by V.M. Sellers, T.A. Dailey, and H.A. Dailey Examination of Ferrochelatase Mutations That Cause Erythropoietic Protoporphyria by V.M. Sellers, T.A. Dailey, and H.A. Dailey Blood Volume 91(10):3980-3985 May 15, 1998 ©1998 by American Society of Hematology

Full-length sequence of human ferrochelatase. Full-length sequence of human ferrochelatase. The full-length amino acid sequence of human ferrochelatase is shown with intron/exon boundaries specified by **. Phenylalanine at position 417 is denoted by the double underline. V.M. Sellers et al. Blood 1998;91:3980-3985 ©1998 by American Society of Hematology

Western blot analysis of exon deletion ferrochelatase mutants. Western blot analysis of exon deletion ferrochelatase mutants. Cell extracts of the exon deletion and wild-type human ferrochelatases containing about 5 μg were separated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and treated with human ferrochelatase antiserum. Procedures are as stated in Materials and Methods. From right to left, lanes 1 and 2, wild-type human ferrochelatase used as control; lanes 3 through 11, ▵exon 3 through ▵exon 11 in sequential order. Exons 3 and 4 are believed to contain the epitope for antibody recognition. The size differences of these proteins are evident as the mature-length wild-type human ferrochelatase (from pHDTF20) has a molecular weight of 42 kD and contains 363 amino acid residues, ▵exon 5 contains 320 residues, ▵exon 6 contains 331 residues, ▵exon 7 contains 333 residues, ▵exon 8 contains 330, ▵exon 9 contains 310, ▵exon 10 contains 347, and ▵exon 11 contains 318 residues. V.M. Sellers et al. Blood 1998;91:3980-3985 ©1998 by American Society of Hematology

UV/visible spectrum of F417S recombinant human ferrochelatase. UV/visible spectrum of F417S recombinant human ferrochelatase. The spectrum of purified recombinant F417S ferrochelatase in elution buffer7 was obtained with a Varian 219 spectrophotometer. Not present are characteristic features resulting from the [2Fe-2S] cluster, which are normally present at 460 nm, 420 nm, and 325 nm in spectra from the wild-type protein.14 A minor absorbance peak at 410 nm is attributed to small amounts of residually bound porphyrin. V.M. Sellers et al. Blood 1998;91:3980-3985 ©1998 by American Society of Hematology

X-band EPR spectrum of F417L recombinant human ferrochelatase. X-band EPR spectrum of F417L recombinant human ferrochelatase. Spectra are from whole cells of E coli DW35 (A) with the plasmid encoding wild-type human ferrochelatase, (B) with the plasmid encoding F417L human ferrochelatase, and (C) alone, containing no plasmid. Cell pellets were washed and suspended anerobically in 0.1 mol/L Tris MOPS, pH 8.1, containing an excess of sodium dithionite. Immediately before freezing, approximately 10% (vol/vol) chloroform was added. EPR conditions: temperature, 35°K; microwave power, 10 mW; microwave frequency, 9.60 Ghz; modulation amplitude, 0.64 mT. V.M. Sellers et al. Blood 1998;91:3980-3985 ©1998 by American Society of Hematology