Study Of Chemically Modified Food Proteins By Vibrational Spectroscopy

This dissertation, "Study of Chemically Modified Food Proteins by Vibrational Spectroscopy" by Hing-wan, Wong, 王慶雲, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled STUDY OF CHEMICALLY MODIFIED FOOD PROTEINS BY VIBRATIONAL SPECTROSCOPY Submitted by Wong Hing Wan for the degree of Doctor of Philosophy at The University of Hong Kong in August 2006 The Raman and Fourier-transform infrared (FTIR) vibrational spectroscopic methods were used to study chemically modified food proteins. Four chemical modification methods: acid deamidation, tryptophan-amidation, sulfitolysis, and trypsin-hydrolysis, and several widely-used food protein products: soy protein isolates (SPI), spray-dried egg white powders (EW), whey protein isolates (WPI), gluten, and casein were selected for study. Raman and FTIR spectra of the chemically modified proteins showed characteristic -1 marker bands. A new Raman C=O stretch vibration band at 1780 cm was observed in deamidated proteins, and was attributed to the γ-carboxyl groups of aspartic and -1 glutamic acids. Similarly, a phenyl stretch vibration at 1552 cm (Raman) was found in - -1 amidated proteins, a -S-SO (thiosulfate) stretch vibration at 1028 cm (in both Raman and FTIR) was found in disulfide bond cleaved samples, and a C=O stretch vibration at -1 -1 1732 cm (Raman) and 1746 cm (FTIR) was observed in trypsin-hydrolyzed proteins. The intensity of these marker bands was found to increase with increases in the level of chemical modification. Calibration curves were constructed by plotting the ratio of the -1 intensity of a particular marker band to the intensity of a Raman 1003 cm -1 phenylalanine stretching band or a FTIR 2116 cm ferricyanide stretching band (used as internal standards) against the extent of modification determined by conventional wet chemistry methods. Linear fits were obtained with correlation coefficients (r) >0.98 and > 0.94 for the Raman and FTIR calibration curves, respectively, indicating strong linear relationships between the marker band intensities and the levels of modification for all the modified protein products. Advantages of the newly developed Raman and FTIR methods over wet chemistry methods are simple and rapid sample preparation, fast determination, and utilization of relatively safe chemicals. Hence, Raman and FTIR spectroscopy have the potential to be further developed for quality control in the food processing industry. The effects of chemical modifications on the conformation and molecular structure of food proteins were studied by vibrational spectroscopy, supplemented by circular dichroism spectroscopy and laser light scattering. Deamidation increased the negative charges in the proteins, resulting in pronounced conformational changes including exposure of hydrophobic residues, increases in disordered conformations and formation of aggregated molecules with compact structures. Amidation also led to increases in disordered structures, possibly due to the attachment of bulky non-polar tryptophan residues. Sulfitolysis breaks up disulfide bonds in proteins, leading to increases in random coil structures and disaggregation of molecules. Hydrolyzed proteins showed marked spectral changes in the amide I and C-H bending vibrations, and progressive increases in random coil structures with concomitant decreases in ordered secondary structure components, suggesting protein denaturation due to cleavage of the peptide bonds. The present study demonstrates the wide application of Raman and FTIR

Chemical and Functional Properties of Food Proteins presents the current state of knowledge on the content of proteins in food structures, the chemical, functional, and nutritive properties of food proteins, the chemical and biochemical modification of proteins in foods during storage and processing, and the mutagenicity and carcinogenicity of nitr

Food Protein Chemistry: An Introduction for Food Scientists discusses food proteins and how they are studied. Proteins are both biological entities and physicochemical compounds, and they will be examined in both contexts in this volume. The chemical and physical properties of proteins will be viewed from the perspective of chemists despite the fact that their use in the food supply emphasizes their biological nature. Key topics discussed include proteins as essential to life; amino acids; protein classification; selected proteins of the most important food systems; and protein structure. The book also includes chapters on protein measurement; protein purification; and spectral techniques for the study of proteins. The book requires readers to have the equivalent of the Institute of Food Technologists requirements for undergraduate food science majors. It also assumes a knowledge of math through calculus. While primarily intended for senior and first-year graduate food science students, the text may also be useful to researchers in allied fields.

Protein chemistry has entered a revolutionary era due to the introduction of genetic engineering for modifying protein structure, as well as the application of advanced computer technology to the study of proteins. By supplementing the traditional ways of studying protein behavior with these newer methods, food processors will be able to resolve difficult problems without using the costly trial-and-error-method so common in the past. This book gives the reader a good foundation in the basics of modern protein chemistry and to show how applications of these concepts to food proteins helps explain their roles in food processing.

Protein research is a frontier field in science. Proteins are widely distributed in plants and animals and are the principal constituents of the protoplasm of all cells, and consist essentially of combinations of a-amino acids in peptide linkages. Twenty different amino acids are commonly found in proteins, and serve as enzymes, structural elements, hormones, immunoglobulins, etc., and are involved throughout the body, and in photosynthesis. This book gathers new leading-edge research from throughout the world in this exciting and exploding field of research.

