Homogeneous preparation of human thymidine-5'-triphosphatase by electroelution from SDS/PAGE with subsequent renaturation.
This paper describes a rapid and inexpensive method for homogeneous enzyme preparation from SDS/polyacrylamide gels with subsequent renaturation. The method was optimized for an enzyme of pyrimidine metabolism, thymidine-5'-triphosphatase (dTTPase), present in human serum in small amounts. After gel electrophoresis, the enzyme was eluted from gel pieces in an elution chamber based on a tube gel electrophoresis system. Renaturation conditions were optimized in preliminary tests. The best results were obtained with an initial acetone precipitation to remove sodium dodecyl sulfate. The precipitate was then dissolved in 8 M guanidine hydrochloride and diluted 50-fold for renaturation. Adding 1.5 mg/ml lauryl maltoside to the renaturation buffer, followed by subsequent dialysis of the renaturating samples, improved the renaturation yield up to 95%. This method was used to purify dTTPase to homogeneity from a partially purified sample, and to determine the molecular mass of the subunits. The procedure can also be applied to other enzymes and could give rise to a general strategy for enzyme purification.
Schultes
B C
BC
Institute of Clinical Biochemistry, University of Bonn, Federal Republic of Germany.
Dahlmann
N
N
D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation; D011755:Pyrophosphatases; D012967:Sodium Dodecyl Sulfate
D003902:Detergents; D004793:Enzyme Reactivators; D005960:Glucosides; D012967:Sodium Dodecyl Sulfate; C040358:dodecyl maltoside; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C023117:thymidine-triphosphatase