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1.
Conf Proc IEEE Eng Med Biol Soc ; 2006: 1862-5, 2006.
Article in English | MEDLINE | ID: mdl-17946075

ABSTRACT

This paper reports on the development of an acoustic-wave biosensor based on integrated MEMS technology that promises high sensitivity and selectively without the need for molecular tagging or external optical equipment. The device works by detecting frequency shifts resulting from the selective binding of target molecules to the surface of a functionalized resonating polymer MEMS-composite membrane. Here, we characterize the frequency response of our metal-oxide MEMS resonators. We show that the structural topology, which includes the amount of void area spacing, total mass of the resonator, and how the mass is distributed on the surface, affects the resonant frequency response in a measurable way. Using a multimodal electrostatic drive, we can either excite or suppress higher order harmonic frequencies. The excitation of higher order harmonics is important for multiple analyte detection or redundancy testing. We use a finite element model to demonstrate how a distributed mass loading affect the frequency responses of our MEMS structures.


Subject(s)
Acceleration , Acoustics/instrumentation , Computer-Aided Design , Models, Theoretical , Transducers , Computer Simulation , Elasticity , Electric Impedance , Equipment Design , Equipment Failure Analysis , Reproducibility of Results , Sensitivity and Specificity , Stress, Mechanical , Vibration
2.
Gene Ther ; 10(22): 1882-90, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14502217

ABSTRACT

We have developed a novel gene delivery scaffold based on DNA plasmid condensation with colloidal gold/polyethylenimine conjugates. This scaffold system was designed to enable systematic study of the relationships between DNA complex physical properties and transfection efficiency. Using an enhanced green fluorescent protein-coding reporter plasmid and a Chinese hamster ovary cell line, we have measured the transfection efficiencies of our complexes using flow cytometry and their cytotoxicities using the trypan blue assay. We have also assayed complex particle morphologies using atomic force microscopy, photon correlation spectroscopy, and a novel plasmon absorbance peak position analysis. We achieved comparable rates of transfection relative to the commonly used polycationic condensation agents calcium phosphate and LipofectAMINE, with comparably low cytotoxicities. In addition, by manipulating colloidal gold concentration, we could partially decouple complex physical properties including charge ratio, size, DNA loading, and polyethylenimine concentration. Our morphological analyses showed that complexes with a diameter of a few hundred nanometers and a charge ratio of approximately 8 perform best in our transfection efficiency assays. The use of colloidal gold as a component in our delivery system provides a versatile system for manipulating complex properties and morphology as well as a convenient scaffold for planned ligand conjugation studies.


Subject(s)
DNA , Genetic Engineering , Genetic Vectors/genetics , Gold Colloid , Polyethyleneimine , Animals , CHO Cells , Cricetinae , Flow Cytometry , Microscopy, Atomic Force , Microscopy, Fluorescence , Transfection/methods
3.
Anal Biochem ; 285(1): 33-49, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-10998261

ABSTRACT

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique combines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent background subtraction. Previous attempts to generate structural reference spectra from a basis set of reference protein spectra have had limited success because of inaccuracies arising from sequential background subtractions and spectral normalization, arbitrary spectral band truncation, and attempted resolution of spectroscopically degenerate structure classes. We eliminated these inaccuracies by defining a single mathematical function for protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with the sequential nature of previous methods, especially with regard to removing the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unordered helix, sheet, turns, and unordered structures from a basis set of spectra of well-characterized proteins. Structural reference spectra were generated in the amide I and amide III bands, both of which have been shown to be sensitive to protein secondary structure content. We accurately account for overlaps between amide and nonamide regions and allow different structure types to have different extinction coefficients. The agreement between our structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good.


Subject(s)
Protein Structure, Secondary , Animals , Humans , Solutions , Spectroscopy, Fourier Transform Infrared , Water
4.
Anal Biochem ; 269(2): 255-72, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10221997

ABSTRACT

We have developed a holistic protein structure estimation technique using amide I band Raman spectroscopy. This technique combines the superposition of reference spectra for pure secondary structure elements with simultaneous aromatic, fluorescence, and solvent background subtraction, and is applicable to solution, suspension, and solid protein samples. A key component of this technique was the calculation of the reference spectra for ordered helix, unordered helix, and sheet, turns, and unordered structures from a series of well-characterized reference proteins. We accurately account for the overlap between the amide I and non-amide I regions and allow for different scattering efficiencies for different secondary structures. For hydrated samples, we allowed for the possibility that bound water spectra differ from the bulk water spectra. Our computed reference spectra compare well with previous experimental and theoretical results in the literature. We have demonstrated the use of these reference spectra for the estimation of secondary structures of proteins in solution, suspension, and dry solid forms. The agreement between our structure estimates and the corresponding determinations from X-ray crystallography is good.


