Spectroscopic studies on the gemini surfactant mediated refolding of human serum albumin.

Refolding of guanidinium hydrochloride (GdCl) denatured human serum albumin (HSA) using a combination of cationic gemini surfactants; pentanediyl-α,ω-bis(cetyldimethylammonium bromide) (C16H33(CH3)2N+-(CH2)5-N+(CH3)2C16H33)2Br- designated as G5 and methyl- ß-cyclodextrin, is attempted in the present study. The studies were carried out in an aqueous medium (pH 7.4) using dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy. A careful study of the DLS data indicates that against the hydrodynamic radius (Rh) of 3.5nm in native human serum albumin (HSA), hydrodynamic radius after attempting refolding by simple dilution increases to 33.8nm. The large Rh values of the diluted protein sample is associated with the formation of aggregates as dilution is an aggregation prone pathway. Hydrodynamic radii equal to 5.4nm, that is very near to the native protein (3.5nm), is obtained on the sequential addition of G5 and methyl- ß-cyclodextrin to the denatured protein. The results obtained from the multi-technique approach are associated with the presence of two charged head-groups and two hydrocarbon tails in the gemini surfactants resulting in very strong electrostatic and hydrophobic interactions.The present study suggests that gemini surfactants may be utilised in the protein refolding studies and may prove to be inexpensive and efficient folding agents.

Inhibitory effect of copper nanoparticles on rosin modified surfactant induced aggregation of lysozyme.

Protein aggregation is associated with many serious diseases including Parkinson's and Alzheimer's. Protein aggregation is a primary problem related with the health of industrial workers who work with the surfactants, metal ions, and cosolvents. We have synthesized rosin-based surfactants, i.e., quaternary amines of rosin diethylaminoethyl esters (QRMAE), which is an ester of rosin acid with polyethylene glycol monomethyl ether. Here, we report the thermal aggregation of lysozyme induced by QRMAE at 65 °C and pH 7.4 for a given time period in which amorphous aggregates are formed and confirm that copper-nanoparticles have the ability to inhibit QRMAE-induced aggregation compared with zinc and silver-nanoparticles. Aggregation experiments was evaluated using several spectroscopic methods and dye binding assay, such as turbidity, Rayleigh light scattering, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) and circular dichroism (CD), that was further supported by scanning electron microscopy (SEM) and SEM with EDX. The therapeutic use of nanoparticles and the fact that rosin possesses excellent film-forming properties, and that its derivatives have pharmaceuticals application such as micro encapsulation, coating and film forming, it's matrix materials are used for sustained and controlled release tablets, renders importance and application to the present study.

Secondary structural changes in guanidinium hydrochloride denatured mammalian serum albumins and protective effect of small amounts of cationic gemini surfactant pentanediyl-α,ω-bis(cetyldimethylammonium bromide) and methyl-ß-cyclodextrin: A spectroscopic study.

In the present study the cationic gemini surfactant assisted refolding of guanidinium hydrochloride (GdCl) denatured mammalian serum albumins viz. sheep serum albumin (SSA), rat serum albumin (RSA) and porcine serum albumin (PSA) using a combination of cationic gemini surfactants, pentanediyl-α,ω-bis(cetyldimethylammonium bromide) (C16H33(CH3)2N(+)-(CH2)5-N(+)(CH3)2C16H33)⋅2Br(-) designated as G5 and methyl-ß-cyclodextrin in the artificial chaperone assisted two step method, is attempted. The studies were carried out in an aqueous medium (pH 7.4) using dynamic light scattering (DLS), circular dichroism (CD), and fluorescence spectroscopy. A perusal of DLS data indicates that against the native hydrodynamic radius (Rh) of 4.3nm in SSA, 3.9nm in PSA and 3.5nm in RSA, the Rh of the said proteins, when refolding is attempted by simple dilution, increases to 21.7nm, 36.6nm and 37.2nm, respectively. Hydrodynamic radii very near to the native protein, i.e., 4.0nm, 4.1nm and 4.4nm for RSA, PSA and SSA respectively, is obtained on the sequential addition of G5 and methyl-ß-cyclodextrin to the denatured protein. Circular dichroism studies corroborate with the DLS data. The results obtained from the multi-technique approach are ascribed to the presence of two charged head-groups and two hydrocarbon tails in the gemini surfactants resulting in a very strong electrostatic and hydrophobic interactions. Based on the present study it is suggested that the gemini surfactants may be utilized in the protein refolding studies and thus may address one of the most pressing demand of biotechnology industry for the development of efficient and inexpensive folding aides.

