Renaturation of Myoglobin Denatured by Sodium Dodecyl Sulfate by Removal of Dodecyl Sulfate Ions Bound to the Protein Using Sodium Cholate.

The secondary and tertiary structures of myoglobin were disrupted by sodium dodecyl sulfate (SDS) but were hardly affected by the bile salt, sodium cholate (NaCho). This disruption was induced by the binding of dodecyl sulfate (DS) ions to the protein. In this study, the removal of DS ions bound to the protein was attempted using NaCho. The extent of removal of DS ions was estimated by the restoration of the secondary and tertiary structures of the protein disrupted by SDS. The secondary structural change was followed by monitoring mean residue ellipticity at 222 nm, [θ]222, which was frequently used as a measure of α-helical content. The tertiary structural change was followed by examining the Soret band absorbance of the protein. Evidently, the magnitude of [θ]222 of myoglobin in the SDS solution initially decreased and then increased back to almost its original value as the NaCho concentration increased. The initial decrease in [θ]222 indicated the cooperation of NaCho and SDS in disrupting the secondary structure at low concentrations of both surfactants. This cooperation was also observed in the tertiary structural change as a shift of the Soret band maximum wavelength, λmax, and a decrease in the molar absorption coefficient, εmax, at λmax. Above a certain NaCho concentration, the position of λmax and the magnitude of εmax were also restored to their original states. The secondary and tertiary structures were simultaneously restored by adding NaCho. These recoveries were attributed to removal of the DS ions bound to the protein. This removal might be due to the ability of cholate anions to strip DS ions bound to the protein. The stripped DS ions are more likely to form SDS-NaCho mixed micelles in bulk than SDS-NaCho mixed aggregates on the protein.

Removal of Dodecyl Sulfate Ions Bound to Human and Bovine Serum Albumins Using Sodium Cholate.

The secondary structures of human serum albumin (HSA) and bovine serum albumin (BSA) were disrupted in the solution of sodium dodecyl sulfate (SDS), while being hardly damaged in the solution of the bile salt, sodium cholate (NaCho). In the present work, the removal of dodecyl sulfate (DS) ions bound to these proteins was attempted by adding various amounts of NaCho. The extent of removal was estimated by the restoration of α-helical structure of each protein disrupted by SDS. Increases and decreases in α-helical structure were examined using the mean residue ellipticity at 222 nm, [θ]222, which was frequently used as a measure of α-helical structure content. The magnitudes of [θ]222 of HSA and BSA, weakened by SDS, were restrengthened upon the addition of NaCho. This indicated that the α-helical structures of HSA and BSA that were disrupted by the binding of DS ions were nearly reformed by the addition of NaCho. The NaCho concentration at which the maximum restoration of [θ]222 of each protein was attained increased nearly linearly with SDS concentration. These results indicated that most of the bound DS ions were removed from the proteins but the removal was incomplete. The removal of DS ions, examined by means of the equilibrium dialysis, was also incomplete. The α-helical structure restoration and the DS ion removal by NaCho were considered to be due to the ability of cholate anions to strip the surfactant ions bound to HSA and BSA. These stripped DS ions appeared to be more likely to form SDS-NaCho mixed micelles in bulk rather than SDS-NaCho mixed aggregates on the proteins.

Secondary Structural Changes of Intact and Disulfide Bridges-Cleaved Human Serum Albumins in Thermal Denaturation up to 130°C - Additive Effects of Sodium Dodecyl Sulfate on the Changes.

