Spectral analysis of QC-308 binding to hHO-1.

<p>(A) Heme-conjugated hHO-1 (10 µM) in 20 mM potassium phosphate (pH 7.4) was incubated with increasing concentrations of <b>QC-308</b> at room temperature. Absorbances were measured over a range of 300-700 nm at intervals of 1 nm, and values were corrected for buffer (20 mM potassium phosphate, pH 7.4). The assays were performed in duplicate and the values averaged. The Soret peak gradually shifted from 404 to 410 nm with increasing concentrations of <b>QC-308</b>. Secondary peaks centered at 535 and 560 nm were amplified with increasing concentrations of inhibitor, while a third minor peak at 630 nm decreased until no longer detectable at high inhibitor concentrations (B) Heme degradation rates in the presence of <b>QC-308</b>. Heme degradation was subsequently initiated by the addition of 1 mM l-ascorbic acid and allowed to proceed for 90 min at room temperature. Absorbances were measured at 404, 406, 408 and 410 nm at 1 min intervals and normalized to the initial absorbance (t = 0) for the respective condition. Graph is representative of one replicate for each condition at the wavelength of its Soret peak (indicated in legend). (C) Initial rates were determined for each condition over a period of 1 min (from t = 2–3 min) at the wavelength corresponding to its respective Soret peak as indicated in the legend in (B). For each replicate, values were normalized to the respective control condition, and subsequently averaged. Parallel reactions were also performed for heme-conjugated hHO-1 in the absence of both inhibitor and electron donor (l-ascorbic acid) as a negative control (i.e., no oxidative degradation). (D) Spectral analysis following heme degradation. Absorbances were measured and analyzed as described in (A). Heme degradation corresponded to the disappearance of the Soret peak. Increasing concentrations of inhibitor resulted in increased attenuation of the loss of the Soret peak as well as of the secondary peaks at 535 and 560 nm but the appearance of a peak at 699 nm. Inset depicts the fraction of heme still undegraded after the 90 min reaction, relative to that present at t = 0 for each condition.