Refolding of bovine serum albumin via artificial chaperone protocol using gemini surfactants.

Surfactants prevent the irreversible aggregation of partially refolded proteins, and they are also known to assist in protein refolding. A novel approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and cyclodextrin, was proposed by Rozema and Gellman (D. Rozema, S.H. Gellman, J. Am. Chem. Soc. 117 (1995) 2373). We report the refolding of bovine serum albumin (BSA) assisted by these artificial chaperones, utilizing gemini surfactants for the first time. A combination of cationic gemini surfactants, bis(cetyldimethylammonium)pentane dibromide (C(16)H(33)(CH(3))(2)N(+)-(CH(2))(5)-N(+)(CH(3))(2)C(16)H(33)·2Br(-) designated as G5 and bis(cetyldimethylammonium)hexane dibromide (C(16)H(33)(CH(3))(2)N(+)-(CH(2))(6)-N(+)(CH(3))(2)C(16)H(33)·2Br(-) designated as G6 and cyclodextrins, was used to refold guanidinium chloride (GdCl) denatured BSA in the artificial chaperone assisted two step method. The single chain cationic surfactant cetyltrimethylammonium bromide (CTAB) was used for comparative studies. The studies were carried out in an aqueous medium at pH 7.0 using circular dichroism, dynamic light scattering and ANS binding studies. The denatured BSA was found to get refolded by very small concentrations of gemini surfactant at which the single chain counterpart was found to be ineffective. Different from the single chain surfactant, the gemini surfactants exhibit much stronger electrostatic and hydrophobic interactions with the protein and are thus effective at much lower concentrations. Based on the present study it is expected that gemini surfactants may prove useful in the protein refolding operations and may thus be effectively employed to circumvent the problem of misfolding and aggregation.

Unfolding of rabbit serum albumin by cationic surfactants: surface tensiometry, small-angle neutron scattering, intrinsic fluorescence, resonance Rayleigh scattering and circular dichroism studies.

Unfolding of rabbit serum albumin (RSA) by cationic surfactants cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethylammonium bromide (TTAB) was studied by exploiting surface tensiometry, small-angle neutron scattering (SANS), intrinsic fluorescence, resonance Rayleigh scattering (RRS) (also referred as turbidity at 350/350), and circular dichroism (CD) techniques. Surface tension measurements revealed the formation of highly surface-active complexes occurring as a consequence of RSA-surfactants interactions. SANS measurements show that, in the low surfactant concentration regime (0-10 mM), increase in the dimension of the ellipsoidal protein occurs. Conversely, at higher concentrations (20-80 mM), the surfactant molecules result in the formation of a fractal structure representing a 'necklace model' of micelle-like clusters randomly distributed along the polypeptide chain. The overall size of the complex increases and the fractal dimension decreases on increasing the surfactant concentration. The size of the micelle-like clusters decreases while the number of such clusters and their aggregation number increase with increasing CTAB concentration. Taken all observant together, the fluorescence, RRS, and CD studies were found to be consistent with the SANS measurements. Both CTAB and TTAB were found to behave likewise and the effect of hydrophobicity was clearly visible in the CD, RRS, and intrinsic fluorescence results. The Rayleigh scattering study shows that TTAB was more skilled to solubilize the serum albumin and may be more convenient than CTAB to isolate proteins from inclusion bodies.

Interaction of bovine serum albumin with cationic single chain+nonionic and cationic gemini+nonionic binary surfactant mixtures.