Subject(s)
Protein Structure, Secondary , Spectrum Analysis, Raman/methods , Algorithms , Animals , Crystallography, X-Ray , Data Interpretation, Statistical , Humans , Proteins/chemistry , Proteins/standards , Reference Standards , Software Design , Spectrum Analysis, Raman/standards
5.
Biotechnol Bioeng ; 62(3): 247-58, 1999 Feb 05.
Article in English | MEDLINE | ID: mdl-10099536

ABSTRACT

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.


Subject(s)
Carbon Dioxide/chemistry , Proteins/isolation & purification , Alkaline Phosphatase/isolation & purification , Catalase/isolation & purification , Chemical Fractionation , Chemical Precipitation , Dimethyl Sulfoxide , Insulin/isolation & purification , Muramidase/isolation & purification , Ribonucleases/isolation & purification , Solubility , Solutions , Trypsin/isolation & purification
6.
Anal Chem ; 71(19): 4272-7, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10660438

ABSTRACT

We developed electrochemical hardware and media targeted for protein chromatography. Two types of stationary phases were investigated. The first comprised gold-plated stainless 316L beads coated with a self-assembled monolayer of 6-mercaptohexan-1-ol and was expected to behave like an ion-exchange resin in the presence of an electric field. The secondary stationary phase comprised the first stationary phase with further functionalization with immobilized heme moieties and was expected to behave like immobilized metal affinity resin. We tested apparatus with both stationary phases using ribonuclease A as a model protein and applied potentials from -0.3 to +0.3 V versus the saturated calomel electrode. Despite low binding capacities, we demonstrated that protein retention on both stationary phases could be controlled with an applied potential. The greatest extent of electromodulation was achieved with the mercaptohexanol-based ion-exchange media.


Subject(s)
Chromatography/methods , Proteins/analysis , Electrochemistry , Proteins/chemistry
7.
Pharm Res ; 14(10): 1370-8, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9358549

ABSTRACT

PURPOSE: The long-term and high-temperature storage of dry, micron-sized particles of lysozyme, trypsin, and insulin was investigated. Subsequent to using supercritical carbon dioxide as an antisolvent to induce their precipitation from a dimethylsulfoxide solution, protein microparticles were stored in sealed containers at -25, -15, 0, 3, 20, 22, and 60 degrees C. The purpose of this study was to investigate the suitability of supercritical antisolvent precipitation as a finishing step in protein processing. METHODS: Karl Fisher titrations were used to determine the residual moisture content of commercial and supercritically-processed protein powders. The secondary structure of the dry protein particles was determined periodically during storage using Raman spectroscopy. The proteins were also redissolved periodically in aqueous buffers and assayed spectrophotometrically for biological activity and by circular dichroism for structural conformation in solution. RESULTS: Amide I band Raman spectra indicate that the secondary structure of the protein particles, while perturbed from that of the solution state, remained constant in time, regardless of the storage temperature. The recoverable biological activity upon reconstitution for the supercritically-processed lysozyme and trypsin microparticles was also preserved and found to be independent of storage temperature. Far UV circular dichroism spectra support the bioactivity assays and further suggest that adverse structural changes, with potential to hinder renaturation upon redissolution, do not take place during storage. CONCLUSIONS: The present study suggests that protein precipitation using supercritical fluids may yield particles suitable for long-term storage at ambient conditions.


Subject(s)
Insulin/chemistry , Muramidase/chemistry , Trypsin/chemistry , Animals , Carbon Dioxide , Cattle , Chemical Precipitation , Circular Dichroism , Drug Storage , Particle Size , Powders , Protein Structure, Secondary , Solutions , Spectrum Analysis, Raman , Temperature
8.
Biotechnol Bioeng ; 52(2): 193-203, 1996 Oct 20.
Article in English | MEDLINE | ID: mdl-18629888

ABSTRACT

We describe a new protein characterization technique called self-interaction chromatography (SIC), which exploits the specificity of protein-protein interactions that is common to protein aggregates and enables the rapid screening of protein formulation additives as physical stabilizers against aggregation. This technique also enables the identification of specific interaction sites and the determination of their relative importance for self-association. Mannitol, glycine, and dextran 40 were tested for their stabilizing effect toward the model protein lysozyme. Dextran 40 exhibited a poor stabilizing effect. While mannitol stabilized both the native and acid-denatured forms of lysozyme, glycine stabilized the native form with respect to the denatured species. These results are in good agreement with findings in the formulation literature. The SIC shows tremendous potential as a rapid formulation development tool. We also screened two putative interaction sites for involvement in the self-association of lysozyme and estimated the associated binding energies using a binding isotherm model that we developed. The sites screened consisted of residues 41-48 and 125-128 and were selected based on their apparent importance in forming crystal contacts in several different crystal forms of lysozyme. Of the two sites, only residues 125-128 were found to influence self-association under the conditions we employed. Because the success of this technique depends on the exploitation of self-interactions between native species, several important applications are also suggested such as separating native from misfolded or variant species and probing site utilization in aggregation versus crystallization phenomena.

