Econo pack 10dg desalting column

Holoenzymes (wild-type OAM, OAM variants or DAPDH), reagent solution preparation and anaerobic measurements were performed in a Belle Technology anaerobic glove box (Belle Technology, Weymouth, UK) (O₂ levels < 1 p.p.m.) under very dim light (to minimize photolysis of AdoCbl), unless otherwise stated. Buffer solutions [100 mm NH₄-EPPS (4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid), pH 8.5] were purged for 3 h with nitrogen, and then brought into the glove box and allowed to equilibrate for 18 h prior to use. Solid AdoCbl, PLP, d-ornithine and inhibitor (DAB) were introduced into the glove box and dissolved in anaerobic buffer. A concentrated protein sample was introduced into the glove box and gel-filtered using a 10 mL Econo-pack 10DG desalting column (Bio-Rad, Hertfordshire, UK) pre-equilibrated with anaerobic buffer. Excess oxygen was scavenged from eluted protein using glucose oxidase (13 units·mL⁻¹) and glucose (10 mm) to prepare anaerobic apoenzyme samples prior to use.

In this study, holoenzymes (wild-type OAM, OAM variants, or DAPDH), reagent solution preparation, and anaerobic measurements were conducted in a Belle Technology anaerobic glove box (O₂ levels < 1 ppm) under very dim light (to minimize photolysis of AdoCbl) unless otherwise stated. Buffer solution (100 mm NH₄-EPPS, pH 8.5) was purged for 3 h with nitrogen and then brought into and allowed to equilibrate for 18 h in the glove box to prepare anaerobic buffer. Solid AdoCbl, PLP, d-ornithine, and inhibitors (DABA and DAPA) were introduced into the glove box and dissolved in anaerobic buffer. A concentrated protein sample was introduced into the glove box and gel-filtered using a 10-ml Econo-pack 10DG-desalting column (Bio-Rad) pre-equilibrated with anaerobic buffer. In preparing samples for protein labeling, 1 mm DTT (final concentration) was added to the concentrated enzyme to reduce the cysteine disulfides before passing through a desalting column. Excess oxygen was scavenged from eluted protein by glucose oxidase (13 units ml⁻¹) and glucose (10 mm) to prepare anaerobic apoenzyme prior to use.