Biochemical properties of soybean leaf lipoxygenases: Presence of soluble and membrane-bound forms

Abstract

Lipoxygenases (EC 1.13.11.12, LOX) extracted from soybean leaves (Glycine max [L.] Merrill cv. IAC-100) at pH 6.5 showed low stability. Given the importance of correlating the biochemical roles with the physiological characteristics of each LOX isoenzyme, this work evaluates biochemical characteristics and stability conditions of these enzymes in order to plan a purification procedure. LOX activity (A234 at pH 6.0) increased four to five times when 0.25 % (v/v) Triton X-100, 1 % (w/v) polyvinylpyrrolidone, and 1 mM phenylmethylsulfonyl fluoride were added to leaf macerates. Fe2+ (1 mM) stabilised LOX (70.3 % of activity recovered after 48 h storage). Ammonium sulphate fractionation (35–65 % saturation) increased specific LOX activity five times and stabilised the enzymes. Two optimum LOX activities were observed at pH 6.0–6.5 and 4.0–5.0, and the greater storage stability was at pH 6.5 (after 24–28 h storage at different pH values). The results suggest the presence of at least two different forms of the enzyme. The forms of LOX that are active at acidic pH are more stable than the ones that are active at neutral pH. These stable forms were extracted in absence of detergents (soluble forms), while the forms of LOX that are active at pH 6.0–6.5 are unstable forms specially extracted in presence of Triton X-100, and possibly correspond to membrane-bound proteins.