased Asn fraction and decrease with increased His fraction. On the other hand, amino acids were divided into eight groups according to their side-chain properties and the conformational preference for each of the amino acid groups of C proteins was calculated using a simple assumption.

Critical Temperature of Secondary Structural Change of Myoglobin in Thermal Denaturation up to 130 °C and Effect of Sodium Dodecyl Sulfate on the Change

The secondary structural change of horse heart myoglobin was examined in the thermal denaturation up to 130 degrees C. The original helicity of 82% gradually decreased to 67% with rise of temperature until 75 degrees C. Thereafter, it suddenly decreased to 24% at 90 degrees C and then slightly decreased to 14% at 130 degrees C. The helices of this protein were mostly destroyed between 75 and 100 degrees C. On the other hand, upon cooling to 25 degrees C from temperatures below 75 degrees C, the helicity completely recovered to the original value, but it did not after heating to temperatures above 80 degrees C. Thus, myoglobin maintains the reversibility of the structural change up to a temperature as high as 75 degrees C. This protein had another critical temperature around 90-100 degrees C in addition to 75 degrees C in the present thermal denaturation. Upon cooling to 25 degrees C after heating to temperatures above 80 degrees C, the extent of recovered helicity decreased with rise of temperature before cooling. The additive effect of sodium dodecyl sulfate (SDS) on the structural change of myoglobin differed below and above the critical temperature at 75 degrees C. In the temperature range below 75 degrees C where the structural change was reversible, the presence of SDS cooperated with the thermal denaturation to disrupt the structure. On the contrary, the presence of the surfactant more or less restrained the decrement of helicity at high temperatures above 85 degrees C. The helicity decreased and increased with an increase of SDS concentration upon cooling to 25 degrees C after heating to temperatures below 75 degrees C and after heating to temperatures above 85 degrees C, respectively. Then, upon cooling to 25 degrees C from any temperature, the helicity settled to a magnitude around 60% in the presence of the surfactant above 0.6 mM.

Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.

Secondary structural changes of homologous proteins, lysozyme and α-lactalbumin, in thermal denaturation up to 130 °C and sodium dodecyl sulfate (SDS) effects on these changes: comparison of thermal stabilities of SDS-induced helical structures in these proteins.

The thermal stability of two homologous proteins, lysozyme and α-lactalbumin, was examined by circular dichroism. The present study clearly showed two different aspects between the homologous proteins: (1) the original helices of lysozyme and α-lactalbumin were unchanged at heat treatments up to 60 and 40 °C, respectively, indicating a higher thermal stability of lysozyme, and (2) upon cooling to 25 °C, the original helices of lysozyme were never reformed after they were once disrupted, while those of α-lactalbumin, disrupted at a particular temperature range between 40 and 60 °C, were completely reformed. In addition, the structural changes were also examined in the coexistence of sodium dodecyl sulfate (SDS), which induced the formation of helical structures in these proteins at 25 °C. A distinct difference appeared in the thermal stabilities of the SDS-induced helices. All of the SDS-induced helices of lysozyme were disrupted below 60 °C, while those of α-lactalbumin at 10 mM SDS were unchanged up to 130 °C. A similarity was also fixed. Not only the SDS-induced helices but also the original helices of the two proteins were reformed upon cooling to 25 °C after the thermal denaturation below 100 °C in the coexistence of 10 mM SDS.

Secondary structural changes in the intact and the disulfide Bridges cleaved β-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of α-helix, β-sheet, and unordered form in β-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of β-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% α-helix and 41% β-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased β-sheet up to 48% but did not affect the α-helical proportion. The α-helical proportions of nonreduced β-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the α-helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The β-sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.