Trypsin and alpha-chymotrypsin were immobilized on nylon pellets or porous glass by covalent methods to change molecular properties and functional characteristics of soybean proteins. The amount of trypsin immobilized on nylon pellets using the glutaraldehyde method was high when the pellets were treated with methanolic solution and 6 - 8% glutaraldehyde as well as high concentrations of soluble trypsin. Immobilized trypsin and chymotrypsin had uniform pKm and were stable at high temperatures. The optimum pH for activity of immobilized enzymes could be changed by using different supports and different methods of immobilization. A multi-enzyme system with immobilized trypsin and chymotrypsin was designed to produce an efficient hydrolysis and various desirable products of hydrolysis. Controlled hydrolysis of soybean proteins by immobilized enzyme(s) increased water holding capacity, oil holding capacity, and relative viscosity, and improved emulsifying and foaming characteristics. Hydrolysis by immobilized protease(.s) increased solubility, relative viscosity and foaming ability of partially purified fractions. Succinylated soybean proteins had high oil holding capacity, viscosity, emulsifying ability, emulsion stability, and foaming ability. The order in which succinylation and hydrolysis by immobilized enzymes were done, conferred on soybean proteins various functional properties. Evaluation of the molecular size of modified soybean proteins with sodium dodecyl sulfate (SDS) indicated that immobilized trypsin and chymotrypsin preferentially hydrolyzed specific protein components, and that succinylation enhanced hydrolysis, expanded protein molecules, and dissociated subunits. Measurement of molecular size and charge of the modified soybean proteins without SDS showed that immobilized trypsin hydrolyzed the intermediate subunits relatively fast, and succinylation separated the intermediate subunits. Succinylation increased the average molecular charge of soybean proteins, while hydrolysis decreased their average molecular size and their average molecular charge. The ratio of the average molecular weight to the average molecular charge could explain various functional properties. When the ratio was less than 5.0 x 10⁵, the modified soybean proteins had high soluble amino groups, high foaming ability, low water holding capacity and low foam stability. When the ratio was 9.0 x 10⁵, oil holding capacity, emulsifying ability, and emulsion stability were maximum. Relative viscosity was high at a constant value of 2.5 x 10⁵. / Ph. D.

Variable-temperature Fourier transform infrared (VT-FTIR) spectroscopy in conjunction with 2D correlation analysis was employed to study the unfolding of aspartate transcarbamylase (ATCase) and its individual subunits. The regulatory subunit (RSU) was uniformly labeled with 13C/15N and then reconstituted with the unlabeled catalytic subunit (CSU) to form the holoenzyme. The activity of the holoenzyme was shown to be unaffected by the isotopic labeling of the RSU. The VT-FTIR investigation of the isolated CSU and the CSU in the holoenzyme revealed that the CSU is more thermally stable when bound to the RSU (i.e., in the holoenzyme). The RSU also showed more thermal stability when bound to the CSU. The sequences of events leading to the unfolding of the isolated CSU and RSU and the CSU in the holoenzyme were deduced by 2D correlation analysis of the VT-FTIR spectra. The results for the isolated CSU demonstrated that beta-sheets unfold first, followed by a-helices and then turns, and finally aggregates form. The sequence of unfolding of the RSU showed an increase of turns followed by a loss of intramolecular beta sheets, then a loss of alpha-helices and the formation of aggregates. The CSU in the holoenzyme exhibited a slightly different unfolding pathway and was observed to unfold subsequent to the unfolding of the RSU, consistent with the two thermal transitions observed by differential scanning calorimetry.

Although the native conformation of a protein is thermodynamically its most stable form, this stability is only marginal. As a consequence, globular proteins have a certain amount of flexibility in their backbones which allows for conformational changes in the course of their biological function. In the course of this thesis, we study protein models at the edge of stability in different contexts: (1) First, we use molecular dynamics to determine the force needed to rupture a chain molecule (an unfolded protein) being stretched at constant loading rate and temperature. When all energy bonds of the molecule are identical, we find that the force F depends on the pulling rate r and temperature T according to F ~ const -- T 1/3|ln(r/T)|1/3 When a single weak bond is introduced, this result is modified to F ~ const -- T2/3|ln(r/ T)|2/3 This scaling, which is model independent, can be used with force-spectroscopy experiment to quantitatively extract relevant microscopic parameters of biomolecules. (2) Second, we study the structural stability of models of proteins for which the selected folds are unusually stable to mutation, that is, designable. A two-dimensional hydrophobic-polar lattice model is used to determine designable folds and these folds were investigated under shear through Langevin dynamics. We find that the phase diagram of these proteins depends on their designability. In particular, highly designable folds are found to be weaker, i.e. easier to unfold, than low designable ones. This is argued to be related to protein flexibility. (3) Third, we study the mechanism of cold denaturation through constant-pressure simulations for a model of hydrophobic molecules in an explicit solvent. We find that the temperature dependence of the hydrophobic effect is the driving force for cold denaturation. The physical mechanism underlying this phenomenon is identified as the destabilization of hydrophobic contact in favor of solvent separated configurations, the same mechanism seen in pressure induced denaturation. A phenomenological explanation proposed for the mechanism is suggested as being responsible for cold denaturation in real proteins.

