Solubilization technique of membrane-bound enzyme: Sialidase from horse liver

Abstract

Two different buffers containing salt were determined to bring sialidase in 100,000 g supernatant, while some detergents like cholic acid, taurocholic acid and Triton X-100 were tried as solubilizing agents. Acetate buffer could be solubilized sialidase activity of about 5 %, this was increased to 25 % with the addition of 0.15 M NaCl. Fifteen percent of the sialidase activity was recovered with the addition 1 % Triton X-100, and 40 % when 0.15 M NaCl and 1 % Triton X-100 were added. In the phosphate buffer system about 20 % of the sialidase activity was found in the supernatant, and was increased to 30 and 35 % with the addition of 0.15 M NaCl and 1 % Triton X-100, respectively. The sialidase activity found in the supernatant was increased of approximately 45 % with addition of both 0.15 M NaCl and 1 % Triton X-100. Cholic acid, taurocholic acid and Triton X-100 showed the same effect in solubilizing the sialidase. The amount of detergent required in solubilization of sialidase from membrane preparation could be reduce from 1 to 0.5 % with addition of 0.25 M sucrose in the solubilization buffer. From the result above, phosphate buffer showed specific effect on sialidase solubilization from horse liver, and this solubilization effect increase when salt was added. But detergent showed no specific effect in solubilizing sialidase.