Interaction between proteins and cationic gemini surfactant

Surface tension, fluorescence, and circular dichroism (CD) methods have been used to investigate the interaction between cationic gemini surfactant 1,2-ethane bis(dimethyldodecylammonium bromide) (C12C2C12) and proteins including bovine serum albumin (BSA) and gelatin. Surface tension measurements show that the complexes of gelatin--C12C2C12 form more easily than that of BSA--C12C2C12. Addition of C12C2C12 has a different effect not only on the polarity of the microenvironment in BSA and gelatin systems but also on their fluorescence spectra. It can be seen from far-UV CD spectra that the alpha-helical network of BSA is disrupted and its content decreases from 41.7% to 27.6% while the random coil content of gelatin increases from 53.0% to 55.9% with increasing C12C2C12 concentration. The results from near-UV CD spectra show that the binding of C12C2C12 induces changes of the microenvironment around the aromatic amino acid residues and disulfide bonds of BSA at high C12C2C12 concentrations.     

Unfolding and refolding of bovine serum albumin induced by cetylpyridinium bromide

The interaction of bovine serum albumin (BSA) with cationic surfactant cetylpyridinium bromide (CPB) in aqueous solution (pH 7.00) was studied quantitatively with ultraviolet (UV)-visible, far-UV, and near-UV circular dichroism, fluorescence, small angle x-ray scattering, and nuclear magnetic resonance measurement. It was found that CPB at low and high concentrations could induce the unfolding and refolding of BSA, respectively. We suggest that in the unfolding process, there existed BSA-CPB complex with the "necklace and bead" structure in which the unfolded BSA wrapped around CPB micelles, and that the hydrophobic interaction between the complexes led to the formation of large aggregates. The aromatic headgroup of CPB interacted with the tryptophan residues of BSA, resulting in the aromatic ring stacking between BSA and CPB. During the refolding process, the BSA molecule was penetrated into the rod micelle of CPB and the hydrophobic moiety of the BSA molecule was exposed outside while its hydrophilic part was hidden inside, thereby disrupting the aromatic ring stacking.     

Aggregation behaviors of gelatin with cationic gemini surfactant at air/water interface

The dilational rheological properties of gelatin with cationic gemini surfactant 1,2-ethane bis(dimethyl dodecyl ammonium bromide) (C(12)C(2)C(12)) at air/water interface were investigated using oscillating barriers method at low frequency (0.005-0.1 Hz), which was compared with single-chain surfactant dodecyltrimethyl ammonium bromide (DTAB). The results indicate that the maximum dilational modulus and the film stability of gelatin-C(12)C(2)C(12) are higher than those of gelatin-DTAB. At high concentration of C(12)C(2)C(12) or DTAB, the dilational modulus of gelatin-surfactant system becomes close to that corresponding to pure surfactant, suggesting gelatin at interface is replaced by surfactant. This replacement is also observed by surface tension measurement. However, it is found that gelatin-C(12)C(2)C(12) system has two obvious breaks but gelatin-DTAB has not in surface tension isotherms. These phenomena are ascribed to the double charges and strong hydrophobicity of C(12)C(2)C(12). Based on these experimental results, a mechanism of gelatin-surfactant interaction at air/water interface is proposed.     

Thermal stability and enzymatic activity of RNase A in the presence of cationic gemini surfactants

The thermal stability and enzymatic activity of bovine pancreatic ribonuclease A (RNase A) have been investigated in the presence of a homologous series of cationic gemini surfactants (alkanediyl-α,ω-bis(hydroxyethyl methyl hexadecyl ammonium bromide)). UV, circular dichorism and fluorescence spectroscopies have been used for this study. The denaturation curves at various surfactant concentrations were analyzed on basis of a two-transition model to obtain values of T(m) (temperature at the midpoint of denaturation) and ΔH(m) (enthalpy change at T(m)) of each transition. The main conclusion of this study is that these cationic gemini surfactants slightly activate and stabilize RNase A below their critical micelle concentrations at pH 5.0. The cationic gemini surfactant with the shorter spacer interacts more efficiently with RNase A than those with longer spacers.     

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.     

