1. Weigh out the cholesterol into a 50-mL round bottom flask. Then add the phos-pholipids (phospatidylcholine and stearylamine) dissolved in 3 mL chloroform and allow the cholesterol to dissolve by gentle swirling. The lipid concentration is 18 |imol with a molar ratio of phospatidylcholine:cholesterol:stearylamine 7:2:0.25 (see Note 4).
2. Dry the lipid mixture in a rotary evaporator (Rotavapor Buchi B-480) under an N2 stream to maintain an inert atmosphere.
3. Add slowly 1 mL of the L-asparaginase solution (0.03%) with gentle stirring in order to hydrate the thin film of lipid formed and form multilamellar vesicles. The stirring is continued for 2 h and should not be too vigorous in order to avoid heat generation (see Note 5).
4. The liposome suspension is lyophilized in a freeze-dryer (Cooling HetoTrap® HETO Model CT 110) overnight.
5. Following freeze-drying, the preparation is rehydrated with 0.1 mL of 0.3 M man-nitol and the mixture is gently stirred in a vortex (see Note 6).
6. Leave the suspension at room temperature for 30 min.
7. The volume of the suspension is brought to 1 mL by adding 0.154 MNaCl.
8. Nonencapsulated enzyme is removed by three cycles of 30-fold dilution at 38,000g for 30 min.
9. Finally, the liposome pellet is resuspended in 1 mL of 0.154 MNaCl (see Note 7).
3.2.2. Determination of L-Asparaginase Recovery in Liposomes
Protein recovery is the liposome-associated protein in the final liposome suspension related to the total protein initially added, and indicates the yield of the process.
final liposome-associated protein (Pf)
total initial protein (R)
1. Two milliliters of liposome suspension are mixed with 2 mL of 2% (v/v) Triton X-100 and 0.5% SDS aqueous solution in order to disrupt the membranes and release the enzyme.
2. After that, the protein is determined using the method described by Lowry (37).
3.2.3. Determination of L-Asparaginase Encapsulation Efficiency
Encapsulation efficiency is the percentage of the ratio between the final pro-
tein-to-lipid (P/L) ratio and the initial P/L ratio.
R / Lr in liposome suspension
total Pf / Lf in initial mixture
1. Two milliliters of liposome suspension are mixed with 2 mL of 2% (v/v) Triton X-100 aqueous solution in order to disrupt the membranes.
2. After that, the protein is determined using the method described by Lowry (37) and lipid determinations are performed using the method described by Rouser et al. (39).
The biological activity of the enzyme is determined by the method described by Jayaram (38) after disruption of the liposomes with Triton X-100 as described in the previous section. The enzymatic activity of one unit L-asparaginase corresponds to formation of 1 mmol NAD/min at 37°C.
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