To this end, I sincerely thank a few of the many special people who inspired this work. My parents instilled in me a love of knowledge and a desire to persevere. 8-fold increases were correlated with the proportion of charged residues and the surface area of the native protein.
Chapter 1
Background
Salt-induced precipitation is the oldest and perhaps most widely used protein purification procedure in the separation repertoire of the bioprocess industry. The reduction of protein solubility in aqueous solutions associated with the addition of neutral salts was first described at the end of the nineteenth century by Hofmeister [1,2]. The role of precipitation in bioprocesses varies and is reflected in the means used to generate and recover protein precipitates.
Bioprocess Separation Operations
In addition, up to 70% of the total capital cost of bioprocess equipment can be related to separation. If the processing conditions deviate significantly from the conditions of the protein's natural environment, there are significant losses in form yield. Although the organization of subsequent processing operations strongly depends on the initial composition of the process stream and the nature of the desired protein [9], four generic operations common to most separation arrays [10] can be identified: particle removal, primary isolation, purification and final product isolation.
Thesis Scope
Experimental data were used to assess the predictive ability of current equilibrium theory for protein salting out. The effects of the type and amount of salt on elements of secondary and tertiary structure were analyzed via spectroscopic techniques. The secondary structure content was examined using the Raman spectroscopy technique described in Chapter 3. The generality of the results found for o:CT was examined and extended.
Chapter 2
Figures
Data for myoglobin, ovalbumin, hemoglobin and albumin were taken from Melander and Horvath.6 The horizontal dashed line represents the RSH estimated for o:CT with CHF = 0.12. NaCl and x Na2SO4 • The solid line is the theoretical prediction for 1-1 salts, the dashed line is for 2-1 salts. The solubility curves are plotted as the natural logarithm of the o:CT concentration in grams per kilogram of solvent versus the salt molality.
The solid line represents the fit of equation (20) to the data, and the dashed line represents the fitted saltation constant. Symbols: + KSCN, D KBr, Q NaBr, 6 NaCl and x Na2SO4 • Figures 6 a-e o: CTactivity, active fraction and specific activity as a function. Note that all data are based on soluble precipitation and expressed as a proportion of natural value.
KSCN, D KBr, Q NaBr, 6 NaCl and x Na2S04 • Error bars represent the standard deviation of the mean. The type of salt is defined in terms of the corresponding increase in molal surface tension. The performance parameter is the product of the salinity constant and the active fraction and is a measure of the efficiency of a salt in
The salts, in order of increasing u values, are KSCN, KBr, NaBr, NaCl, and Na2SO4. • Error bars indicate the standard deviation of the mean.
Chapter 3
Results
The resulting structure determinations were within a percent structure of the lysozyme reference values contained in F;. Some of the structure estimates are beyond the content range found in the reference protein set for individual elements (see Table 2); this is because the overlap vector x is implicitly unbounded in system (7). The position of the outer hyperfine resonances is indicative of the polarity of the spin label environment.
Precipitation does not appear to cause any changes in the polarity of the active site environment. The precipitation spectra for all salts used were similar to those of the native, labeled enzyme. If the spin label were to sample altered active site environments in precipitate samples, the resulting change in spin-lattice relaxation times would affect the line shape of ST-EPR spectra [35,36].
Random helix segments are consumed to form the {3-sheet and are produced by the termination of the C-terminal helix. The helix and sheet formation potentials of the primary structure [39] of the C-terminal helix imply amphomorphic character [42]. This does not rule out the unexplored possibility that part of the C-terminal helix acquires {3-sheet character.
The hydrophobic surface of the modified structure was 26% greater than that of the native protein; the dipole moment decreased by 22%.
Discussion
Some of the assembled structures had increased parallel rather than antiparallel B-sheet content; this may depend on the orientation of individual protein molecules in the quaternary structure of the precipitate. Alternatively, the similarity of the structural reference spectra for the pure antiparallel and pure parallel B-sheet in the AF+ matrix (see equation 7) [10] may make the distinction between the two structures somewhat artificial [48]. For the reference set used in this work (13 proteins from Table 5 of [11], excluding avidin, phage fd and the helical form of poly-L-lysine), the AF+ state number was approximately 22.2 and the smallest single value was about 0.0303.
