Triplet-state kinetics of Zn-porphyrin cytochrome c in micellar media. Measurement of intermicellar exchange rates

The interactions of protein molecules with surfactant assemblies in aqueous and hydrocarbon media have been studied via the triplet-state kinetics of Zn-porphyrin cytochrome c in solutions containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] or a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. In aqueous solution, the observed triplet state decay is single exponential with a lifetime of 8 ms. In aqueous solutions of AOT and in AOT-reversed micellar solutions, biexponential triplet state decays were observed, indicating that interactions between the surfactant and the protein occur, resulting in a change in protein conformation near the porphyrin ring. In CTAB-reversed micellar solutions, quenching of the Zn-porphyrin cytochrome c triplet state by ferricyanide and methyl viologen was studied. Because the quenching is exchange-limited under the conditions used, the exchange rate constants for the water pools can be obtained from these experiments. The observed exchange rate constants are in the range (1-5) x 10(7) M-1 S-1, depending on the water content of the reversed micelles and on the type of quencher used. These values are three orders of magnitude lower than the calculated collision rate of the reversed micelles.     

Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.     

Photophysical and photodynamic properties of Pyronin Y in micellar media at different temperatures

In this study, photophysics and photodynamical properties of Pyronin Y (PyY) in different liquid media were investigated. Interactions of PyY, which is a positively charged pigment compound pertaining to the xanthene derivatives with surfactants possessing distinct charges, were determined by using the molecular absorption and fluorescence spectroscopy techniques. It was observed that band intensities of absorption and fluorescence spectra belonging to PyY increase in proportion to the water when compared to three micelle systems, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X‐100 (TX‐100). This suggests that interactions in micelle systems are different from those in deionized water, and solvation and surface interactions modify. It is determined that the strongest interaction occurs between PyY dye and SDS, anionic surfactant, and this interaction arises from the electrostatic forces. Calculated photophysical parameters indicated that the microenvironment of PyY in SDS micelle is different to that of other systems. In temperature studies, it was reported that increasing the temperature of the samples increased non‐radiative transitions. Steady‐state fluorescence anisotropy values were calculated by using fluorescence intensities of PyY compound in pre‐micellar, micellar and post‐micellar systems. Once the PyY fluorescence probe is added to the surfactant containing solutions below the critical micelle concentrations, the measured anisotropy values were found to be low because the probe remains in the deionized water phase. When the surfactant concentration of the medium becomes closer to the critical micelle concentrations, the steady‐state anisotropy value prominently increases. This is because of the restrictions on the rotational diffusion of the probe in micellar solution. It is observed that positively charged PyY shows a higher affinity to the negatively charged SDS compared with the positively charged CTAB and neutral TX‐100 surfactants. This can be explained by Coulombic interactions.     

Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization

Experiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease-a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di-2-ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P](rm,max) was found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase-transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P](rm). In this work, it was found to be possible to dissociate the protein-surfactant complex and recover the precipitated protein. The kinetics of protein-surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head-groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein-surfactant complex formation as [P](rm) --> [P](rm,max). In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein-surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co-partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P](rm). (c) 1995 John Wiley & Sons Inc.     

Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are proposed to interact preferentially with glycosphingolipids and cholesterol to form microdomains, which may play an important role in apical targeting and signal transduction. The objective of the present study was to investigate the interaction of the GPI-anchored protein Thy-1 with phospholipids and a glycosphingolipid. Purified Thy-1 was reconstituted into lipid bilayer vesicles of dimyristoyl-phosphatidylcholine (DMPC) alone or in combination with galactosylceramide (GC). The ability of Thy-1 to perturb the gel to a liquid-crystalline phase transition of DMPC was examined by differential scanning calorimetry. As the mole fraction of Thy-1 increased, the phase transition enthalpy, deltaH, declined. Analysis indicated that each molecule of Thy-1 perturbed over 50 phospholipids, suggesting that, in addition to the anchor insertion into the bilayer, the protein itself may interact with the membrane surface. Inclusion of 5% w/w GC in the bilayer resulted in a striking change in the interaction of Thy-1 with phospholipids. At low Thy-1 content, there was a reduction in the phase transition temperature and an increase in phospholipid cooperativity, suggesting the formation of Thy-1/GC-enriched domains. DeltaH initially decreased with increasing Thy-1 content of the bilayer; however, at higher Thy-1 mole ratios, deltaH rose again. These results are interpreted in terms of a model whereby, at low protein:lipid mole ratios, Thy-1 preferentially sequesters GC to form enriched microdomains. At high protein:lipid mole ratios, Thy-1 may alter its conformation in response to steric crowding within these domains such that its interaction with the bilayer surface is reduced.     