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Fourier transform infrared (FTIR) spectroscopy is a powerful tool for probing protein structure-function relationships. With the use of isotope editing, it can also be employed to elucidate protein-nucleic acid interactions. This technique was used to study the sequence of heat-induced unfolding of the uniformly labeled 13C regulatory subunit (RSU) of E. coli aspartate transcarbamylase (ATCase) with its inhibitor CTP. The absorption of CTP in the amide I' region limits our ability to detect protein conformational changes upon binding of CTP. Therefore, by labeling the protein with 13C shifts the amide I' band ~ 40 cm -1 and clearly separates the protein bands from those of CTP. Variable-temperature (VT) FTIR spectroscopy was then employed to monitor the thermal unfolding of the labeled RSU in the presence and absence of CTP. / In addition, isotope editing was further explored to probe domain-domain interactions of the two domains of RSU using intein technology. Intein technology provides a novel means by which isotope editing can be performed to extract information on protein inter-domain and inter-subunit interactions by spectroscopic analysis but has not yet been exploited in Fourier transform infrared (FTIR) spectroscopy. The objective of this project is to present for the first time the feasibility of segmental labeling through intein-mediated protein ligation (IPL) for the purpose of studying conformational changes by FTIR spectroscopy, using ATCase as a model enzyme. In the first phase of this project, the RSU of ATCase, which houses a Zn-binding domain and a nucleotide binding domain, was reconstructed from its isolated domains using commercially available intein-base expression vectors. As steps towards obtaining an isotope labeled RSU, we have fused each domain to separate inteins. Following affinity purification, the intein tags were chemically cleaved and the reactive ends of the two RSU domains were ligated together to form a peptide. Although ligation was successful, improved yields are required for the FTIR spectroscopic studies.

Photocopy of typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 174-182. / Microfiche. / xvi, 182 leaves ill. 29 cm

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Spores of foodborne pathogens such as Clostridium botulinum, Clostridium perfringens and Bacillus cereus are widely distributed in nature. Presence of those spores in food products, particularly C. botulinum spores in vacuum packed, ready-to-eat low-acid products, is a great safety concern. The research here described is a first effort towards understanding the role of the spore coat proteins in the inactivation of bacterial spore using high pressure processing. This study proposes a coat protein solubilization methodology using non-ionic detergents minimizing protein damage and compatible with spectroscopy methods. The methodology developed here was compared with approaches proposed in the literature with respect to protein yield, protein fractions identified, amino acid composition and suitability with spectroscopy techniques for the further analysis of coat proteins. Bacillus subtilis ATCC 6633 spore coat proteins were solubilized (n=3) using octyl-β-D-glucopyranoside (OGP) at room temperature and urea/sodium dodecyl sulphate (UDS) at 37C and 70C. Analysis of variance (ANOVA) showed no significant (95% confidence) differences between the three repetitions of the three spore coat protein solubilization methods. Protein yield was significantly larger (95% confidence) when using UDS at 70C as compared to UDS at 37C. OGP gave the lowest protein yield but allowed circular dichroism (CD) analysis of the spore coat protein solution with minimum blank signal. SDS-PAGE revealed that the UDS-70C coat protein solutions consisted of five major and six minor proteins ranging 6 to 65 kD while the OGP solution appear to consist of four major and nine minor bands in the same mw range. Amino acid analysis of the protein extracted by the OGP method was conducted using reverse phase HPLC (RP-HPLC) and compared with published information. The OGP spore coat protein solution showed a higher proportion of aspartate, glutamate, alanine and tyrosine. Pressure, heat and time effects were studied on spore coat proteins obtained from untreated and pressure-treated B. subtilis ATCC 6633 spores. Pressure treatments of spores, and of extracted spore coat protein solutions, at 50 kpsi (345 mPa) and 85 kpsi (586 mPa) for 10 and 30 min at constant 85C along with appropriate heat- and pressure-only controls and untreated sample, were used to study the effect of pressure, heat and time on spore coat proteins. Both spore coat protein solubilization procedures showed a significant reduction in protein yield for pressure-only, heat-only and pressure/heat treated spores when compared with untreated spores. When OGP-solubilized proteins from untreated spores were pressure treated, SDS-PAGE profile showed an increasing overall band intensity with increasing pressure and time. In the case of protein solution obtained from pressure-treated spores the electrophoretic pattern showed the loss of higher molecular weight proteins. The significance of this study is that for the first time we have observed extensive changes on spore coat proteins caused by pressure, as well as heat treatments. Future studies will examine what is the probable physiological role of the proteins damaged by these physical treatments. An advantage of the protein solubilization here developed will allow the application of spectroscopy techniques to characterize changes in spore coat proteins. / Graduation date: 2003