1. 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):
May 15, 1998
©1998 by American Society of Hematology

2. 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:
©1998 by American Society of Hematology

3. 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:
©1998 by American Society of Hematology

4. 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:
©1998 by American Society of Hematology

5. 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:
©1998 by American Society of Hematology