was treated with 4 04 micrograms of carboxypeptidase b for 40 minutes at 25 u00b0 c the reaction was terminated by adding 1 0 ml of 7m urea 0 2m acetic acid the solution was chromatographed on a 0 9 u00d7 40 cm g25m sephadex column equilibrated with 2 acetic acid and then lyophilized solid material 1 91 mg was purified on a 5 u00d7 50 mm pharmacia mono s column equilibrated with 7m urea 0 1m acetic acid a linear salt gradient from 0 07 to 0 3m nacl over 30 minutes was used to elute the protein the main peak material was recovered and stored at 4 u00b0 c crude approximately 70 pure by hplc 65 al split hpi 10 46 mg wet wt in 2 5 ml of 0 1m tris final ph 7 1 was treated with 20 1 micrograms of carboxypeptidase b for 40 minutes at 25 u00b0 c the reaction was terminated by acidifying to ph 3 4 with 7m urea 0 1m acetic acid and 1m hcl the main peak solution from part a described above was pooled with this reaction product for the following g50sf step the resulting des 64 65 hpi was applied to a 2 5 u00d7 125 cm g50sf sephadex column and eluted at 6 u00b0 c with 1m acetic acid fractions containing des 64 65 hpi were pooled and lyophilized to obtain 8 07 mg of the desired product as determined by u v analysis the solids from the g50sf sephadex column were chromatographed on.