Protein based biopharmaceuticals are becoming increasingly popular therapeutic agents. Recent changes to the legislation governing stem cell technologies will allow many further developments in this field. Characterisation of these therapeutic proteins poses numerous analytical challenges. In this work we address several of the key characterisation problems; detecting glycosylation, monitoring conformational changes, and identifying contamination, using vibrational spectroscopy. Raman and infrared spectroscopies are ideal techniques for the in situ monitoring of bioprocesses as they are non-destructive, inexpensive, rapid and quantitative. We unequivocally demonstrate that Raman spectroscopy is capable of detecting glycosylation in three independent systems; ribonuclease (a model system), transferrin (a recombinant biopharmaceutical product), and GFP (a synthetically glycosylated system). Raman data, coupled with multivariate analysis, have allowed the discrimination of a glycoprotein and the equivalent protein, deglycosylated forms of the glycoprotein, and also different glycoforms of a glycoprotein. Further to this, through the use of PLSR, we have achieved quantification of glycosylation in a mixture of protein and glycoprotein. We have shown that the vibrational modes which are discriminatory in the monitoring of glycosylation are relatively consistent over the three systems investigated and that these bands always include vibrations assigned to structural changes in the protein, and sugar vibrations that are arising from the glycan component. The sensitivity of Raman bands arising from vibrations of the protein backbone to changes in conformation is evident throughout the work presented in this thesis. We used these vibrations, specifically in the amide I region, to monitor chemically induced protein unfolding. By comparing these results to fluorescence spectroscopy and other regions of the Raman spectrum we have shown that this new method provides improved sensitivity to small structural changes. Finally, FT-IR spectroscopy, in tandem with supervised machine learning methods, has been applied to the detection of protein based contaminants in biopharmaceutical products. We present a high throughput vibrational spectroscopic method which, when combined with appropriate chemometric modelling, is able to reliably classify pure proteins and proteins 'spiked' with a protein contaminant, in some cases at contaminant concentrations as low as 0.25%.

Vibrational Spectroscopy in Protein Research offers a thorough discussion of vibrational spectroscopy in protein research, providing researchers with clear, practical guidance on methods employed, areas of application, and modes of analysis. With chapter contributions from international leaders in the field, the book addresses basic principles of vibrational spectroscopy in protein research, instrumentation and technologies available, sampling methods, quantitative analysis, origin of group frequencies, and qualitative interpretation. In addition to discussing vibrational spectroscopy for the analysis of purified proteins, chapter authors also examine its use in studying complex protein systems, including protein aggregates, fibrous proteins, membrane proteins and protein assemblies. Emphasis throughout the book is placed on applications in human tissue, cell development, and disease analysis, with chapters dedicated to studies of molecular changes that occur during disease progression, as well as identifying changes in tissues and cells in disease studies. Provides thorough guidance in implementing cutting-edge vibrational spectroscopic methods from international leaders in the field Emphasizes in vivo, in situ and non-invasive analysis of proteins in biomedical and life science research more broadly Contains chapters that address vibrational spectroscopy for the study of simple purified proteins and protein aggregates, fibrous proteins, membrane proteins and protein assemblies

Theoretical Vibrational Spectroscopy of Proteins Lu Wang Under the supervision of Professor James L. Skinner At the University of Wisconsin-Madison Vibrational spectroscopy, such as linear and two-dimensional infrared (IR) spectroscopy, is widely utilized to study the structure and dynamics of peptides and proteins. Interpretation of the experiment, or a direct assignment of the complex experimental spectra to the underlying protein structure, can be difficult. Molecular dynamics (MD) simulations offer a complementary approach to provide high-resolution structural and temporal information of proteins, although they are limited by factors such as force field accuracy and are not directly comparable to spectroscopic experiments. We have developed vibrational frequency maps for proteins that generate instantaneous site frequencies directly from MD simulations. We combine the frequency maps with established nearest-neighbor frequency shift and coupling schemes and a mixed quantum/classical framework to form a theoretical strategy for calculating protein linear and 2D IR spectra in the amide I region. This theoretical method provides a means to bridge spectroscopic experiments and molecular simulations, which allows a critical assessment of MD simulations by comparison to experiment, and enables the interpretation of experimental spectra at the molecular level. In this dissertation, we present the development of the vibrational frequency maps and provide the theoretical protocol that allows the calculation of protein vibrational spectra directly from MD simulations. We validate the theoretical method by applying it to peptides with various secondary structures in aqueous solution, and apply it to a few biologically relevant problems. For instance, we have studied the thermal unfolding transition of the villin headpiece subdomain (HP36) using IR spectra calculations. We follow the unfolding process of HP36 by monitoring its spectral changes as a function of temperature. With the help of isotope labeling, we are able to capture the feature that helix 2 of HP36 loses its secondary structure before global unfolding occurs, in agreement with experiment. In collaboration with the Zanni group and the de Pablo group at University of Wisconsin, we have also carried out studies on IAPP, a peptide closely related to type 2 diabetes. By combining theoretical modeling with extensive computer simulations and spectroscopic experiments, we have investigated the structure and dynamics of IAPP in aqueous solution, in the fibril form and in the vicinity of lipid membranes.