9.
J Pharm Sci ; 85(6): 586-94, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8773954

ABSTRACT

Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.


Subject(s)
Carbon Dioxide/chemistry , Protein Structure, Secondary , Proteins/chemistry , Chemical Phenomena , Chemical Precipitation , Chemistry, Physical , Dimethyl Sulfoxide , Muramidase/chemistry , Solutions , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Trypsin/chemistry
10.
Biophys J ; 70(6): 2888-902, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8744327

ABSTRACT

We simulated the structure of reversible protein aggregates as a function of protein surface characteristics, protein-protein interaction energies, and the entropic penalty accompanying the immobilization of protein in a solid phase. These simulations represent an extension of our previous work on kinetically irreversible protein aggregate structure and are based on an explicit accounting of the specific protein-protein interactions that occur within reversible aggregates and crystals. We considered protein monomers with a mixture of hydrophobic and hydrophilic surface regions suspended in a polar solvent; the energetic driving force for aggregation is provided by the burial of solvent-exposed hydrophobic surface area. We analyzed the physical properties of the generated aggregates, including density, protein-protein contact distributions, solvent accessible surface area, porosity, and order, and compared our results with the protein crystallization literature as well as with the kinetically irreversible case. The physical properties of reversible aggregates were consonant with those observed for the irreversible aggregates, although in general, reversible aggregates were more stable energetically and were more crystal-like in their order content than their irreversible counterparts. The reversible aggregates were less dense than the irreversible aggregates, indicating that the increased energetic stability is derived primarily from the optimality rather than the density of the packing in the solid phase. The extent of hydrophobic protein-protein contacts and solvent-exposed surface area within the aggregate phase depended on the aggregation pathway: reversible aggregates tended to have a greater proportion of hydrophobic-hydrophobic contacts and a smaller fraction of hydrophobic solvent-exposed surface area. Furthermore, the arrangement of hydrophobic patches on the protein surface played a major role in the distribution of protein contacts and solvent content. This was readily reflected in the order of the aggregates: the greater the contiguity of the hydrophobic patches on the monomer surface, the less ordered the aggregates became, despite the opportunities for rearrangement offered by a reversible pathway. These simulations have enhanced our understanding of the impact of protein structural motifs on aggregate properties and on the demarcation between aggregation and crystallization.


Subject(s)
Proteins/chemistry , Biophysical Phenomena , Biophysics , Crystallization , In Vitro Techniques , Macromolecular Substances , Models, Chemical , Molecular Structure , Monte Carlo Method , Solvents , Surface Properties , Thermodynamics
11.
Proc Natl Acad Sci U S A ; 93(11): 5460-5, 1996 May 28.
Article in English | MEDLINE | ID: mdl-8643597

ABSTRACT

The physical stability of pharmaceutical proteins in delivery environments is a critical determinant of biological potency and treatment efficacy, and yet it is often taken for granted. We studied both the bioactivity and physical stability of interleukin 2 upon delivery via continuous infusion. We found that the biological activity of the delivered protein was dramatically reduced by approximately 90% after a 24-hr infusion program. Only a portion of these losses could be attributed to direct protein deposition on the delivery surfaces. Analysis of delivered protein by size exclusion chromatography gave no indication of insulin-like, surface-induced aggregation phenomena. Examination of the secondary and tertiary structure of both adsorbed and delivered protein via Fourier-transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated that transient surface association of interleukin 2 with the catheter tubing resulted in profound, irreversible structural changes that were responsible for the majority of the biological activity losses.


Subject(s)
Infusion Pumps , Interleukin-2/administration & dosage , Interleukin-2/chemistry , Acrylamide , Acrylamides , Adsorption , Animals , Biological Assay , Cell Line , Circular Dichroism , Cloning, Molecular , Drug Stability , Escherichia coli , Humans , Infusions, Intravenous , Interleukin-2/biosynthesis , Lymphocyte Activation , Mice , Potassium Iodide , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/administration & dosage , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Spectroscopy, Fourier Transform Infrared , Time Factors
12.
Biotechnol Bioeng ; 48(4): 324-32, 1995 Nov 20.
Article in English | MEDLINE | ID: mdl-18623493

ABSTRACT

Previous work by us and others has shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, we probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation as our model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control the effects contacting conditions on precipitate yield, purity, and particle size distribution. First, we studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, we examined the role of the molecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine gamma-globulins, carried out at a nominal 1-L scale; glycine was examined as a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle size. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations.

