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.