Biophysical characterization of complexation of DNA with oppositely charged Gemini surfactant 12-3-12

The interaction between DNA and cationic gemini surfactant trimethylene-1, 3-bis (dodecyldimethylammonium bromide) (12-3-12) has been investigated by the measurements of fluorescence, surface tension, UV spectrum and circular dichroism (CD). Micelle-like structure of 12-3-12 induced by DNA appears at critical aggregation concentration (CAC), which is much lower than critical micelle concentration (CMC) of 12-3-12 in DNA-free solution. CAC is independent of DNA concentration, but the CMC of the mixed solutions of DNA and 12-3-12(CMC(mix)) increases with the increasing of DNA concentration. The surface tensions of the mixed system are higher than that of the pure surfactant solution, much different from the so-called synergistic lowering of the surface tension for other polymer-surfactant systems. Phase separation occurs after the neutralization point and the precipitate redissolves with superfluous 12-3-12. Cationic surfactant 12-3-12 can exclude ethidium bromide (EB) from the DNA/EB complex, and this process does not depend on the DNA concentration but on the charge ratio of 12-3-12 to DNA. The binding constant of EB to DNA decreases sharply at the charge ratio from 0.5 to 1.0. Circular dichroism (CD) spectra show that DNA undergoes a conformational transition from native B-form to chiral psi-phase with increasing of 12-3-12.     

Structural and transfection properties of amine-substituted gemini surfactant-based nanoparticles

BACKGROUND: Increases in DNA transfection efficiencies for non-viral vectors can be achieved through rational design of novel cationic building blocks. Based on previous results examining DNA condensation by polyamines, novel gemini surfactants have been designed that incorporate aza or imino substituents within the spacer group in order to increase interactions with DNA and potentially improve their DNA transfection ability. METHODS: Transfection efficiencies and cell toxicity of gemini nanoparticles constructed from plasmid DNA, gemini surfactant, and a neutral lipid were measured in COS7 cells using a luciferase assay. Structural properties of nanoparticles were examined by using circular dichroism, particle size, zeta potential, and small-angle X-ray scattering (SAXS) measurements. RESULTS: The incorporation of aza and imino substituents within the spacer group was observed to enhance the transfection ability of gemini surfactants. Incorporation of an imino group in the structure of the 1,9-bis(dodecyl)-1,1,9,9-tetramethyl-5-imino-1,9-nonanediammonium dibromide surfactant (12-7NH-12) resulted in a statistically significant (p < 0.01) 9-fold increase in transfection compared to an unsubstituted gemini surfactant and a 3-fold increase compared to the corresponding aza-substituted compound. A pH-dependent transition in size and zeta potential was observed to occur at pH 5.5 for complexes formed from the 12-7NH-12 compound. SAXS results show weakly ordered structures and the presence of multiple phases. CONCLUSIONS: The incorporation of a pH-active imino group within the spacer of the gemini surfactant results in a significant increase in transfection efficiency that can be related to both pH-induced changes in nanoparticle structure and the formation of multiple phases that more readily allow for membrane fusion that may facilitate DNA release.     

Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

Background

Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency.

Results

Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-??) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression.

Conclusion

Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.     

Tuning the fluorescence response of surface modified CdSe quantum dots between tyrosine and cysteine by addition of p-sulfonatocalix[4]arene.

A simple identification method of L-tyrosine (Tyr) and L-cysteine (Cys) using gemini surfactant coated CdSe quantum dots by using a fluorescent spectroscopic technique is proposed. The gemini surfactant modified QDs show a selective fluorescence response between Tyr and Cys by addition of p-sulfonatocalix[4]arene (pSCA). The CdSe QDs coated with gemini surfactant [C(12)H(25)N(+)(CH(3))(2)(CH(2))(4)(CH(3))(2)N(+)C(12)H(25)].2Br(-) (GS) obviously responds to Tyr. While in the presence of pSCA, it shows selectivity to Cys due to the cooperation of gemini surfactant coated QDs (GS-QDs) and pSCA. Under optimal conditions, it is found that the luminescence of the GS-QDs enhanced by Tyr in a concentration-dependent fashion is described by a Langmuir binding isotherm equation in the range 5 x 10(-8)-10(-5) M. In the presence of pSCA, the luminescence of the GS-QDs enhanced by Cys in a concentration-dependent fashion can also be described by a Langmuir binding isotherm equation in the range 10(-8)-10(-4) M. The possible mechanism is discussed.     

