Title:Some Minor Characteristics of Spectrophotometric Determination of
Antioxidant System and Phenolic Metabolism Enzyme Activity in Wood
Plant Tissues of Pinus sylvestris L.
Volume: 29
Issue: 8
Author(s): Maria A. Ershova*, Kseniya M. Nikerova, Natalia A. Galibina, Irina N. Sofronova and Marina N. Borodina
Affiliation:
- Forest Research Institute of the Karelian Research Centre of the Russian Academy of Science (FRI KarRC RAS), 11
Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia
Keywords:
Pinus sylvestris L., xylem, phloem, superoxide dismutase, catalase, peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase.
Abstract:
Introduction: A comprehensive study of enzymes of the antioxidant system (AOS) and
phenolic metabolism is an actual subject of biochemical research; changes in the activity of these
enzymes can be used as a diagnostic sign. At the same time, practically little attention has been paid
to describing the regularities of these enzymatic reactions. The article presents the chemical kinetics
study of reactions catalyzed by superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and
phenylalanine ammonia-lyase in Scots pine trunk tissues (Pinus sylvestris L.). The dependence of
the enzyme reaction rate on the enzyme concentration and the substrate concentration is presented,
and the pH-optimum for each reaction is established.
Background: Determination of AOS enzyme activity and PAL activity in woody plants has many
difficulties. The chemical composition of pine trunk tissues affects determining AOS enzyme
activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL
activity gives perfect results when considering all additional controls by taking into account minor
characteristics.
Objective: This study aimed at determining the AOS enzyme activity in 40-year-old Scots pine
(Pinus sylvestris L.) plants growing in the Karelian (Russia) forest seed plantation.
Methods: Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4 °C
in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM
MgCl2, and 0.5 mM PMSF. After 15-min of extraction, the homogenate was centrifuged at 12000 g
for 10 min (MPW-351R centrifuge, Poland). The supernatant was purified on 20 cm3 columns with
Sephadex G-250. Aliquots with the highest protein amount were collected. In tissues, the protein
concentration was 10-50 μg/ml. Proteins in the extracts were quantified by a Bradford assay. The
enzyme activity was determined spectrophotometrically on a SpectroStar Nano plate
spectrophotometer (BMG Labtech, Germany).
Results: Our study made it possible to modify the methods for determining the activity of
superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase
in Scots pine trunk tissues. The enzymatic reaction rate dependence on the enzyme concentration
and the substrate concentration was determined, and pH-optimum was also noted. This
methodological article also provides formulas for calculating the activities of the enzymes.
Conclusion: We found that determining AOS enzyme activity and PAL activity in woody plants is
challenging. The chemical composition of the xylem and phloem of pine affects determining AOS
enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and
PAL activity gives perfect results when considering all additional controls by taking into account
minor characteristics.