The secondary structural changes of human serum albumin with the intact 17 disulfide bridges (HSA) and the disulfide bridges-cleaved human serum albumin (RCM-HSA) in thermal denaturation were examined. Most of the helical structures of HSA, whose original helicity was 66%, were sharply disrupted between 50 and 100°C. However, 14% helicity remained even at 130°C. The temperature dependence of the degree of disrupted helical structures of HSA was discussed in connection with questions about a general protein denaturation model. When HSA lost the disulfide bridges, about two-thirds of the original helices were disrupted. Although the helices of RCM-HSA remaining after the cleavage of the disulfide bridges were relatively resistant against the heat treatment, the helicity changed from 22% at 25°C to 14% at 130℃. The helicity of RCM-HSA at 130°C agreed with the helicity of HSA at the same temperature, indicating that the same helical moieties of the polypeptides remained unaffected at this high temperature. The additive effects of sodium dodecyl sulfate (SDS) on the structural changes of HSA and RCM-HSA in thermal denaturation were also examined. A slight amount of SDS protected the helical structures of HSA from thermal denaturation below 80°C. Upon cooling to 25°C after heat treatment at temperatures below 70°C with the coexistence of SDS of low concentrations, the helical structures of HSA were reformed to the original level at 25°C before heating. A similar tendency was also observed after heat treatment at 80°C. In contrast, the helical structures of the RCM-HSA complexes with SDS are completely recovered upon cooling to 25°C even after heat treatment up to 100°C. Similar investigations were also carried out on bovine serum albumins which had the intact 17 disulfide bridges and lost all of the bridges.

Kinetic Aspects of Surfactant-Induced Structural Changes of Proteins - Unsolved Problems of Two-State Model for Protein Denaturation -.

The kinetic mechanism of surfactant-induced protein denaturation is discussed on the basis of not only stopped-flow kinetic data but also the changes of protein helicities caused by the surfactants and the discontinuous mobility changes of surfactant-protein complexes. For example, the α-helical structures of bovine serum albumin (BSA) are partially disrupted due to the addition of sodium dodecyl sulfate (SDS). Formation of SDS-BSA complex can lead to only four complex types with specific mobilities depending on the surfactant concentration. On the other hand, the apparent rate constant of the structural change of BSA increases with an increase of SDS concentration, indicating that the rate of the structural change becomes fast as the degree of the change increases. When a certain amount of surfactant ions bind to proteins, their native structures transform directly to particular structures without passing through intermediate stages that might be induced due to the binding of fewer amounts of the surfactant ions. Furthermore, this review brings up a question about two-state and three-state models, N⇌D and N⇌D'⇌D (N: native state, D: denatured sate, D': intermediate between N and D), which have been often adopted without hesitation in discussion on general denaturations of proteins. First of all, doubtful is whether any equilibrium relationship exists in such denaturation reactions. It cannot be disregarded that the D states in these models differ depending on the changes of intensities of the denaturing factors. The authors emphasize that the denaturations or the structural changes of proteins should be discussed assuming one-way reaction models with no backward processes rather than assuming the reversible two-state reaction models or similar modified reaction models.

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.

Protein structural change in a mixed system of ionic and zwitterionic surfactants.

The secondary structure of bovine serum albumin (BSA) in the binary surfactant system of anionic sodium dodecyl sulfate (SDS) and zwitterionic N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DDAPS) was examined at 25°C. The helicity of BSA decreased from 66% to 55% in a solution of DDAPS alone and decreased to 50% in a solution of SDS alone. However, the late addition of DDAPS reformed the helical structure of BSA, which was initially disrupted by SDS. The reformation required higher DDAPS concentrations as the initial SDS concentration increased. A maximum helicity of 63% was attained by this reformation. On the other hand, the helical structure of the protein, which was first affected by DDAPS denaturation, was also reformed to the same degree by the late addition of certain amounts of SDS. Although attention was paid to the additive order of these two surfactants to BSA, the final helicity of the protein depended on the final concentrations of these two surfactants, irrespective of the additive order. These phenomena may be attributed to the predominance of mixed micelle formation over complex formation between BSA and the two surfactants below the mixing ratio of DDAPS ([DDAPS]/([DDAPS]+[SDS])) of 0.95. The predominance of the mixed micelle formation distinctly appeared in mixing ratios between 0.50 and 0.75. In this range, the mixed micelle formation accompanied the removal of dodecyl sulfate (DS) ions bound to BSA upon the late addition of DDAPS to the BSA-SDS mixture, whereas, upon the late addition of SDS to the BSA-DDAPS mixture, the mixed micelle formation was accelerated by the coexistence of DDAPS which disturbed the binding of DS ions to the protein.

