28 



Biochemical Characterization of Conju ga tes 



Spectroscopy 



As previously mentioned all conjugates were analyzed 

 for protein content on the basis of their uv absorption at 

 280nm and for their a-amanitin content at 384nm (ADH conju- 

 gates) or 395nm (ADGG conjugates) . A Beckman dual beam 

 spectrophotometer and recorder were used for spectral studies 

 with standard 1cm pathlength quartz cuvettes. Measurements 

 were made with aqueous solutions unless noted otherwise. 

 Protein Determination 



Protein determinations were made by minor modification 

 of the Lowry-Folin assay (Lowry et al., 1951). Crystalline 

 BSA (Sigma, lyophilized, crystallized) or affinity purified 

 Con A were used as standards. The assay was performed at 

 20-22°C with 0.1ml of protein sample containing between 20 

 and 300yg of protein per milliliter. Results were obtained 

 by sample absorption at 600nm, a wavelength at which no 

 interference from the chromogenic diazo linkage of the con- 

 jugates was found. Duplicate or triplicate determinations 

 were made for each assay point and standard. 

 Analysis of Conjugate Linkages 



Conjugate bond formation . The nature of the chemical 



bond formed between the protein (BSA or Con A) and the EDC 



14 

 coupled moiety was examined with radioactively labeled C- 



■3 



HA-Con A and H-DM-ADGG-Con A conjugates. The stability of 

 the bond to hydrolysis by hydroxylamine was determined by 

 exposure of the conjugates to 0.5M hydroxylamine at 37°C 