Role of surfactant on the proteolysis of aqueous bovine serum albumin

Nonionic and ionic surfactants diminish the initial rate of proteolysis of aqueous bovine serum albumin (BSA) by subtilisin Carlsberg. Surfactants studied include: nonionic tetraethylene glycol monododecyl ether (C12E4); anionic sodium dodecyl sulfate (SDS), anionic sodium dodecylbenzenesulfonate (SDBS), and cationic dodecyltrimethylamonium bromide (DTAB). Kinetic data are obtained using fluorescence emission. Special attention is given to enzyme kinetic specificity determined by fitting initial-rate data to the Michaelis-Menten model. All surfactants reduce the rate of proteolysis, most strongly at concentrations near and above the critical micelle concentration (CMC). Circular dichroism (CD), tryptophan/tyrosine fluorescence spectra, and tryptophan fluorescence thermograms indicate that BSA partially unfolds at ionic surfactant concentrations near and above the CMC. Changes in BSA conformation are less apparent at ionic surfactant concentrations below the CMC and for the nonionic surfactant C12E4. Subtilisin Carlsberg activity against the polypeptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, decreased due to enzyme-surfactant interaction. At the concentrations and time frames studied, there was no enzyme autolysis. Importantly, aqueous proteolysis rates are significantly reduced at high surfactant concentrations where protein-micellar-surfactant aggregates occur. To explain the negative effect of surfactant on subtilisin Carlsberg proteolytic activity against BSA, we propose that micelle/protein complexes hinder enzyme access.     

Photocontrol of protein folding: the interaction of photosensitive surfactants with bovine serum albumin

The photoresponsive interaction of light-sensitive azobenzene surfactants with bovine serum albumin (BSA) at neutral pH has been investigated as a means to control protein folding with light irradiation. The cationic azobenzene surfactant undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, with the visible-light (trans) form of the surfactant being more hydrophobic than the UV-light (cis) form. As a consequence, the trans form exhibits enhanced interaction with the protein compared to the cis form of the surfactant, allowing photoreversible control of the protein folding/unfolding phenomena. Small-angle neutron-scattering (SANS) measurements are used to provide detailed information on the protein conformation in solution. A fitting of the protein shape to a low-resolution triaxial ellipsoid model indicates that three discrete forms of the protein exist in solution depending on the surfactant concentration, with lengths of approximately 90, 150, and 250 A, respectively, consistent with additional dynamic light-scattering measurements. In addition, shape-reconstruction methods are applied to the SANS data to obtain relatively high-resolution conformation information. The results confirm that BSA adopts a heart-shaped structure in solution at low surfactant concentration, similar to the well-known X-ray crystallographic structure. At intermediate surfactant concentrations, protein elongation results as a consequence of the C-terminal portion separating from the rest of the molecule. Further increases in the surfactant concentration eventually lead to a highly elongated protein that nonetheless still exhibits some degree of folding that is consistent with the literature observations of a relatively high helical content in denatured BSA. The results clearly demonstrate that the visible-light form of the surfactant causes a greater degree of protein unfolding than the UV-light form, providing a means to control protein folding with light that, within the resolution of SANS, appears to be completely reversible.     

In vivo cutaneous interferon-gamma gene delivery using novel dicationic (gemini) surfactant-plasmid complexes

