Polymerization of dissolved humic substances catalyzed by peroxidase. Effects of pH and humic composition

Abstract

High performance size exclusion chromatography (HPSEC) was used to follow the changes in molecular size distribution of three dissolved humic materials of various origins brought about by oxidative coupling of humic constituents under the combined action of hydrogen peroxide and horseradish peroxidase (HRP). Increase in weight-average molecular weight ( M w) occurred invariably for all humic substances with the oxidative polymerization catalyzed by HRP. Polymerization was found to occur to a further extent at pH 7 than at pH 4.7. This was attributed to the larger mobility of reacting molecules in the hydrated and smaller humic associations stabilized only by weak dispersive forces at pH 7. Conversely, hydrogen bonds confer a conformational rigidity to humic associations at pH 4.7 and depress molecular reactivity. Comparison with chromatograms and M w values obtained by treating humic solutions with acetic acid to pH 3.5 before HPSEC injection confirmed that the increase in molecular size by HRP catalysis was stable and due to the formation of covalent bonds among reacting humic molecules. Humic polymerization was somewhat inhibited when humic substances were rich in alkyl carbons and poor in carboxyl carbons. These molecular characteristics are conducive to thermodynamically stable hydrophobic domains in aqueous solution and may limit the mobility of potentially reactive humic molecules towards the catalytic sites of HRP. Control of the conformational structure of dissolved humic substances may be of importance in regulating their reactivity in the environment.