Occurrence and Role of Growth Hormones 69 



tained. Because of this a certain amount of reserve should be exercised 

 in interpreting such molecular weight determinations. Before con- 

 clusions about the identity of the auxins being measured are drawn, 

 steps should be taken to purify the auxin and re-determine its molec- 

 ular weight, using the method of W^ildinan and Bonner (1948) and 

 Kramer and Went (1949). 



A method often used for distinguishing between the hypothetical 

 auxins a and b and indoleacetic acid is by observing the stability to hot 

 acid or alkali. The indole ring is destroyed by hot acid, auxin a is 

 destroyed by hot alkali, and auxin b is destroyed by either the acid 

 or the alkali. This means of identifying auxins is confused, however, 

 by the fact that one would expect the immediate precursor to in- 

 doleacetic acid, viz., indoleacetaldehyde, to be destroyed by either 

 the hot acid or the alkali. Refluxing with acid should destroy the 

 indole ring just as happens with indoleacetic acid, and refluxing with 

 alkali would probably produce a variety of destructive combinations 

 of the aldehyde by reactions similar to the well-known aldol condensa- 

 tion reaction. Indoleacetonitrile, another naturally occurring com- 

 pound which shows auxin activity in some tests, is destroyed by alkali 

 and is somewhat resistant to acids (Bentley and Housley, 1952). It is 

 easily conceivable, then, that indoleacetaldehyde could be confused 

 with auxin b and the nitrile could be confused with auxin a if in- 

 stability to acid and alkali were used as the criteria for establishing 

 auxin identity. 



A comparison of dilution curves, as measured by any of the 

 standard assay procedures, is a helpful means of distinguishing between 

 auxins. The dilution curve for indoleacetic acid shows a less rapid 

 decline in activity upon dilution than for indolepyruvic or indole- 

 butyric acid, for example, but a much more rapid dilution effect than 

 for indole sulfonic acid (Went and Thimann, 1937, p. 135). 



The dissociation characteristics of acid auxins have been used at 

 times as evidence for the identity of the material. Indoleacetic acid, 

 for example, has a pK of 4.75 and the known parallelism of the effects 

 of pH changes upon dissociation and growth promotion has been 

 used as evidence in identifying indoleacetic acid as a growth hormone 

 (Bonner, 1934). 



Aside from the actual crystallization of a pure derivative of an 

 auxin, the best means of identification is certainly by chromatography. 

 Precise separation of compounds can be achieved, and the identity 

 can be established in a variety of ways, including the Rf values, fluo- 

 rescence and color reactions as described in chapter II. 



Indoleacetic acid was first definitely shown to be an auxin in plant 



