47 



by LII-2 chromatography in methanol. Yields were generally 

 69% or greater. The purified ADBH chromatographed as a 

 single spot in TLC in methanol :methyl ethyl ketone (1:1) 

 with an R f of 0.67. NMR spectroscopy by Drs. E. Gabbay and 

 W. Brey, Jr. of the Department of Chemistry, University of 

 Florida, Gainesville, Florida indicated that the hydrogen 

 atom resonance spectra of the ADBH were consistant with the 

 proposed structure. The uv and visible spectra presented 

 for ADBH in Figure 3 show ratios of extinctions at 384nm 



to 304nm of 0.85, identical to the ratio of the extinction 



2 

 coefficient for the azo dye at 384nm, 14000cm /mmole to that 



2 

 of free a-amanitin at 304nm, 16400cm /mmole. This would 



imply a stoichiometric relationship between a-amanitin and 



the diazo linked spacer molecule. 



Bovine serum albumin was chosen for the initial con- 

 jugate studies based on reports in the literature of selec- 

 tive toxicity of BSA-3 _ amanitin conjugates for macrophages 

 (Fiume and Barbanti-Brodano , 1974). Following removal of 

 the BOC group from ADBH and carbodiimide coupling to BSA, 

 the conjugate was purified from the reaction mixture by 

 chromatography on Sephadex G-75 (Figure 4) . 

 Characterization 



The conjugate peak eluted in the void volume of the 

 G-75 column and contained significant absorption at 384nm 

 as well as 280nm (Figure 3) indicative of covalent associa- 

 tion of the diazo compound and BSA. The ADH-BSA contained 

 a molar ratio of a-amanitin (as azo compound) to BSA of 1.2 



