Fruit ripening is accompanied by increased production of reactive oxygen species (ROS) and oxidative stress. Ascorbate peroxidase (APX) plays a key role in the detoxification of hydrogen peroxide. The present study aimed to investigate the kinetic properties of APX extracted from a local Iranian pear cultivar at two developmental stages (unripe and ripe) and to evaluate the effects of nitrate salts on its catalytic behavior. Pear samples were collected at the unripe and ripe stages, and crude enzyme extracts were prepared from the pericarp tissue. The effects of pH, temperature, and different concentrations of nitrate salts, including AgNO₃, NH₄NO₃, KNO₃, NaNO₃, and Ca(NO₃)₂, on APX activity and kinetic parameters were determined spectrophotometrically. APX activity in ripe fruits was significantly higher (approximately 47.5%) than in unripe fruits. The optimum pH was 6.8 for unripe fruits and 7.0 for ripe fruits. The highest enzyme activity, maximum reaction velocity (Vmax), and catalytic efficiency were observed at 35°C, whereas elevated temperatures (60°C) markedly reduced the enzyme's catalytic capacity. Among the nitrate salts tested, the chaotropic salt silver nitrate exhibited the strongest inhibitory effect, showing the lowest IC₅₀ value (0.2 mM) and causing a substantial decrease in Vmax and catalytic efficiency. In contrast, calcium nitrate enhanced enzyme activity by up to 135% at concentrations of 5–10 mM and exhibited the weakest inhibitory effect. The type of nitrate salt had no significant influence on the Km value of APX. Ripe fruits displayed greater sensitivity to ionic inhibition than unripe fruits. These findings indicate that pear fruit ripening enhances the stability and catalytic capacity of APX, thereby improving its ability to counteract oxidative stress.
EXTENDED ABSTRACT
Introduction
Pear (Pyrus communis L.) is an important climacteric fruit whose ripening is associated with increased respiration, ethylene production, and reactive oxygen species (ROS) accumulation. Excessive ROS accelerate oxidative damage and reduce postharvest fruit quality. Ascorbate peroxidase (APX), a key enzyme of the ascorbate–glutathione cycle, scavenges hydrogen peroxide and plays a vital role in maintaining cellular redox balance. APX activity is influenced by factors such as pH, temperature, and ionic environment. According to the Hofmeister theory, kosmotropic and chaotropic ions differentially affect protein stability and enzyme function. However, limited information is available on the kinetic properties of APX during pear ripening and its response to nitrate salts. Therefore, this study investigated the kinetic characteristics of APX extracted from unripe and ripe fruits of a local Iranian pear cultivar and evaluated the effects of different nitrate salts on its catalytic activity.
Materials and Methods
Fresh pear fruits were collected from orchards in Baneh County, Kurdistan Province, Iran, at two developmental stages (unripe and ripe). Crude APX extracts were prepared from pericarp tissues using cold phosphate buffer containing PVPP, EDTA, and PMSF. Soluble protein concentration was determined by the Bradford method. APX activity was measured spectrophotometrically following the method of Nakano and Asada by monitoring the decrease in absorbance of ascorbate at 290 nm. The effects of pH were evaluated over the range of 4–9 using appropriate buffer systems, whereas temperature dependence was investigated at 4, 25, 35, 50, and 60°C. Kinetic parameters (Km and Vmax) were estimated by nonlinear regression using Michaelis–Menten kinetics with ascorbate concentrations ranging from 1 to 40 mM. The influence of AgNO₃, NH₄NO₃, KNO₃, NaNO₃, and Ca(NO₃)₂ was examined at concentrations between 0 and 50 mM, and IC₅₀ values were calculated using four-parameter logistic regression. Statistical analyses were performed using one-way and two-way ANOVA followed by Tukey's multiple comparison test at P < 0.05.
Results and Discussion
APX activity increased significantly during fruit ripening. Ripe fruits exhibited approximately 47.5% higher enzyme activity than unripe fruits, indicating enhancement of the antioxidant defense system in response to increased ROS production during ripening. The enzyme exhibited a bell-shaped pH profile, with optimum activity at pH 6.8 in unripe fruits and pH 7.0 in ripe fruits. Two-way ANOVA showed that pH was the major factor affecting APX activity, while ripening stage also had a significant effect. The broader pH stability observed in ripe fruits suggests improved enzyme stability during maturation. Temperature markedly influenced APX catalytic properties. Enzyme activity increased progressively from 4°C to 35°C, where the highest values of Vmax and catalytic efficiency (Vmax/Km) were recorded. At temperatures above 35°C, particularly at 60°C, enzyme activity declined sharply, indicating thermal destabilization of the protein. The higher catalytic efficiency observed in ripe fruits at all temperatures further demonstrates the enhanced antioxidant capacity developed during fruit maturation. The nitrate salts produced distinct effects on APX activity. Silver nitrate (AgNO₃) showed the strongest inhibitory effect, exhibiting the lowest IC₅₀ value (0.2 mM) and causing substantial reductions in Vmax and catalytic efficiency without significantly affecting Km. This kinetic pattern suggests that Ag⁺ mainly decreases catalytic turnover rather than substrate affinity, probably through interaction with sulfhydryl groups or disruption of the heme environment. In contrast, calcium nitrate [Ca(NO₃)₂] enhanced APX activity by approximately 135% at concentrations of 5–10 mM and exhibited the weakest inhibitory effect. Sodium, potassium, and ammonium nitrates produced intermediate concentration-dependent inhibition. The inhibitory potency followed the order AgNO₃ > NH₄NO₃ > KNO₃ > NaNO₃ > Ca(NO₃)₂.
Kinetic analyses further demonstrated that the nitrate salts significantly reduced Vmax and catalytic efficiency, whereas Km remained nearly constant among treatments, indicating that the salts primarily affected catalytic capacity rather than substrate binding. Moreover, APX extracted from ripe fruits was more sensitive to ionic inhibition than that from unripe fruits, as reflected by lower IC₅₀ values. Heatmap analysis confirmed that maximum enzyme activity occurred at near-neutral pH and ascorbate concentrations of approximately 20–24 mM, whereas lower or higher substrate concentrations resulted in reduced activity. In conclusion, fruit ripening significantly enhanced APX catalytic capacity and antioxidant potential in pear fruits. Among the nitrate salts investigated, calcium nitrate effectively preserved enzyme activity and catalytic efficiency, whereas silver nitrate acted as a potent inhibitor. The absence of significant changes in Km together with pronounced reductions in Vmax indicates that nitrate salts mainly influence enzyme turnover rather than substrate affinity. These findings provide new insights into the biochemical regulation of APX during pear ripening and suggest that calcium nitrate could be considered as a promising postharvest treatment to maintain antioxidant capacity and improve storage quality of pear fruits.