BACKGROUND: Localized scleroderma (morphea and linear scleroderma) is a connective tissue disease, accompanied by excessive proliferation and deposition of collagen within the skin, inflammation, vasculopathy and a deranged immune system. Interferon gamma (IFNgamma), an inhibitor of collagen synthesis and an immunomodulator, could be a potential therapeutic agent if it could be delivered into or expressed locally in affected skin in a non-invasive manner. In this study, the feasibility of topical delivery of the IFNgamma gene and expression of IFNgamma were investigated in mice. METHODS: Novel dicationic (gemini) surfactant (spacer length n=2-16; alkyl chain m=12 or 16)-DNA complexes were formulated and characterized by circular dichroism and atomic force microscopy to select gemini analogues with the highest transfection efficiency (TE). Transfection and cellular expression of IFNgamma from the bicistronic pGTmCMV.IFN-GFP plasmid were evaluated in PAM 212 keratinocyte culture by ELISA and fluorescence microscopy. Topical delivery of plasmid using liposomal and nanoemulsion systems, based on gemini surfactant 16-3-16, was evaluated in mice by IFNgamma expression analysis. RESULTS: In vitro TE was found to be dependent on the spacer length of the gemini surfactant, with the C3 spacer showing the highest activity (both 12-3-12 and 16-3-16). Both gemini cationic liposomes and gemini nanoemulsion (3x25 microg DNA/animal) produced significantly higher levels of IFNgamma in the skin (359.4 and 607.24 pg/cm2) compared to naked DNA (135.69 pg/cm2) or a liposomal Dc-chol formulation (82.15 pg/cm2). IFNgamma expression in the lymph nodes was higher in the animals treated with gemini liposomes (422.74 pg/animal) compared to the nanoemulsion formulation (131.27 pg/animal) or the Dc-chol formulation (82pg/animal). CONCLUSIONS: The feasibility of topical delivery of pGTmCMV.IFN-GFP plasmid in mice using gemini cationic surfactant based delivery systems was demonstrated. IFNgamma expression after treatment with gemini-DNA formulations in the skin was 3-5-fold higher compared to the treatment with naked DNA (p<0.05), and 4-6-fold higher than the Dc-chol-DNA complex, indicating a significant advance in topical DNA delivery across intact skin in vivo.     

The structure of DNA-DLPC-cationic gemini surfactant aggregates: a small angle synchrotron X-ray diffraction study

The structure of aggregates formed by interaction of DNA with unilamellar dilauroylphosphatidylcholine (DLPC) vesicles (DNA:DLPC=1:1 base/mol) in the presence of gemini surfactant butane-1,4-diyl-bis(dodecyldimethylammonium bromide) (C12GS) was investigated using synchrotron small angle X-ray diffraction. In the concentration range C12GS+:DLPC< or =1 mol/mol, a condensed lamellar Lalphac phase was found with a repeat period of lipid bilayer stacking in the range d approximately 5.70-6.53 nm and the DNA interhelical distance d(DNA) approximately 3.52-3.99 nm, depending on the concentration of C12GS. At molar ratio C12GS+:DLPC> or =0.35:1, the diffractograms have shown the presence of a second lamellar phase with the repeat period d approximately 5.31 nm which slightly decreases with increasing concentration of C12GS+. The increasing fraction of this phase in the aggregates with increasing concentration of C12GS supports the association of this phase with microscopic domains enriched by surfactant molecules. The temperature behaviour of aggregates was investigated in the range 25-60 degrees C and the transversal thermal expansivities of the observed phases were determined.     

Complexation of beta-lactam antibiotic drug carbenicillin to bovine serum albumin: energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 831-840, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Affinity extraction of BSA with reversed micellar system composed of unbound Cibacron Blue

Affinity Cibacron Blue 3GA (CB) dye in aqueous phase was directly transferred to the reversed micelles due to electrostatic interaction between anionic CB and cationic cetyltrimethylammonium bromide (CTAB). The bovine serum albumin (BSA) transfer to the reverse micelles increases significantly in a wide range of pH by the addition of a small amount of CB ( approximately 1.0-7.0% of the total surfactant concentration) to the aqueous phase. For pH < pI, the selectivity can be significantly improved with the presence of affinity CB because no BSA was extracted in the absence of CB. For backward extraction of BSA from the micellar phase with stripping aqueous solution, the addition of 2-propanol to the aqueous phase can recover almost all BSA (98.5%) extracted into the reverse micelles.     

