Studies on native and modified peroxidases
Ryan, Orlaith (1992) Studies on native and modified peroxidases. Master of Science thesis, Dublin City University.
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The aim of the project was to chemically modify horseradish peroxidase (HRP) in an attempt to enhance the stability of the enzyme. Initially a colorimetric microassay for horseradish peroxidase was developed. Chemical modification of the enzyme was then carried out using various homo- and hetero- bifunctional crosslinking reagents in an attempt to improve the stability and hence the shelf life and range of applications of horseradish peroxidase. Stabilized derivatives were produced using bis-imidates and N-hydroxy bis-succinimide esters. Bis-imidates are amino specific homobifunctional crosslinking reagents. Successful crosslinking was seen using dimethyl suberimidate (molecular length = l.lnm), dimethyl adipimidate (0.77nm) and to a lesser extent for dimethyl pimelimidate (0.92nm). Increased thermostability was used as an index that crosslinking had occurred. Crosslinking prevents unfolding of the protein backbone and so a crosslinked enzyme should not unfold when heated. Thermostability was assessed by incubating native or modified enzyme at 72.5°C for 60 minutes. Enzyme activity of samples withdrawn onto ice over the 60 minute incubation period was determined using standard conditions to see if thermostability had been conferred by crosslinking. Although increased thermostability was seen for bis-imidate treated samples, after 60 minutes at 72.5°C modified samples were only slightly more active than native enzyme. N-hydroxysuccinimide esters were also used to modify HRP. Very successful results were seen with these homobifunctional reagents. Increased thermostability was seen to the level that modified samples only lost a small degree of activity after 60 minutes at 72.5°C. Characterization of modified HRP was carried out to determine the effects of modification on the properties and structure of the enzyme. In all cases there was no adverse effect on catalytic activity and no physical alterations to the enzyme were evident. Thus, chemically-stabilized derivatives of HRP have been produced.
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