Fig. 1.

Purified HCCS is a heme protein. (A) Coomassie blue staining of purified GST-tagged HCCS showing 57-kDa full-length GST-HCCS. (B) Anti-GST immunoblot of purified GST-tagged HCCS showing 57-kDa full-length GST-HCCS, 44-kDa GST-HCCS** and 32-kDa GST-HCCS* degradation products, and 29-kDa GST. (C) Heme staining of purified 57-kDa full-length GST-HCCS. For A–C abbreviations are CS, crude sonicate; E, elution; EC, concentrated elution; FT, flow through; L, load (DDM-solubilized membranes); M, molecular weight standards; S, soluble fraction; W1, wash 1; W2, wash 2; W3, wash3. (D) Heme staining of cytoplasmic extracts (B-PER) showing synthesis of the human holocytochrome c as a function of HCCS expression and arabinose concentration. The 12-kDa holocytochrome c is indicated by an arrow. One hundred micrograms of total protein was loaded in each lane. In C and D, prestained molecular weight standards were overlaid in red onto the heme stains. (E) UV-Vis absorption spectra of purified GST-HCCS (black line), reduced with sodium dithionite (red line), or oxidized with ammonium persulfate (blue line). (Inset) Sodium dithionite-reduced pyridine hemochrome spectrum of purified GST-HCCS from 500–600 nm. (F) UV-Vis absorption spectra of purified GST-HCCS (black line) in the presence of imidazole (purple line) and in the presence of imidazole reduced with sodium dithionite (red line). In E and F, the region from 500–700 nm has been multiplied by a factor of three. Absorption maxima are indicated by arrows.