Critical temperature of secondary structural change of myoglobin in thermal denaturation up to 130 degrees 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.

Protective effect of gemini surfactant on secondary structural change of bovine serum albumin in thermal denaturation up to 130 degrees C.

The effect of gemini surfactant, sodium dilauramidoglutamide lysine (DLGL), on the secondary structure of bovine serum albumin (BSA) was examined at 25 degrees C and at high temperatures up to 130 degrees C. The helicity (66%) of the protein decreased to 53% in the DLGL solution at 25 degrees C and it also decreased to 16% with rise of temperature. Although approximately half of the original helical structures were destroyed upon heating up to 75 degrees C, most of the structures were maximally protected in the coexistence of 0.30 mM DLGL at 75 degrees C (the protein concentration was 0.010 mM). At temperatures below 75 degrees C, the protected helicity became maximal at such low DLGL concentrations. In the thermal denaturations above 80 degrees C, the protective effect did not appear at low DLGL concentrations, but monotonously enlarged with the surfactant concentration. On the other hand, upon cooling to 25 degrees C after the thermal denaturations below 75 degrees C, the helicity was maximally recovered to about 60% in the presence of DLGL below 0.30 mM. Upon cooling to 25 degrees C from high temperatures above 85 degrees C, the recovered helicity gradually increased with the surfactant concentration. The present novel effect, especially observed at low DLGL concentrations, might be fulfilled by the monomer ions of the gemini surfactant, since actual binding numbers of DLGL onto BSA are necessarily smaller than the mixing ratios around 30 mol/mol.

Immunochromatographic assay using gold nanoparticles for measuring salivary secretory IgA in dogs as a stress marker.

The concentration of salivary secretory immunoglobulin A (sIgA) is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA) for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2) containing non-ionic surfactant (3 wt% Tween 20). The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.

Secondary structural change of bovine serum albumin in thermal denaturation up to 130 degrees 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 effects of small amounts of bis(2-ethylhexyl)sulfosuccinate on the helical structures of human and bovine serum albumins in their thermal denaturations.

The protective effect of an anionic double-tailed surfactant, sodium bis(2-ethylhexyl)sulfosuccinate (AOT), on the structures of human serum albumin (HSA) and bovine serum albumin (BSA) in their thermal denaturations was examined by means of circular dichroism measurements. The structural changes of these albumins were reversible in the thermal denaturation below 50 degrees C, but became partially irreversible above this temperature. The effect was observed in the thermal denaturation above 50 degrees C. Although the helicity of HSA decreased from 66% to 44% at 65 degrees C in the absence of the surfactant, the decrement of it was restrained in the coexistence of AOT of extremely low concentrations. When the HSA concentration was 10 muM, the maximal protective effect appeared at 0.15 mM AOT. In the coexistence of the surfactant of this concentration, the helicity was maintained at 58% at 65 degrees C, increasing to the original value upon cooling to 25 degrees C. Beyond 0.15 mM AOT, the helicity sharply decreased until 3 mM AOT. A particular AOT concentration required to induce the maximal protective effect ([AOT]REQ) was examined at different HSA concentrations. [AOT]REQ shifted to higher values with an increase of the protein concentration. From the protein concentration dependences of [AOT]REQ, the maximal protection was estimated to require 8.0 and 5.0 AOT ions per a molecule of HSA and BSA, respectively. The AOT concentration, where the protective effect was observed, was too low to form its micelle-like aggregate. Then the protein structures might be stabilized by a cross-linking of surfactant monomers bound to specific sites. These specific sites might exist between a group of nonpolar residues and a positively charged residue located on several sets of amphiphilic helical rods in the proteins. Such a unique function of the double-tailed ionic surfactant is first presented by its characteristic nature as an amphiphilic material.

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.