13.
J Pharm Sci ; 83(12): 1651-6, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7891290

ABSTRACT

The secondary structure content of microparticulate insulin powders produced by the supercritical antisolvent (SAS) precipitation technique was investigated via Raman spectroscopy. Precipitate samples were generated at 25 and 35 degrees C processing temperatures. Both precipitate samples gave amide I band spectra that were shifted roughly +10 cm-1 relative to the commercial powder. The corresponding secondary structure estimates had significantly increased beta-sheet contents with concomitant decreases in alpha-helix contents relative to the commercial protein; the sum of beta-turn and random coil content remained essentially unchanged. The magnitude of the perturbation was slightly greater for the 35 degrees C sample. Dissolution of the commercial powder and precipitates in 0.01 M HCl yielded solution structure estimates similar to that of the commercial powder. An analysis of insulin in dimethyl sulfoxide, the suspending solvent in the SAS process, indicated that some, but not all, of the structural change observed for the precipitate samples may be attributed to solvent exposure. These results are suggestive of extensive beta-sheet-mediated intermolecular interactions in precipitate states, consistent with analyses of irreversible protein aggregate/fibril states. Interestingly, unlike irreversible protein aggregates, the insulin powders recover essentially full biological activity on reconstitution.


Subject(s)
Insulin/chemistry , Protein Structure, Secondary , Chemical Phenomena , Chemical Precipitation , Chemistry, Pharmaceutical/methods , Chemistry, Physical , Dimethyl Sulfoxide , Feasibility Studies , Hydrochloric Acid , Particle Size , Powders , Solutions , Spectrum Analysis, Raman
14.
Biophys J ; 66(5): 1274-89, 1994 May.
Article in English | MEDLINE | ID: mdl-8061184

ABSTRACT

We have simulated the structure of kinetically irreversible protein aggregates in two-dimensional space using a lattice-based Monte-Carlo routine. Our model specifically accounts for the intermolecular interactions between hydrophobic and hydrophilic protein surfaces and a polar solvent. The simulations provide information about the aggregate density, the types of inter-monomer contacts and solvent content within the aggregates, the type and extent of solvent exposed perimeter, and the short- and long-range order all as a function of (i) the extent of monomer hydrophobic surface area and its distribution on the model protein surface and (ii) the magnitude of the hydrophobic-hydrophobic contact energy. An increase in the extent of monomer hydrophobic surface area resulted in increased aggregate densities with concomitant decreased system free energies. These effects are accompanied by increases in the number of hydrophobic-hydrophobic contacts and decreases in the solvent-exposed hydrophobic surface area of the aggregates. Grouping monomer hydrophobic surfaces in a single contiguous stretch resulted in lower aggregate densities and lower short range order. More favorable hydrophobic-hydrophobic contact energies produced structures with higher densities but the number of unfavorable protein-protein contacts was also observed to increase; greater configurational entropy produced the opposite effect. Properties predicted by our model are in good qualitative agreement with available experimental observations.


Subject(s)
Computer Simulation , Models, Chemical , Proteins/chemistry , Biophysical Phenomena , Biophysics , Macromolecular Substances , Molecular Structure , Monte Carlo Method , Protein Conformation , Solutions , Solvents , Surface Properties , Thermodynamics
15.
Protein Eng ; 7(1): 131-6, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8140090

ABSTRACT

The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.


Subject(s)
Inclusion Bodies/ultrastructure , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , beta-Lactamases/chemistry , Escherichia coli , Inclusion Bodies/chemistry , Recombinant Fusion Proteins/biosynthesis , Spectrum Analysis, Raman , beta-Lactamases/biosynthesis
16.
Biochim Biophys Acta ; 1076(1): 103-11, 1991 Jan 08.
Article in English | MEDLINE | ID: mdl-1986783

ABSTRACT

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na2SO4, were studied for twelve different proteins. alpha-helix and beta-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of beta-sheet structures with a concomitant loss of alpha-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of alpha-helix and beta-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting beta-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in alpha-helix content were influenced by the initial helical content and the dipole moment. The enhanced beta-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.


Subject(s)
Protein Conformation , Proteins/chemistry , Chemical Precipitation , Spectrum Analysis, Raman , Sulfates , Thiocyanates
17.
Biochim Biophys Acta ; 995(3): 231-45, 1989 May 01.
Article in English | MEDLINE | ID: mdl-2539865

ABSTRACT

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.


Subject(s)
Chemical Precipitation , Chymotrypsin , Potassium Compounds , Protein Conformation , Salts , Sodium Compounds , Structure-Activity Relationship , Animals , Bromides , Crystallography , Electron Spin Resonance Spectroscopy , Models, Molecular , Potassium , Sodium , Sodium Chloride , Spectrum Analysis, Raman , Sulfates , Thiocyanates
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