mixing after each addition the following components 0 5 ml of 0 2 m tris hcl ph 7 8 8 9 ml of ethylene glycol and 0 25 ml of 1 m hcl twenty microliters of a salt amino acid energy mixture saem consisting of 0 375 m kcl 10 mm mg ch 3 co 2 2 15 mm glucose 0 25 10 mm amino acids excluding leucine 5 mmatp 1 mmgtp 50 mmtris hcl ph 7 6 10 ul creatinine phosphate creatinine phosphokinase 8 ul 14 c leucine amersham 348 mci mmol and adding 1 5 ul of solutions containing varying concentrations of the gelonin mixture the mixture was incubated for 60 minutes at 30 u00b0 c 14 c leucine incorporation was monitored in an aliquot of the mixture by precipitating synthesized protein on glass fiber filters washing in 10 tca and acetone and monitoring the radioactivity in a beta counter using aquasol scintillation fluid utilizing this assay purified gelonin had a specific activity of 4 u00d7 10 9 u mg protein a unit of gelonin activity is the amount of gelonin protein which causes 50 inhibition of incorporation of 14 c leucine into protein in the cell free assay the gelonin amino acid sequence was determined by the edman degradation method using an automated amino acid sequencer as described in european patent application no ep 257735 large peptides and unfragmented protein were applied to the.