Dynamic interactions between enzyme activity and the microstructured environment

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 °C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 °C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Spectroscopic properties of horse liver alcohol dehydrogenase in reversed micellar solutions

Catalytic and spectroscopic properties of alcohol dehydrogenase from horse liver, incorporated in reversed micellar media, have been studied. Two different reversed micellar systems have been used, one containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT], the other containing a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. With 1-hexanol as substrate the turnover number of the enzyme in AOT-reversed micelles is strongly dependent on the water content of the system. At low wo ([H2O]/[surfactant]) (wo less than 20) no enzymatic activity can be detected whereas at high wo (wo = 40) the turnover is only slightly lower than in aqueous solution. In CTAB-reversed micelles the dependence of the turnover number on wo is much less. The enzymatic activity is in this case significantly lower than in aqueous solution and increases only slightly with an increasing water content of the reversed micelles. Possible interactions of the protein with the surfactant interfaces in the reversed micellar media were studied via circular dichroism and fluorescence measurements. From the circular dichroism of the protein backbone it is observed that the protein secondary structure is not significantly affected upon incorporation in the reversed micelles since the far-ultraviolet spectrum is not altered. Results from time-resolved fluorescence anisotropy experiments indicate that, especially in AOT-reversed micelles, interactions between the protein and the surfactant interface are largely electrostatic in nature, as evident from the dependence on the pH of the buffer used. In CTAB-reversed micellar solutions such interactions appear to be much less pronounced than in AOT.     

A novel method for dissolution and stabilization of non-mulberry silk gland protein fibroin using anionic surfactant sodium dodecyl sulfate

The importance of silk protein has increased because of its potential use as a natural biopolymer for tissue engineering and biomedical applications. In this report we show a novel and ecofriendly method for dissolution of gland silk protein fibroin. Non-mulberry silk fibroin from mature fifth instar larvae of Antheraea mylitta was found to be optimally soluble in 1% (w/v) anionic surfactant sodium dodecyl sulfate (SDS). Regenerated fibroin showed distinct bands of approximately 395 and 197 kDa on electrophoresis in non-reducing and reducing conditions, respectively. Enhanced fibroin dissolution via internalization of hydrophobic amino groups inside a hydrophilic amino acid core in the form of micelles was observed. Prolonged storage stability without gelation of SDS-extracted fibroin was seen. Atomic force microscopy showed micellar aggregation with mean micellar aggregation size of 8 nm. Circular dichroism spectroscopy revealed predominantly helical conformation due to surfactant addition with internal protein conformational changes as revealed by fluorescence spectroscopic studies.     

Short-chain phospholipids as detergents

The physico-chemical properties of short-chain phosphatidylcholine are reviewed to the extent that its biological activity as a mild detergent can be rationalized. Long-chain diacylphosphatidylcholines are typical membrane phospholipids that form preferentially smectic lamellar phases (bilayers) when dispersed in water. In contrast, the preferred phase of the short-chain analogues dispersed in excess water is the micellar phase. The preferred conformation and the dynamics of short-chain phosphatidylcholines in the monomeric and micellar state present in H(2)O are discussed. The motionally averaged conformation of short-chain phosphatidylcholines is then compared to the single-crystal structures of membrane lipids. The main conclusion emerging is that in terms of preferred conformation and motional averaging short-chain phosphatidylcholines closely resemble their long-chain analogues. The dispersing power of short-chain phospholipids is emphasized in the second part of the review. Evidence is presented to show that this class of compounds is superior to most other detergents used in the solubilization of membrane proteins and the reconstitution of the solubilized proteins to artificial membrane systems (proteoliposomes). The prominent feature of the solubilization/reconstitution of integral membrane proteins by short-chain PC is the retention of the native protein structure and hence the protein function. Due to their special detergent-like properties, short-chain PC lend themselves very well not only to membrane solubilization but also to the purification of integral membrane proteins. The retention of the native protein structure in the solubilized state, i.e. in mixed micelles consisting of the integral membrane protein, intrinsic membrane lipids and short-chain PC, is rationalized. It is hypothesized that short-chain PC interacts primarily with the lipid bilayer of a membrane and very little if at all with the membrane proteins. In this way, the membrane protein remains associated with its preferred intrinsic membrane lipids and retains its native structure and its function.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 degrees C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 degrees C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Protein extraction using the sodium bis(2-ethylhexyl) phosphate (NaDEHP) reverse micellar system

