Hormones and hormone precursors 



a red chromogenic reaction typical of lAA, prompted the examination of the 

 syrup in another solvent system, n-butanol/ammonia. The result {Figure l{b)) 

 differed from that shown in Figure l{a) in that the first peak of activity no 

 longer corresponded to lAA though activity again occurred in the IAN zone. 

 Results with chromogenic sprays gave yellow at Rf 0-0-2 and mauve at IAN. 



These results, in which the pattern of growth promotion varied with the 

 solvent system used, suggested that the growth-active zones at lAA 

 [Figure l{a)) and at the starting line [Figure 1(b)) should be examined further 

 to see whether they are identical. 



Two 28 cm chromatograms were each loaded with 200 mg syrup and 

 chromatographed in wopropanol/ammonia. U. v. -fluorescent zones corre- 

 sponding to 1 and 2 in Figure l{a) were removed and eluted at 5°C with 

 4 ml water. 0-75 ml of this eluate was pipetted on each of two papers, and 

 chromatographed in ammoniacal z.s'opropanol or w-butanol (Figures 2(a) and 

 (b)). The histogram of the ?.ropropanol chromatogram is essentially the same 

 as that of Figure 1(a), while that in ?i-butanol differs but slightly from that of 

 Figure 1(b). In Figure 2(b), the chromatogram division at Rf 0-0-08 was 

 according to u.v. fluoresence, but it was too uneven to warrant the conclusion 

 that two growth promoters had been separated. Before discussing this 

 experiment further, data of the complementary experiment are presented. 



Two chromatogram papers were prepared as in the preceding experiment 

 and chromatographed in «-butanol/ammonia. Two zones were removed, 

 viz., the area of initial loading and the following zone to Rf 0-18 (corre- 

 sponding to u.v. -fluorescent zones 1-3 in Figure 1(b)). These were eluted with 

 1 ml and 2 ml water respectively. 0-4 ml of each eluate was rechromato- 

 graphed in ammoniacal isopropanol or «-butanol. The histogram of the 

 starting line eluate run in uopropanol/ammonia (Figure 2(c)) resembles 

 Figure 1(a), except for differences in relative position of IAN and the growth 

 promoter near the solvent front. In «-butanol/ammonia (Figure 2(d)) the 

 histogram is basically similar to Figure 1(b), but there is an ill-defined peak 

 at Rf 0-8 and continuous tailing behind it. It is probable that growth pro- 

 motion at Rf 0-06-0-19 does not actually overlap the lAA control and that 

 division of the chromatogram has caused this effect. 



Histograms of the remaining eluate differ slightly from those described 

 above. Chromatography in uopropanol/ammonia (Figure 2(e)) gave an 

 active region at Rf 0-12-0-25, which may correspond with the Rf 0-0-02 zone 

 on the «-butanol chromatogram (Figure 2(f)). Uneven chromatogram 

 division, however, prevents one from stating this with certainty. 



The results shown in Figures 2(a)-(f) indicate that the substance at the 

 lAA zone on /j^opropanol chromatograms is identical with that near the 

 starting line on «-butanol chromatograms. Thus, differences in relative 

 position of this substance and lAA result from chromatographic factors, and 

 not from molecular change under the influence of these different solvent 

 systems. From Figures 1(a) and (b), it is clear that chromatography in 

 w-butanol gives better separation of active zones, which in this solvent may 

 be complete. One may deduce from Figures 2(c)-(f) that promotion at the 

 IAN zone originates from a precursor on and near the starting line of 

 n-butanol chromatograms; in wopropanol this precursor occurs at the lAA 

 zone and/or in a zone of smaller Rf range (see Figures 2(a) and (b)). 



43 



