Graduate Thesis Or Dissertation
 

Reactions of horseradish peroxidase with the isothiocyanates of horseradish oil

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  • Horseradish peroxidase (HRP) has been investigated extensively and for many years. In spite of this it still exhibits unexplained anomalies. Among these is variation in the reported number of isozymes (from 7 to 42), and reports of instability of the isozyme patterns. Since horseradish is a rich source of organic isothiocyanates (mustard oils), it seemed likely that artifactual chemical modification of the enzyme might occur during or after extraction. The present investigation demonstrates that isothiocyanates can in fact cause extensive modification of HRP without corresponding loss of catalytic activity. Incubation of HRP with isothiocyanates at alkaline pH and 4°C altered the isozyme pattern. Peroxidase activity was shifted from bands of higher p1 to bands of lower p1, some of them preexisting bands and some of them newly generated. With time, the basic "C" band, initially the principal isozyme, virtually disappeared, and enzyme activity shifted to bands of lower pr. These were discrete bands, presumably formed by stepwise blocking of successive amino groups. Modification of HRP was found to be dependent on pH, reaction time, and isothiocyanate concentration. Amino acid analysis showed loss of 50 % of the lysine residues after reaction of HRP with allylisothiocyanate at pH 10. Reaction of phenyl- (¹⁴C)isothiocyanate with HRP at pH 10 resulted in an average of five phenylisothiocyanates incorporated per enzyme molecule. Autoradiography showed four radioactive HRP bands , corresponding to a shift of enzyme activity into these bands. Incubation of HRP with allylisothiocyanate in the presence of L-lysine at pH 10 showed little protective effect of the lysine, although the concentration of lysine was 800 times the enzyme concentration. This suggested that hydrophobic sites that bind isothiocyanates to the enzyme surface enhance the reaction of the isothiocyanates with the enzyme. Extraction of HRP from horseradish roots in the presence of adsorbent pclystyrene resulted in extracts that were colorless and odorless, in contrast to the brown color and pungent odor of untreated extracts. The initial extracts showed no difference in isozyme pattern between polystyrene-treated and untreated extracts. After ammonium sulfate precipitation, however, the polystyrene-treated extract retained the original isozyme pattern, while the untreated extract produced new bands of lower pI.
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