Premium
When Reduction Leads to Expansion: A Simple Native PAGE Gel Enzyme Assay Experiment as a Starting Point for Other Structure‐Function Investigations
Author(s) -
Hazzard James Taylor,
Pasalic Dario,
Edwards Sarah,
Friedrich Kenneth
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.531.2
Subject(s) - denaturation (fissile materials) , chemistry , circular dichroism , enzyme , reagent , tryptophan , function (biology) , disulfide bond , fluorescence , chromatography , biochemistry , nuclear chemistry , amino acid , organic chemistry , biology , physics , quantum mechanics , evolutionary biology
A simple assay on native PAGE gel for two E. coli enzymes, purified by undergraduates in a biochemistry laboratory, provides an excellent springboard for more sophisticated investigations on protein structure. They perform colorimetric assays on b‐galactosidase (b‐gal) using ortho‐nitrophenyl‐b‐galactosidase in a buffer containing b‐mercaptoethanol (BME) and on alkaline phosphatase (AP) using FAST RED TR/Naphthol AS‐MX reagent. Half of the class assays for b‐gal first and AP second; the other half of the class reverses the order. Results differ because the former group reduces two disulfide bonds which are essential for AP activity, only observing activity for b‐gal, while the latter sees activity for both enzymes. This simple PAGE experiment leads the students to the question: Is loss of activity due to protein denaturation? Answering this question raises more sophisticated investigations, including BME dialysis, intrinsic fluorescence spectroscopy of AP tryptophan residues, ANS fluorescence spectroscopy, and circular dichroism. These can be offered as special projects to laboratory groups who report their findings to the entire class, providing a holistic approach to investigating the relationship between loss of activity and protein denaturation. Initial data and results from our laboratory course will be presented. This work supported in part by a grant from NSF (DUE 0837398 to JTH).