Purification and electrospray mass spectrometry of aldose reductase from pig lens.
Aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified from pig lens to homogeneity by a rapid and efficient three-step procedure involving poly(ethylene glycol) fractionation, ion-exchange chromatography and chromatofocusing. The homogeneity of the purified enzyme was examined by polyacrylamide gel electrophoresis under native and denaturing conditions, by isoelectric focusing and by high-performance liquid chromatography on a size-exclusion column. The highly purified enzyme is a monomeric protein with a molecular mass of 35,775 +/- 3 Da as determined by electrospray mass spectrometry (ESMS). This purification procedure is particularly suited for the preparation of triclinic single crystals of pig lens aldose reductase, which are currently used in X-ray studies of this enzyme.
Reymann
J M
JM
Laboratoire de Chimie Organique Biologique, Université Louis Pasteur, Strasbourg, France.
Rondeau
J M
JM
Barth
P
P
Jaquinod
M
M
Van Dorsselaer
A
A
Biellmann
J F
JF
D000449:Aldehyde Reductase / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D007908:Lens, Crystalline / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D013552:Swine
D000449:Aldehyde Reductase