Mechanism of Enhancement of IAA Oxidation by 2,4-Dichlorophenol

Abstract

WVe recenitly proposed a series of reaction's to accouint for the observed formlaition of peroxidase comp)ound's I and II during the aerobic oxidation of indole-3-acetic acid (IAA) by horseradish peroxidase (HRP) (3): 02 + HRP HRP-O. (i) HRP-O2 + IAA Per I (ii) Per I + e Per II +HO-IAA (iii) P,er II + IAA HRP + IAA (iv) (HRP-O.,: a complex betw een ferriperoxidase anid the biradical form of oxygeii; Per I anid Per II: intermefdiate peroxidas,e compoundis I and II; HO-IAA: hydroxyindolenineacebic acid; IAA*: an JAA free radical.) T'he interaction between IAA and HRP led to enzyme inactivaition or destruotion, and iit was postulated tfhiat this inactivation resulted from reactions between IAA free radical's and the enzyme (3). We subsequently repoxited a reaction time course 'in which 2 linear p'hases of IAA disappearance were observed (2). The first phase represented the enzyme caltalyzed oxidaltion of IAA, whiile the second was interpreted as t'he oxidation of IAA by a free radical ch'a'in mecihanism. Other datia have been interpreted as suggesting the presence of free radicals (5,9,12), anid the peroxidase caitalyzed production of s'ub9tralte free iradicals is consistent with dalta obtained from o1ther oxidative and peroxidative sysitem's (6, 7, 10,11). 2,4-Dichlo,rophenoll (DCP) i's widefly employed as a promoter of 'the peroxidase ca'talyzed oxid,ation of IAA; however, the mechani,sm o'f 'promotion has niot been elucidated. We now propose a mechanism of DCP action which is consistent wiith that shown a'bove for the IAA-HRP interaction and wilth other mechanism's suggesting the formation of substrate free redicais in peroxidase catalyzed oxida-tion's. IAA anid DCP were obtained from K and K La,boratories (New York) and analytical reagent grade HRP from Mann Rese,a,r;ch Laboratories (New York). HRP concentration was calculated by methods described previouAly (2, 3). IAA oxidaition was assayed in 2 ways. For the spectrophotometric assay, a Beckman DK-2A. ratio rec,ording spectrophotometer with a w,avelength scani attachment was used. Scans were made from 340 to 230 nm. The Salkowski assay employed the Giordon-Weber modified reagent (4). At vardiolls timeis, 2 ml aliquots of the reaction mixture were added to 4 mil modified Salkowski reagent. Color was allllowed to develop in the dark for 30 mnintutes, anid the optica,l density at 530 nm was measuired wi;th a Beckman DB spectrophotometer. Absorbance ohanges during the co-urse of 1AA oxidation are sh-own in fiiigu1re 1. A,fter 75 m,inutes the spectrum showed maxima at 254 and 248 nim. This suggesited the presence of methyleneoxindole, the likely final produet of peroxidase cata'lyzed IAA oxidation in vitro (5). The conversion of IAA to methyleneoxindole w,as niot a 1-step reaction, since specitral changes con'tinued to occur after all TAA