High-performance metal chelate affinity chromatography of cytochromes P-450 using Chelating Superose

Abstract

High-performance metal chelate affinity chromatography [immobilized metal ion affinity chromatography (IMAC)] using Chelating Superose (iminodiacetic acid adsorbent) was investigated for its suitability in purifying phenobarbital- induced rat liver microsomal cytochrome P-450 isozymes (P450) and optimized for preparative purposes. Starting with an 8-aminooctyl-Sepharose fraction of partially purified P450, it was found that only Ni²⁺ - and Cu²⁺ -charged columns could bind P450. No binding was ever observed when Zn²⁺, Co²⁺, Mn²⁺, Cd²⁺, Fe³⁺, Fe²⁺ or Tl³⁺ ions were employed. Of eight commonly used elution buffers, imidazole and tryptamine were found to cause some denaturation of P450. For desorption of proteins bound to Ni²⁺ -charged columns, the following order of decreasing elution buffer strength was determined: cysteine ; ≈ histidine > glycine > histamine > tryptophan > ammonium chloride. During protein desorption with some of these buffers, metal ions were found to bleed from the gel, resulting in P450 denaturation. This could be eliminated by prebleeding the charged columns prior to sample application and had an effect on product recovery and homogeneity. Ni²⁺ and glycine were chosen as a standard for further optimization involving sample adsorption conditions as influenced by equilibration buffer, detergent, load capacity and flow, gradient and temperature conditions. In this way, potassium phosphate (pH 7.75) and 0.4% Emulgen 911 were used to equilibrate a 1.6-ml column and purify 20–50 nmol of P450 (5–15 mg of protein) within 15 min. One gradient fraction consisted of a single sodium dodecyl sulphate-polyacrylamide gel electrophoresis band as judged by silver staining and represented about 25% of the total P450 applied to the column; total recoveries were usually more than 80%. Comparison with the molecular weights and spectral, catalytic and immunological properties of P450 forms isolated according to established procedures indicated that the form isolated here using Chelating Superose comprises mainly P450 2B1 (PB-B). A method is described for fully automated, programmable column regeneration and sample runs.