Journal: Scientific Reports

Article Title: Heme-dependent Inactivation of 5-Aminolevulinate Synthase from Caulobacter crescentus

doi: 10.1038/s41598-018-32591-z

Figure Lengend Snippet: Spectroscopic characterizations of the PLP form of cALAS. ( A ) UV/Vis absorption spectra of the PLP form of cALAS (10 μM; based on monomeric state) in the presence of 0, 2.5, 7.4, 15, 29, 48, 74, and 107 mM of glycine (lines 1–8, respectively) at 25 °C. The buffer system was 50 mM HEPES-NaOH (pH 7.5) containing 150 mM KCl and 0.1 mM EDTA. The inset shows a titration curve of the molar extinction coefficient at 422 nm. ( B ) CD spectra of the PLP form of cALAS in the absence (line 1) and presence (line 2) of 100 mM glycine.

Article Snippet: Each purified cALAS (4.5 nmol) was incubated with 1.0 mM glycine and 0.5 mM succinyl-CoA in 100 μL of 50 mM HEPES-NaOH pH 7.4 containing 150 mM KCl at 37 °C for 1 h. A 5 μL aliquot of each reaction mixture was applied to a RP-18 WF 254 s HPTLC plate (Merck) and developed in a methanol-water-TFA (60:40:0.001 v/v) solvent system.

Techniques: Titration

Journal: Scientific Reports

Article Title: Heme-dependent Inactivation of 5-Aminolevulinate Synthase from Caulobacter crescentus

doi: 10.1038/s41598-018-32591-z

Figure Lengend Snippet: Spectroscopic characterization of the heme form of cALAS. ( A ) UV/Vis absorption spectra of the heme form of purified cALAS (solid line (line 1)), the reduced form (dashed line (line 2)), and the reduced CO-bound form (dotted line (line 3)). The inset shows an enlarged image in the region 490–625 nm. ( B ) CD spectra in the visible region for the heme form of cALAS. CD spectra of the heme form of cALAS were measured in the absence (upper) and presence (lower) of 100 mM glycine. ( C ) The absorption spectra of the heme-form of cALAS (line 1) and the dithionite-reduced pyridine-hemochrome (line 2). For A–C, the buffer system was 50 mM HEPES-NaOH (pH 7.5) containing 150 mM KCl and 0.1 mM EDTA. The measurements were performed at 25 °C at a protein concentration of 3 μM (based on monomeric state). The concentration of heme was estimated from line 2 of panel C was 2.6 μM.

Article Snippet: Each purified cALAS (4.5 nmol) was incubated with 1.0 mM glycine and 0.5 mM succinyl-CoA in 100 μL of 50 mM HEPES-NaOH pH 7.4 containing 150 mM KCl at 37 °C for 1 h. A 5 μL aliquot of each reaction mixture was applied to a RP-18 WF 254 s HPTLC plate (Merck) and developed in a methanol-water-TFA (60:40:0.001 v/v) solvent system.

Techniques: Purification, Protein Concentration, Concentration Assay