Fluorescence spectroscopic studies on the interaction of Gemini surfactant 14‐6‐14 with bovine serum albumin

The interaction of the cationic Gemini surfactant hexamethylene‐1,3‐bis (tetradecyldimethylammonium bromide) (14‐6‐14) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra and three‐dimensional (3D) fluorescence spectra. The Stern–Volmer quenching constants K_SV and the corresponding thermodynamic parameters ΔH, ΔG and ΔS have been estimated by the fluorescence quenching method. The results indicated that hydrophobic forces were the predominant intermolecular forces between BSA and the surfactant. Competitive experiments and the number of binding sites calculation show that 14‐6‐14 can be inserted in site‐II (in subdomain IIIA) of BSA. The effect of 14‐6‐14 on the conformation of BSA was evaluated by synchronous fluorescence spectroscopy and 3D fluorescence spectral methods. The results show that the conformation of BSA was changed dramatically in the presence of 14‐6‐14, by binding to the Trp and Try residues of BSA. The investigation provides interaction between BSA and 14‐6‐14 as a model for molecular design and industrial research. Copyright © 2011 John Wiley & Sons, Ltd.     

Combining time‐resolved fluorescence with synchronous fluorescence spectroscopy to study bovine serum albumin‐curcumin complex during unfolding and refolding processes

We investigated the complex interaction between bovine serum albumin (BSA) and curcumin by combining time‐resolved fluorescence and synchronous fluorescence spectroscopy. The interaction was significant and sensitive to fluorescence lifetime and synchronous fluorescence characteristics. Binding of curcumin significantly shortened the fluorescence lifetime of BSA with a bi‐molecular quenching rate constant of k_q = 3.17 × 10¹² M^‐1s^‐1. Denaturation by urea unfolded the protein molecule by quenching the fluorescence lifetime of BSA. The tyrosine synchronous fluorescence spectra were blue shifted whereas the position of tryptophan synchronous fluorescence spectra was red shifted during the unfolding process. However, denaturation of urea had little effect on the synchronous fluorescence peak of tyrosine in curcumin‐BSA complex except in the low concentration range; however, it shifted the peak to the red, indicating that curcumin shifted tryptophan moiety to a more polar environment in the unfolded state. Decreases in the time‐resolved fluorescence lifetime and curcumin‐BSA complex during unfolding were recovered during refolding of BSA by a dilution process, suggesting partial reversibility of the unfolding process for both BSA and curcumin‐BSA complex. Copyright © 2012 John Wiley & Sons, Ltd.     

The interaction between ribonuclease A and surfactants

1. U.v. difference spectra show that the anionic surfactant sodium n-dodecyl sulphate unfolds ribonuclease A at pH7.3 and 10.3, but that the cationic surfactant n-dodecyltrimethylammonium bromide does not affect the conformation of the enzyme. 2. Equilibrium-dialysis experiments show that sodium n-dodecyl sulphate binds to ribonuclease A, but no binding of n-dodecyltrimethylammonium bromide could be detected at pH7.3. 3. The enzymic activity of ribonuclease A is unaffected by n-dodecyltrimethylammonium bromide up to a concentration of 0.03m at 25 degrees C. 4. Ultracentrifuge studies support the conclusion that n-dodecyltrimethylammonium bromide does not interact significantly with ribonuclease A. 5. The enthalpy change as measured by microcalorimetry on binding of sodium n-dodecyl sulphate to ribonuclease A is consistent with an exothermic enthalpy of binding occurring simultaneously with an endothermic enthalpy of chain unfolding.     

Surfactant-induced unfolding of cellulase: kinetic studies

Surfactant-induced unfolding is a significant degradation pathway for detergent enzymes. This study examines the kinetics of surfactant-induced unfolding for endoglucanase III, a detergent cellulase, under conditions of varying pH, temperature, ionic strength, surfactant type, and surfactant concentration. Interactions between protein and surfactant monomer are shown to play a key role in determining the kinetics of the unfolding process. We demonstrate that the unfolding rate can be slowed by (1) modifying protein charge and/or pH conditions to create electrostatic repulsion of ionic surfactants and (2) reducing the amount of monomeric ionic surfactant available for interaction with the enzyme (i.e., by lowering the critical micelle concentration). Additionally, our results illustrate that there is a poor correlation between thermodynamic stability in buffer (DeltaG(unfolding)) and resistance to surfactant-induced unfolding.     

Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins

The effect of surfactants on fluorescein isothiocyanate (FITC)-bovine serum albumin (BSA)-hypochlorite (ClO(-)), FITC-human serum albumin (HSA)-ClO(-), FITC-ovoconalbumin (OVA)-ClO(-), FITC-hemoglobin (Hb)-ClO(-) systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.