The reverse micellar system of sodium bis(2-ethylhexyl) phosphate (NaDEHP)/isooctane/brine was used for liquid-liquid extraction of proteins. We investigated the solubilization of cytochrome-c and alpha-chymotrypsin into the NaDEHP reverse micellar phase by varying the pH and NaCl concentration in the aqueous phase. At neutral pH and relatively low ionic strength, the proteins are extracted into the micellar phase with high yield. By contacting the micellar phase with a divalent cation (e.g., Ca(2+)) aqueous solution, the reverse micelles are destabilized and release the protein molecules back into an aqueous solution for recovery. This method separates the proteins from the surfactant with very high overall efficiencies. (c) 1996 John Wiley & Sons, Inc.     

Reverse micellar extraction and precipitation of lysozyme using sodium di(2-ethylhexyl) sulfosuccinate

Sodium di(2-ethylhexyl) sulfosuccinate, referred to as Aerosol-OT or AOT, was used to remove lysozyme from an aqueous phase via reverse micellar extraction and precipitation method. For both methods, when the surfactant was in excess, a complete removal of lysozyme from the aqueous phase was obtained at the values of pH below the pI of lysozyme. However, for the reverse micellar method, a solubilization limit of lysozyme in the organic phase was observed, and a white precipitate was formed at the aqueous-organic interface. This observation suggested using AOT directly as a precipitating ligand. The lysozyme precipitated with AOT was fully recovered, with its original enzymatic activity, using acetone as a recovery solvent. A mechanism is suggested to explain the solubilization of lysozyme in an AOT reverse micellar system. It is shown that a direct precipitation method can be used with advantage instead of using the reverse micellar extraction method to recover lysozyme from an aqueous phase.     

Time-resolved fluorescence and circular dichroism of porphyrin cytochrome c and Zn-porphyrin cytochrome c incorporated in reversed micelles

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Zn-porphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrime-thylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces. Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (wo = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved. Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions. In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.     

Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems

Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain.     

Solubilization and insertion into reverse micelles of the major myelin transmembrane proteolipid

The Folch-Pi proteolipid has been isolated from bovine white matter and characterized with respect to phospholipid and glycolipid composition. The protein-lipid complex has been solubilized in aqueous reverse micelles of di(2-ethylhexyl) sodium sulfosuccinate and isooctane. Solubilization of this otherwise water-insoluble proteolipid requires small amounts of water, the percent of solubility being maximum for a low molar ratio of water to surfactant (Wo = 5.6). Unlike hydrophilic proteins, the extent of incorporation into the micellar system is negligible at 50 mM surfactant and reaches 90Vo only at 300 mM. However, the conformation of the proteolipid in reverse micelles as studied by fluorescence emission spectroscopy and circular dichroism was not affected by variations of the surfactant concentration. These results are consistent with the peculiar properties of the aqueous environment of the proteolipid within the reverse micelles and may reflect the membrane-like character of these bio-assemblies.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Effect of a commercial alcohol ethoxylate surfactant (C<Subscript>11-15</Subscript>E<Subscript>7</Subscript>) on

