Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: (A) The absorbance spectra of the ferric forms of purified Drosophila melanogaster, Oncopeltus fasciatus and Bombyx mori E75 LBDs in the 250–700 nm. The spectra have been vertically displaced and the α/β bands (500–700 nm) magnified for clarity. (B) Circular dichroism in the near UV and visible regions of the purified E75 LBDs of Drosophila melanogaster, Oncopeltus fasciatus and Bombyx mori.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques: Purification

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: The Drosophila melanogaster (A), Oncopeltus fasciatus (B) and Bombyx mori (C) E75 LBDs purified in the ferric state were subsequently reduced with sodium dithionite and the ·NO and CO complexes were formed anaerobically. The ferrous form is depicted with a solid line, the ferrous-CO complex with a dotted line and the ferrous-NO complex with a dashed line. The spectra have been vertically displaced and the α/β bands (500–700 nm) magnified for clarity.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques: Purification

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: The elution of the heme moiety (bottom traces) was determined at 400 nm whereas the protein (upper traces) was detected at 214 nm as described in the Materials and Methods section. An acetonitrile gradient was used to determine the elution position of free heme (A) as well as the elution of the Drosophila melanogaster (B), Oncopeltus fasciatus (C) and Bombyx mori (D) E75 LBD hemoproteins.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques:

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: The purified E75 LBDs of Drosophila melanogaster (solid line), Oncopeltus fasciatus (dotted line) and Bombyx mori (dashed line) were allowed to react with pyridine as described in the Materials and Methods section and the absorbance difference spectra were recorded between 500 and 580 nm. The spectra are vertically displaced for clarity.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques: Purification

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: (A) The absorbance spectra of ~24 µM O. fasciatus E75 LBD purified from bacteria supplemented with Fe(III) protoporphyrin IX (hemin) (solid line) or supplemented with Fe(III) mesoporphyrin IX (dotted line) are shown in the 350–700 nm range. The HPLC elution profiles (B) show the absorbance at both 214 nm (upper line) and 400 nm (bottom line). The elution profile of the O. fasciatus E75 LBD purified from bacteria supplemented with Fe(III) protoporphyrin IX (hemin) is shown in the upper panel whereas the elution profile of the O. fasciatus E75 LBD purified from bacteria supplemented with Fe(III) mesoporphyrin IX is shown in the bottom panel. Free heme elutes at ~42 ml and free Fe(III) mesoporphyrin IX elutes at ~45 ml.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques: Purification

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: Chimeric constructs of Drosophila melanogaster/Oncopeltus fasciatus E75 LBD were made and purified from a bacterial expression system as reported in the Materials and Methods section (full sequences of the chimeras shown in Fig. S3). (A) The electronic absorbance spectra of the Onc/Dros (dotted line) and Dros/Onc (solid line) ferric form of the chimeras are shown in the 350–700 nm range. The 500–700 nm part of the spectra was magnified for clarity. (B) HPLC elution profiles of the Onc/Dros (upper panel) and Dros/Onc (bottom panel) chimeras both at 214 nm (upper line) and 400 nm (bottom line). In all cases free heme elutes at ~42 ml.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques: Construct, Purification, Expressing

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: Amount of heme not associated with the protein moiety calculated after integration of the peak areas at 400 nm obtained from the HPLC analysis.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques:

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: Electronic absorbance spectra as well as HPLC elution profiles of the O. fasciatus E75 LBD Met245Thr (A) and Glu158Lys (B) mutants. The absorbance spectra are shown in the left panels in the 350–700 nm range. The HPLC elution profiles (right panels) show the absorbance at both 214 nm (upper line) and 400 nm (bottom line). In all cases free heme elutes at ~42 ml.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques:

Journal: Biochemistry

Article Title: Covalent attachment of heme to the protein moiety in an insect E75 nitric oxide sensor

doi: 10.1021/bi300848x

Figure Lengend Snippet: Electronic absorbance spectrum (A) as well as HPLC elution profile (B) of the purified B. germanica E75 LBD. The absorbance spectrum is magnified in the 500–700 nm range for clarity. The HPLC elution profile shows the absorbance at both 214 nm (upper line) and 400 nm (bottom line). Free heme elutes at ~42 ml.

Article Snippet: All the E75 proteins expressed in E. coli were purified by Ni-NTA affinity column chromatography (Qiagen).

Techniques: Purification