The procedure used was modified from that reported earlier by Kimmel and
Smith [4] and Baines and Brocklehurst [6]. Thawed latexwas mixed with 40mM
cysteine at a ratio of 3:1 (w/v) and the suspension was adjusted to pH 5.6 using
6M HCl and then stirred for 15 min at 4 ◦C. The mixture was filtered and pH
of the filtrate was adjusted to 9.0 using 6M NaOH. The insoluble material was
removed by centrifugation at 9000×g for 30 min at 4 ◦C. The protein content in
the supernatantwas determined and then adjusted withwater before precipitation
with (NH4)2SO4 at 45% saturation. The salt-enriched solutionwas slowly stirred
at 4 ◦C for 30 min. The precipitate was collected by centrifugation as above, and
dissolved using 20mM cysteine. The solution was kept at 4 ◦C before adding
sodium chloride (10%, w/v). The mixture was slowly stirred for 30 min before
separating the precipitated papain by centrifugation. The enzyme was dissolved
in water and dialyzed overnight at 4 ◦C against three changes (1 l each) of water.
The dialysate was finally lyophilized (in a freeze-dryer, LY5FM-ULE, Snijders
Scientific BV, Tilburg, The Netherlands) to obtain purified papain powder