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Analysis of the stability of native and chemically modified enzymes

Sheehan, Helen (1991) Analysis of the stability of native and chemically modified enzymes. PhD thesis, Dublin City University.

Bovine heart creatine kinase was purified to homogeneity and then chemically modified with a number of different reagents in an attempt to improve the thermal stability of the enzyme. The bifunctional crosslinking reagent, dimethyl suberimidate, successfully enhanced the stability of the enzyme towards heat denaturation at several concentrations. Following optimization of the procedure, accelerated storage studies were performed and the results used to predict the storage time of the native and modified enzyme at lower temperatures. The crojsslinked derivative was predicted to have a longer shelf-life at 4 C than the native enzyme. The pH profile was altered following crosslinking, but the Michaelis constants were not changed. The modified enzyme also exhibited a marked resistance to the action of some denaturing agents. Salt bridges were implicated as contributing significantly to the intrinsic stability of the native protein. Modifying reagents which interferred with these electrostatic interactions caused destabilization of the enzyme to occur. Analysis of the processes causing thermal inactivation of creatine kinase, ruled out aggregation as a cause of inactivation. It was revealed that conformational, as opposed to covalent processes were responsible for the inactivation of the enzyme. The role of conformational processes in deactivation was confirmed using a number of different approaches. It was shown that heat-induced oxidation of thiol groups gave rise to incorrect structures through disulfide formation, which resulted in the subsequent loss of enzymic activity. A microassay was developed for the determination of creatine kinase activity. The assay utilized a microplate reader and microtitre plates, and compared extremely well with the results obtained using other assay procedures. Following reduction and carboxymethylation, crude creatine kinase was purified by electrophoresis and electroblotting. The purified protein was then sequenced and the first sixteen residues of the N-terminus were determined. The techniques used in the stabilization of creatine kinase were applied to two other enzymes, lactate dehydrogenase and aspartate aminotransferase. While only limited studies were performed, some stabilization was achieved, enabling the results to be used as a basis for further studies on these enzymes.
Item Type:Thesis (PhD)
Date of Award:1991
Supervisor(s):O'Kennedy, Richard
Uncontrolled Keywords:Enzymes Analysis; Enzyme stability
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Enzymology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:19357
Deposited On:23 Sep 2013 15:25 by Celine Campbell . Last Modified 23 Sep 2013 15:25

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