Biodegradation of poorly soluble polycyclic aromatic hydrocarbons (PAHs) has been a challenge in bioremediation. In recent years, surfactant-enhanced bioremediation of PAH contaminants has attracted great attention in research. In this study, biodegradation of phenanthrene as a model PAHs solubilized in saline micellar solutions of a biodegradable commercial alcohol ethoxylate nonionic surfactant was investigated. The critical micelle concentration (CMC) of the surfactant and its solubilization capacity for phenanthrene were examined in an artificial saline water medium, and a type of marine bacteria, Neptunomonas naphthovorans, was studied for the biodegradation of phenanthrene solubilized in the surfactant micellar solutions of the saline medium. It is found that the solubility of phenanthrene in the surfactant micellar solutions increased linearly with the surfactant concentrations, but, at a fixed phenanthrene concentration, the biodegradability of phenanthrene in the micellar solutions decreased with the increase of the surfactant concentrations. This was attributed to the reduced bioavailability of phenanthrene, due to its increased solubilization extent in the micellar phase and possibly lowered mass transfer rate from the micellar phase into the aqueous phase or into the bacterial cells. In addition, an inhibitory effect of the surfactant on the bacterial growth at high surfactant concentrations may also play a role. It is concluded that the surfactant largely enhanced the solubilization of phenanthrene in the saline water medium, but excess existence of the surfactant in the medium should be minimized or avoided for the biodegradation of phenanthrene by Neptunomonas naphthovorans.     

Structure-function relationships in the inorganic salt-induced precipitation of alpha-chymotrypsin

alpha-Chymotrypsin (alpha CT) was used as a model protein to study the effects of salt-induced precipitation on protein conformation. Process parameters investigated included the type and amount of salt used to induce precipitation. The salts studied included Na2SO4, NaCl, NaBr, KBr and KSCN. Precipitate secondary structure content was examined via laser Raman spectroscopy. Conventional and saturation transfer electron paramagnetic resonance spectroscopy were employed to probe the tertiary structure of the active site in spin-labelled alpha CT precipitates. As the molal surface tension increment of the inducing salt increased, the beta-sheet content increased and the alpha-helix content decreased. There was no significant variation in secondary structure with the amount of salt used. The fraction of precipitate that recovered activity on redissolution was correlated with the change in secondary structure content. Spin-labelled precipitate spectra indicated that the active site remains unaltered during precipitation. Molecular modelling was employed to investigate how physical property of alpha CT were affected by these types of conformational change. Estimated physical property changes could not account entirely for observed deviations from current equilibrium theory for salt-induced precipitation. The spectroscopic observations were also combined with activity/solubility results to propose a mechanism for the salt-induced precipitation of globular proteins.     

Observation of complex thermal transitions for mixed micelle solutions containing alkyldimethylphosphine oxides and phospholipids and the accompanying cloud points

The thermal properties of various mixtures of two nonionic surfactants, decyldimethylphosphine oxide (APO10) and dodecyldimethylphosphine oxide (APO12) and two phospholipids, dimyristoylphosphatidyl choline (DMPC) and dipalmitoylphosphatidyl choline (DPPC), were examined by differential scanning calorimetry at various mole fractions. The addition of APO12 to DMPC multilamellar vesicles lowered the temperature of the main transition, produced considerable broadening, and eliminated the pre-transition. Phase separation, as evidenced by the existence of a cloud point, T(cp), occurred when the mole fraction of APO12, with respect to DMPC was 0.58 and above. A small abrupt increase in heat capacity was observed at, or slightly above, the cloud point of APO12 and all mixed micelle solutions. It appeared that mixed micelles coexisted with mixed bilayers when the mole fraction was between 0.58 and 0.75 and perhaps as low as a mole ratio of 0.32. All of the mixtures, except APO12/DMPC, exhibited a clear endotherm below the temperature corresponding to the cloud point, which likely reflects the growth in micellar size. Overlapping chain length dependent endothermic peaks, perhaps resulting from reorganization and/or continued association of the micelles, were observed above the cloud point for all of the mixtures except for APO10/DMPC solutions. However, solutions of mixed micelles consisting of APO10/DMPC with mole fractions of surfactant between 0.81 and 0.93 portrayed a broad unidentified exotherm of about 2+/-1 kcal/mol, which was centered nearly 10-20 degrees C above the cloud point.