Metabolism of indole derivatives 



the segments washed with two further quantities of ether, and the combined 

 ether extract treated as above. 



Chromatography was carried out in all-glass apparatus at 20°C using the 

 descending technique. All extracts were normally run in n-butanol/ammonia 

 (0-880)/water (100 :3 : 18) solvent, though sometimes zjo-propanol/ammonia/ 

 water (10:1 :1) was employed. Chromatograms for chromogenic analysis 

 were spotted with amounts of ether extract equivalent to 200 fig of the 

 compound in the original solution. After development, the paper strips were 

 dried in air and in most cases sprayed with Ehrlich's reagent applied as 

 1 per cent /j-dimethylaminobenzaldehyde in alcohol followed by cone. HCl. 

 Other sprays, such as 2 N HCl containing 0-05 per cent NaNOg, and diazotized 

 /^-nitroaniline, were also used. 



When preparing chromatograms for biological examination, ether 

 extract equivalent to 1000 jug of original substance was evenly distributed 

 over 20 spots on a sheet of Whatman No. 1 paper (8 in. wide). After develop- 

 ment, a longitudinal strip containing 2 spots was removed from one side of 

 the chromatogram and sprayed with Ehrlich's reagent to establish the 

 position of indolyl compounds. The rest of the sheet was divided transversely 

 into strips each corresponding to one tenth of the distance travelled by the 

 solvent front. Two thirds of each strip (^ 600 jug) was placed in a petri 

 dish with 20 ml of water for assay in the pea curvature test. The remaining 

 third (^ 300 /xg) was immersed in 5 ml of water in a 5 cm dish for the wheat 

 elongation test. 



The segments for the curvature test were taken from the third internode of 

 pea plants (var. Alaska) grown for seven days under red light at a constant 

 temperature of 25°C. Five segments were placed in each petri dish. The 

 material for the elongation test was excised from 2 cm wheat coleoptiles 

 (var. Eclipse) grown for 72 hours at 25°C in an atmosphere of high humidity. 

 10 mm segments were used in preliminary experiments to determine the 

 growth-promoting activity of the four indolyl compounds (see Figure 1), but 

 5 mm segments were always used for the assay of chromatogram strips. The 

 segments were threaded on glass rods, two segments per rod, ten segments 

 being placed in each test solution. 



Biological testing was restricted to solution extracts. This procedure was 

 adopted because, although the colour chromatograms of tissue extracts 

 revealed the presence of the same indolyl compounds as those observed on 

 chromatograms of the corresponding solution extracts, the quantities 

 present were always very small. Moreover, the presence of pigments and 

 fatty substances extracted from the tissues tended to interfere with the 

 chromatography. 



EXPERIMENTAL RESULTS 



The four compounds studied in this investigation are all active in the coleop- 

 tile extension test {Figure 1), although the activity of the amide is low, 

 lAA, lAAm, and lAMe are active in the pea curvature test {Figure 2), but 

 IAN is inactive at the usual physiological concentrations. 



Chromatography of the extracts of solutions of all four compovmds 

 incubated for 18 hours at 25°C in the absence of tissue showed that they were 

 effectively stable under these conditions. 



V 235 