The interaction of bovine serum albumin (BSA) with cetyltrimethylammonium bromide (CTAB), C(16)C(4)C(16)Br(2), Brij58, and their binary mixtures has been studied using tensiometry, spectrofluorometry, and circular dichroism at physiological pH and 25 degrees C. The tensiometric profiles of CTAB and C(16)C(4)C(16)Br(2) in the presence of BSA exhibit a single break at a lower surfactant concentration termed as C(1) (concentration corresponding to saturation of the interface) compared to their critical micelle concentration (CMC) in the buffered solution. However, for Brij58, CTAB+Brij58, and C(16)C(4)C(16)Br(2)+Brij58, two breaks were observed, first at the critical aggregation concentration (CAC), corresponding to onset of interaction with BSA and the second at C(1) corresponding to saturation of the interface. The interaction of CTAB+Brij58 and C(16)C(4)C(16)Br(2)+Brij58 mixtures with the BSA solution is discussed in terms of competition between surfactant-surfactant and surfactant-BSA interactions. CTAB+Brij58 and C(16)C(4)C(16)Br(2)+Brij58 mixtures show nonideality with respect to mixed micelle formation, which is reflected in their interaction with the BSA. The interaction of CTAB+Brij58 with BSA decreases with increase in the mole fraction of CTAB in the mixture, whereas in C(16)C(4)C(16)Br(2)+Brij58 the reverse is the case. The results of the present study may prove fruitful in optimizing the properties of surfactant-protein mixtures relevant for many formulations.

Interaction of bovine (BSA), rabbit (RSA), and porcine (PSA) serum albumins with cationic single-chain/gemini surfactants: a comparative study.

The interactions among bovine, rabbit, and porcine serum albumins and single-chain cationic surfactant cetyltrimethylammonium bromide (CTAB) versus its gemini counterpart (designated as G4) have been studied. The studies were carried out in an aqueous medium at pH 7.0 using UV, intrinsic and extrinsic fluorescence spectroscopy, and far-UV circular dichroism techniques. The results indicate that compared to CTAB, G4 interacts strongly with the serum albumins, resulting in a significantly larger unfolding or decrease in alpha-helical content as reflected by the significantly larger decrease in ellipticity in the far-UV range. Unlike CTAB, a remarkable increase in the alpha-helical content of BSA at 625 microM G4 and at 250 microM G4 for RSA and PSA is observed. The appearance of conformational changes and saturation points in the proteins occurs at considerably lower [G4] compared to [CTAB]. The results obtained from the multi-technique approach are ascribed to the stronger forces in G4 owing to the presence of two charged headgroups and two hydrocarbon tails. Keeping the results in view, it is suggested that the gemini surfactants may be effectively used in the renaturation of proteins produced in genetically engineered cells via the artificial chaperone protocol and may also prove useful in drug delivery as solubilizing agents to recover proteins from insoluble inclusion bodies.

Spectroscopic studies on the interaction of cationic surfactants with bovine serum albumin.

The interaction of the cationic surfactant cetyltrimethylammonium bromide (CTAB) with bovine serum albumin (BSA), a globular protein, has been studied by small-angle neutron scattering (SANS), fluorescence and circular dichroism (CD). SANS measurements show that at low [CTAB] the protein shows a native-like behavior. On the other hand, at high [CTAB], surfactant molecules result in the formation of a fractal structure representing a 'necklace model' of micelle-like clusters randomly distributed along the polypeptide chain. The overall size of the complex increases and the fractal dimension decreases on increasing the surfactant concentration. The size of the micelle-like clusters does not show any considerable change while the number of such clusters and their aggregation number increases with increasing [CTAB]. Some extrapolatory experiments were performed with tetradecyltrimethylammonium bromide (TTAB) and the surfactant was found to behave similarly leading to an increase in the size of protein along the semi-major axis at low concentrations and formation of a fractal structure at high concentrations. The fluorescence studies undertaken were found to be consistent with the SANS measurements. Native-like behavior of the protein mixed with low concentration of the surfactant was also concluded from the circular dichroism (CD) spectra where the spectra in presence of high [CTAB] could not be monitored because of high dynode voltage.

Spectroscopic studies on the comparative interaction of cationic single-chain and gemini surfactants with human serum albumin.

