30 

 buffer as above. Samples of each were saved for assay, and 

 the remainder of each pool was used in the second 

 purification step (643 ml, 206 mg of SP-I protein and 348 

 ml, 97 mg of SP-II protein) . 



CM-cellulose chromatography. CM cation exchanger 

 columns were equilibrated with 50 mM Epps-KOH, 20% glycerol 

 and 1 mM DTT at pH 8.6. SP-I was applied to a bed volume of 

 123 ml and SP-II was applied to a bed volume of 80 ml. Both 

 SP-I and SP-II eluted in wash fractions and a small portion 

 was saved for assay. The remaining portions of the 

 preparations were used in the third step of purification. 



DEAE-cellulose-52 chromatography. Two DEAE-52 anion 

 exchanger columns were prepared and equilibrated in the same 

 Epps-KOH buffer as the preceding step. Proteins were 

 applied, SP-I to bed volume of 107 ml and SP-II to a bed 

 volume of 72 ml, and washed with the same buffer. A 4 00 -ml 

 (SP-I) or 300-ml (SP-II) gradient from to 0.6 mM KCl was 

 applied to the columns. Fractions of about 6 . 5 ml were 

 collected. Single peaks of activity, each eluting at about 

 0.3 mAf KCL were individually pooled and dialyzed against 5 

 mM KPO4 buffer containing 20% glycerol and 1 mM DTT at pH 

 7.3. Samples were saved for assay, and the remainder of 

 each protein was used in the fourth step of purification. 



Hydroxylapatite column chromatography. SP-I and SP-II 

 were applied to HA columns equilibrated in the KPO4 buffer 

 of the previous step (100 ml and 50 ml bed volumes. 



