206 R. L. Hinman and P. Frost 



degraded by different pathways under different conditions, and tliat 

 oxygen could be consumed and carbon dioxide evolved in each. Dif- 

 ferences in these pathways could not be detected by assaying for 

 residual I A A, but could be discerned by following the changes in 

 the substrate as the reaction proceeds. For this purpose the spec- 

 trophotometric technique is particularly well suited. 



This paper is a preliminary account of the chemistry of the model 

 system, its relationship to the oxidations effected by peroxidase, and 

 the spectrophotometric techniques employed in following the 

 reactions. 



EXPERIMENTAL 



Changes in the ultraviolet absorption spectra were followed in 

 quartz cuvettes of 1 cm. light path by means of a Beckman DK-2 re- 

 cording spectrophotometer, equipped with the usual accessories for 

 work in the ultraviolet region. The reference cell in each experiment 

 contained the buffer system or solvent used in the sample cell. Correc- 

 tions were made for the weak absorptions above 230 m^u, of peroxidase 

 and hydrogen peroxide by subtraction of the absorbancies of blanks 

 from those of the spectra being recorded. 



The reactions were carried out by pipetting solutions of hydro- 

 gen peroxide or peroxidase in buffer into the cuvette, which con- 

 tained a solution of lAA or some other substrate in the same buffer. 

 The solution was stirred and the spectrum scanned. A typical en- 

 zymatic reaction mixture contained lAA (final concn. 10 -*M) and 

 peroxidase (10*' Af) in acetate buffer (O.IM sodium acetate -]- 0.04M 

 acetic acid) at pH 5. A typical reaction in the peroxide system in- 

 volved lAA (10-^M) and hydrogen peroxide (lO-^Tlf) in a buffer of 

 0.097M hydrochloric acid and 0.05M KCl (pH 1). Reactions were 

 allowed to continue until changes in the spectra had ceased or were 

 very slow, requiring from a few hours to several days depending on 

 the reaction. 



Crystalline horseradish root peroxidase was obtained from Nutri- 

 tional Biochemicals Corporation (Lot no. 7625). 2-Phenylindole-3- 

 acetic acid was synthesized by the method of Bauer and Andersag (1), 

 and 2-phenylskatole by the method of Kissman ct al. (5). Oxindole-3- 

 acetic acid was a gift of Dr. Percy Julian, The Julian Laboratories, 

 Chicago, Illinois. Other chemicals were pinified grades. 



THE PEROXIDASE SYSTEM 



The spectral changes which took place when lAA was treated 

 with peroxidase under the conditions described above are shown in 

 Figure 1. There was a very rapid increase in absorbancy in the 240 



