The effects of lauryl maltoside on the reactivation of several enzymes after treatment with guanidinium chloride.
The present study confirms the previous reports that detergents can facilitate the reactivation of guanidinium chloride (GdmCl) denatured rhodanese (Tandon, S. and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618; Tandon, S. and Horowitz, P. (1987) J. Biol. Chem. 262, 4486-4491). Here, we report the effect of the detergent, lauryl maltoside, on the reactivation of several enzymes other than rhodanese. For this study we used five different enzymes each having a single polypeptide chain, namely: adenosine deaminase; 3-phosphoglyceric phosphokinase; myokinase; 3 alpha-hydroxysteroid dehydrogenase; and phosphoglucomutase. The regain of enzyme activity was used to monitor refolding. Like rhodanese, these enzymes were denatured in 6 M GdmCl and diluted into a buffer containing various concentrations of lauryl maltoside. The effect of lauryl maltoside on reactivating these proteins depended on the specific enzyme used. For example, in the presence of lauryl maltoside, reactivation of adenosine deaminase increased to 98%, while phosphoglucomutase could not be reactivated significantly. The critical micelle concentration (CMC) of lauryl maltoside was measured under the present experimental conditions using 2-(p-toluidinyl)naphthalene 6-sulfonate (TNS) as an apolar fluorescent probe, and gave a value of 0.085 mg.ml-1 in 10 mM sodium phosphate (pH 7.4). The reactivating effect of lauryl maltoside was not generally related to its CMC. In some cases an induction period was observed before the enzyme attained its steady-state velocity. This might suggest the presence of intermediate(s) in the refolding pathway that could have been stabilized by the detergent. These findings indicate that 'non-denaturing' detergents may be useful for assisting reactivation of enzymes, although the optimum conditions will have to be determined for each individual case.
Tandon
S
S
University of Texas Health Science Center, Department of Biochemistry, San Antonio 78284-7760.
Horowitz
P
P
D004791:Enzyme Inhibitors; D004798:Enzymes / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology*; D006027:Glycosides / Q000494:pharmacology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D011489:Protein Denaturation
D004791:Enzyme Inhibitors; D004798:Enzymes; D005960:Glucosides; D006027:Glycosides; D006146:Guanidines; C040358:dodecyl maltoside; D019791:Guanidine