To gain insights into the comparative effect of single-chain/gemini surfactants on proteins and the possible implications, the interaction of human serum albumin (HSA) with cationic single-chain surfactant cetyltrimethylammonium bromide (CTAB) and its gemini counterpart bis(cetyldimethylammonium)butane dibromide with spacer -(CH(2))(4)- (designated as G4) using turbidity measurements, far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and extrinsic fluorescence spectroscopy at pH 7.0 are reported in this contribution. A decrease of 33.5% alpha-helical content at 22.5 microM G4 was monitored compared to a 15% decrease at 2,250 microM CTAB. Against a 3.5% increase at 11,250 microM CTAB, a rise of 21.1% in the alpha-helical content was observed 375 microM G4. The result is related to the stronger electrostatic and hydrophobic forces in G4, owing to the presence of two charged headgroups and two hydrophobic hydrocarbon tails that make it to bind strongly to the protein compared to its single chain counterpart, CTAB, resulting in larger unfolding. The stabilization at higher concentrations is attributed to the highly hydrophobic microdomain of the G4 aggregates formed at such concentrations. The results of the multi-technique approach are consistent with the fact that the gemini surfactants are more efficient than their conventional single-chain counterparts and hence may be used more effectively in the renaturation of proteins produced in the genetically engineered cells via the artificial chaperone protocol, as solubilizing agents to recover proteins from insoluble inclusion bodies and in drug delivery.

Effect of physiological concentration of urea on the conformation of human serum albumin.

We report that the presence of very low concentrations (<0.1 M) of urea, a widely used chemical denaturant, induces structure formation in the water-soluble globular protein human serum albumin (HSA) at pH 7. We have presented results suggesting an almost 8% and 5% increase in alpha-helix in the presence of 10 mM urea (U) and 20 mM monomethylurea (MMU), respectively. Far and near-UV circular dichroism studies along with tryptophan fluorescence and 1-anilino-8-naphthalenesulphonicacid (ANS) binding support our view. We hypothesize that both U and MMU, at such low concentrations, modify the solvent structure, increase the dielectric constant and consequently increase hydrophobic forces resulting in enhanced alpha-helical content. The implications of these results of the lower urea regime are significant because the physiological blood urea ranges from 2.5 to 7.5 mM.

Influence of urea additives on micellar morphology/protein conformation.

The present study highlights the fact that the effect of additives (urea, monomethylurea, thiourea) on the supramolecular assemblies and proteins is strikingly similar. To investigate the effect, a viscometeric study on sphere-to-rod transition (s-->r) was undertaken in a system (3.5% tetradecyltrimethylammonium bromide+0.05 M NaBr + 1-pentanol [P.M. Lindemuth, G.L. Bertand, J. Phys. Chem. 97 (1993) 7769]) in the presence and absence of the said additives. [1-pentanol] needed for s-->r (i.e. [1-pentanol]s-->r) was determined from the relative viscosity versus [1-pentanol] profiles. It was observed that the additives preponed as well as postponed s-->r depending upon their nature and concentrations. These effects are explained in terms of increased polarity of the medium and the adsorption ability of urea/monomethylurea on the charged surfactant monomers of the micelle. In case of thiourea, postponement of s-->r was observed throughout which is attributed to its structure. To derive an analogy between micelles and proteins the additive-induced conformational changes of the protein, bovine serum albumin (BSA) was taken to monitor secondary structural changes and tryptophanyl fluorescence. A marked increase in secondary structure (far-UVCD) and increased tryptophanyl fluorescence with a marked blue shift in lambdamax was observed in presence of low concentrations of urea or alkylurea. This indicates that a more compact environment is created in presence of these additives, if added judiciously. Addition of thiourea to BSA caused a marked quenching without any significant change in lambdamax. The large decrease in tryptophanyl emission in presence of low thiourea concentrations seems to be specific and related to thiourea structure as no corresponding changes were observed in urea/alkylurea. All these effects pertaining to protein behavior fall in line with that of morphological observations on the present as well as surfactant systems studied earlier [S. Kumar, N. Parveen, Kabir-ud-Din, J. Phys. Chem. B 108 (2004) 9588].