engleski

Influence of Storage Temperature on Total Peroxidase Activity in Crude Extracts from Picea abies L. Karst. Needles

Peroxidases are enzymes with numerous functions in plant cells, one of them being the key role in stress neutralization. Therefore, these enzymes are interesting and frequent objects in current biochemical research. The purpose of this work was to investigate the effect of 30 days storage at different temperatures on the total peroxidase activity in crude extracts from the one-year old needles of Picea abies L. Karst. The needles were mechanically destroyed and proteins extracted in Tris-HCl buffer pH=8.0, with the addition of polyvinylpyrrolidone (PVP). Crude extract samples were kept 30 days at different temperatures (-196°C, -20°C, +4°C, +24°C and +37°C). Total peroxidase activity was detected in the reaction of extract aliquots with guiacol and H2O2, and developed color product was quantified by absorbance measurement at 470 nm using Analytik Jena SPECORD 40. Measurements were performed daily during first 10 days, and on 12th, 14th, 16th, 18th and 30th day of experiment. Enzyme extracts stored for 30 days were subjected to polyacrilamide gel electrophoresis, and afterwards incubated with same substrates. The colored bands obtained resulting from enzyme activity, were analyzed and quantified densitometrically using computer program ImageJ. Both methods revealed three different dependences on storage temperature. Regardless to the storage period the extracts kept at -196°C and -20°C displayed no change in activity compared to the isolation day ; samples kept at +24°C and +37°C lost their activities during 30 days of storage, the second more rapid then the first one ; finally, samples kept at +4°C manifested a considerable rise in enzyme activity, which remained constant until the end of experiment. All changes were confirmed statistically. Electropherogram analysis revealed two main izoenzymes in each lane, with total quantity for each storage temperature resembling those obtained by measuring total activity of extracts spectrophotometrically. There was no difference in izoenzyme appearance between samples. The possible reasons of the observed changes are discussed. On the basis of obtained results, some recommendations regarding enzyme extract storage are given.

peroxidase; storage temperature; Picea abies; needles

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