The sub‐zero temperature chromatographic isolation of transient intermediates of a multi‐step cycle: Preparation of the substrate‐free oxy‐ferrous cytochrome P₄₅₀

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

“…Application of aqueous – organic solvents for extending the available temperature range down to -25° - -100° C and appropriate experimental modifications for work at such low-temperatures have been described for isoelectric focusing and electrophoresis (102-104), potentiometry (41), size-exclusion and affinity chromatography (84,105,106), and direct assays of enzymatic catalysis (28,86,88,107). …”

Purification and properties of nitrogenase in ethylene glycol at sub-zero temperatures

“…To investigate this hypothesis we purified nitrogenase from Klebsiella pneumoniae by using an ethylene glycol/ water system at sub-zero temperatures. Such methods have been used to advantage with unstable nuclear enzymes (C. Tsopanakis et al, 1978) as well as for isolation of short-lived enzyme intermediates (Hastings et al, 1975;Balny et al, 1976;Debey et al, 1976). Special apparatus and procedures were developed in our laboratory (A. D. Tsopanakis et al, 1978c), with use of in principle the methodology of Douzou and his co-workers (Douzou, 1977;Douzou & Balny, 1978).…”