pure on the basis of its appearance as a single spot with 

 R f of 0.57 after TLC in the systems described for ADBH. 



Concentrations were determined for its absorption at 395nm 



2 

 with an extinction of 14000cm /mmole. 



ADGG was coupled to BSA for production of a conjugate 

 that could be directly compared with the previous work on 

 ADH-BSA conjugates. After EDC mediated conjugation, the 

 ADGG-BSA conjugate was isolated by chromatography on Sepha- 

 dex G-75 (Figure 13) . The ADGG-BSA eluted as a single peak 

 followed by unreacted ADGG. The elution profile contains 

 significant absorbance at both 384 and 280nm indicating co- 

 valent association of the ADGG and BSA. Individual fractions 

 were further analyzed by inhibition of calf thymus RNA poly- 

 merase II. The inhibition profile closely parallels the 

 absorbance at 384nm demonstrating retention of inhibiting 

 activity of a-amanitin after conjugation. 

 Characterization 



Figure 14 shows the absorption spectra for ADGG and 

 ADGG-BSA. The peak absorbancy at 395nm for the azo moiety 

 was used to determine a-amanitin concentration whereas the 

 protein concentration was obtained from Lowry protein analy- 

 sis. No interference with the Lowry assay was noted by ADGG. 

 The ADGG-BSA conjugate prepared contained 2.9 moles of ADGG 

 per mole of BSA. 



The inhibition of calf thymus RNA polymerase II by 

 ADGG and ADGG-BSA is shown in Figure 15. Free ADGG com- 

 pared favorably to a-amanitin with respect to inhibition of 



