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Title: Distribution of water in proteins in their solid state using spectroscopic methods
Author: Almutawah, Abdulla
ISNI:       0000 0004 2740 2173
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2012
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In many case the preservation of peptides and proteins for pharmaceutical use and food products involves the preservation of the active material in a partially dehydrated state. The nature of hydration of proteins and subsequent implications for functionality is a matter of importance in both pharmaceutical and food applications. Whilst it is fairly straight forward to measure the total water content of a protein, it is considerably more difficult to estimate the way in which the water is distributed in the protein. Most published studies rely on the use of one technique to discuss this matter and few studies whose used combinations of techniques. In this work, combined methods of infrared, dielectric and calorimetric were used to examine the hydration process of wheat gluten, lysozyme, bovine serum albumin (BSA), and human serum albumin (RSA). Samples of the proteins over a range of water contents were examined. The adsorption isotherms of the four proteins were plotted and fitted to the Guggenheim-Anderson-de Boer (GAB) model. The isotherms of the four proteins showed a typical sigmoidal shape. The amide I (1600-1700 cm-l ) and 11 (1500-1600 cm-l ) regions of FTIR spectra of the proteins were examined. In the case of gluten, the peak intensity ratios in amide 11 (N-R bend / CN stretch) region of the spectra, 1548 cm-I to 1515 cm" and 1533 to 1515 cm", showed a linear relationship with the water content of the samples up to about 35g water/lOOg dry protein. In more hydrated samples, the slope of the curve decreased. This behaviour was not seen in the other three proteins that showed a linear relationship all throughout the plots. In contrast the intensity ratios in the Amide I (C=O stretch) region show a maximum at about 15-20% water content for gluten, 20-25% for lysozyme, and 35-40% for both BSA and RSA samples. Low frequency dielectric measurements were carried out over the same range of water contents. A plot of the real component of permittivity ,10' (at 0.01 Hz) against the water content for samples at different temperatures shows a very consistent pattern which has a change in slope at a water content close to that observed in the ration of Amide 11 data. It is postulated that the addition of water affects the Amide 11 band and the dielectric data by changing the hydration and motion of the side chains and amide groups. The changes in Amide I reflect changes in conformation whereas the hydration process is reflected in the Amide 11 and dielectric data. These changes to the side chains are reduced when the system might is fully hydrated in the case of gluten. DSC experiments suggest that the non freezing water content in wheat gluten is dependent on initial water content and ranges from 36 - 45 g waterl1 OOg dry protein. This suggests that the protein hydration and the non freezing water are not necessarily strongly connected. The dielectric responses for all proteins used can be attributed to Maxwell- Wagner dispersion at the highest level of hydration and charge-hopping behaviour for moderate levels of hydration. This study has shown that FTIR and dielectric spectroscopy may be used in conjunction to explore protein hydration and may offer a way of probing protein hydration in situ with no requirement for extensive sample preparation or manipulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available