The difference between the two structure types can be further trivialized by the lower S/N ratios associated with some of the precipitate spectra. Physical properties calculated for a hypothetical precipitate phase monomer can be used to characterize the nature of the discrepancy between the theoretical and experimental performance of salts in a salt-out. Using the values of the hydrophobic surface area and the dipole moment calculated for the native and modified o:CT structures, salting-out constants for KSCN were calculated and compared to the experimental value [2] (see Table 5).
The relative magnitudes of the associated rate and equilibrium constants for various conditions are also given. J./ refers to the native solution conformation of the protein. How far the protein continues horizontally in the scheme depicted in Figure 12 depends on the interaction between the salt in question and the protein. Larry Dalton of the University of Southern California for taking the ST-EPR spectra and helping with their interpretation.
34;The Effects of Neutral Salts on the Structure and Conformational Stability of Macromolecules in Solution," in Structure and Stability of Biological Macromolecules (Timasheff, S.N. en Fasman, G.D., Eds.).
Figures
The dotted lines represent the individual adjusted peaks for subtraction and the sum of the adjusted peaks described in equation (1). The salts in order of increasing a values are: KSCN, KBr, NaBr, NaCl, and Na2SO4 • Error bars represent ± S.D. Note that two orthogonal B-loops, each composed of five strands of antiparallel B-sheets, form the dominant ordered structural motif.
The solid lines represent segments of the backbone that have been altered to rrtlmic the secondary structure content of the KSCN-induced whole precipitate. Note that this orientation is rotated a few degrees to the top of the page with respect to Figure 10. The relative magnitudes of the associated rate and equilibrium constants as a function of the solution conditions are also given.
OEt I
Chapter 4
- Abstract
- Materials and Methods
- Acknowledgements
A statistical analysis of the estimated perturbations in secondary structure content indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix upon precipitation with KSCN. Changes in the α-helix content of the precipitates were influenced by the initial helix content and dipole moment of the native proteins. Estimates of the secondary structure content in precipitates were considered relative to the estimated values for the corresponding freeze-dried protein samples.
The strong dependence of the .8-sheet increase on the charged group frequency is somewhat surprising. Studies of the thermal denaturation of ovalbumin found an increase in both the exposed hydrophobic residues and the content of ,B sheets [25]. Many of the amino acids identified as good o:-helix formers in the secondary structure prediction algorithm of Chou and Fasman [28] also appear high in the ranking of B-sheet formers.
Determination of Isosteric Enthalpies and Entropies of Water Associated with Native Enzyme," Biochim. Statistical Significance of the Hypothesis that Salt-Induced Changes in the Secondary Structure of Precipitated Proteins Are Unrelated to the Primary, Secondary, and Tertiary Structure of the Corresponding Native Conformation , Jl0(pxy = 0). The matrix format is the same as in Table 3 and the symbols used are the same as in Table 4.
RNS and CYT were included in the calculation of the correlation terms regarding the structural properties of native proteins.
Chapter 5
- Abstract
- References
- Figures
This work is similar to studies of the binding of casein to milk coagulation in the group. The mixing efficiency of the stopped-flow device was tested by monitoring an indicative pH response. Variation in turbidity curves between replicates is likely the result of fluctuations in mixing efficiency (Walton, 1967).
Effects on the particle size distribution, as reflected in the estimated mean particle radius, r w , were investigated as a function of total protein. The supersaturation ratio dependence of the conformational change terms appearing in equations (7) to (10) results from the following considerations. The stopped-flow turbidity data gives the weight-averaged molecular weight of the particles (see equation (1)) as a function of time.
In terms of the species included in the model, the reduced weight average is molecular weight. The aim of the modeling work was to use the turbidity data to arrive at reasonable estimates for the rate constants k1, k2 and k3. • Marquardt's nonlinear least squares algorithm MRQMIN (Press et al., 1986) was used to fit these rate constants via equation (21) to the turbidity trajectories (see equation (1)). Negative deviations regarding the order of fault tolerance in the integration scheme were allowed.
The linear parts of the turbidity trajectories were analyzed in terms of Smoluchowski kinetics via equation (4). The conformation of aCT has been shown to be a function of the degree of hydration (Luscher and Ruegg, 1978). The experimental data are given by the solid lines; dotted lines represent the corresponding adjustments of the population balance model.
