54 Fundamentals of Auxin Action 



be made from a measurement of the area of the spot, the logarithms 

 of which are proportional to the concentrations within limits (Bennet- 

 Clark et al, 1952). 



Several other types of techniques have been used for the separa- 

 tion and identification of plant growth hormones and growth inhibi- 

 tors. Column chromatography has been successfully used in some in- 

 stances (Bein et al, 1947; Linser, 1951). Countercurrent distribution 

 apparatus has been employed for the separation of the auxins in 

 cabbage (Holley et al, 1951). Von Denffer et al (1952) have used paper 

 electrophoresis for separation of the end products of auxin destruc- 

 tion as well as for identification of the native auxins in cabbage and 

 Brussels sprouts in conjunction with column chromatography and 

 ordinary paper chromatographic methods. Ion-exchange resins, which 

 have been used successfully in the separation and quantitative estima- 

 tion of amino acids and other compounds, may also be employed for 

 plant growth regulators though they have not been applied as yet for 

 the purpose. 



MOLECULAR WEIGHT DETERMINATIONS 



Determination of the molecular weight of an auxin can some- 

 times be very helpful either in identifying the auxin or in establishing 

 changes in molecular size resulting from biological reactions. The 

 method has been described in detail by Larsen (1944) and a clear 

 presentation of its utilization is given by Kramer and Went (1949). 



The method generally used in determining molecular weights of 

 auxins is based on the determination of the rate of diffusion of the 

 substance in agar (as the diffusion coefficient), which value is inversely 

 proportional to the molecular weight under certain specific conditions. 



PROCEDURE FOR MOLECULAR WEIGHT DETERMINATIONS 



1. Blocks or discs of 1.5% agar containing the auxin are made up so that the 

 final thickness is uniform. A convenient method is to pipette 0.5 ml. of 

 agar into a circular mold 10.6 mm. in diameter, making the final thickness 

 1.42 mm. 



2. One block containing auxin is placed upon a pile of 3 plain agar blocks 

 of identical shape. 



3. Diffusion is permitted to continue for 1.5 hours at 26° C in a moist atmos- 

 phere. 



4. The blocks are then separated and the content of auxin in each block is 

 assayed by the Avena test, the ferric chloride test, or other assay. 



The distribution of the auxin in each of the four blocks is then 

 converted into a percentage of the total auxin. For each block, an "x" 

 value is obtained from the tables of Kawalki, reproduced graphically 



