Fig. 4.

Heme and hemoglobin quantification by indirect methods. With the pyridine hemochromogen assay, heme can be quantified in the range of 7.5–37.5 μM (A) and hemoglobin in the range of 1.3–12.7 μM (B). Depicted is the evaluation at the absorbance maximum of the reduced sample at 556 nm. Other commonly used evaluation techniques are displayed in Supplementary Fig. 8 (C). Mixtures of heme (10–15 μM) and hemoglobin (2–10 μM) show the additive effect of both components. Data are evaluated using the linear heme calibration equation (y = 0.034 x - 0.161). (D) With the Hemastix^® test strips, both components are detected by a green to blue color change in the range of 10–1000 nM (heme) and 2.5–250 nM (hemoglobin) through their peroxidase-like activity. Examples for the analysis of mixtures are found in Supplementary Fig. 9 (E). With the apoHRP-based assay, heme was detected in the range of ~33–43 nM by using TMB as the substrate (blue) and ~24–42 nM by using o-dianisidine as the substrate (orange). (F) For hemoglobin, also a concentration-dependent effect could be observed in the range of ~3–5 nM by using TMB as the substrate (blue) and ~9.5–36 nM by using o-dianisidine as the substrate (orange). (G) Combining 1:10 mixtures of hemoglobin and heme with the apoHRP-based assay using TMB as the substrate did not show any additive effect. (H) In contrast, using analyte combinations in the concentration range of the o-dianisidine-based assay, hemoglobin addition increased the determined heme concentration. However, the heme detection level is the same in the heme-hemoglobin combinations and the respective pure hemoglobin solutions. (I) With the modified, hemoglobin detection SLS method, the absorbance at 413 nm was used to quantify hemoglobin in standard solutions, which worked for 0.5–6 μM hemoglobin. (J) With the modified SLS method, the absorbance at 395 nm was used to quantify heme in standard solutions (10–45 μM). (K) Heme (10–20 μM) significantly increased the hemoglobin (2–4 μM) result from hemoglobin-heme mixtures. (L) With the commercially available Heme Assay Kit^® heme could be quantified at 400 nm within the range of 8–32 μM. (M) Hemoglobin could be quantified with the Heme Assay Kit^® at 400 nm within the range of 0.5–10 μM. (N) Using the Heme Assay Kit^® for hemoglobin-heme mixtures revealed again a significant additive effect when determining the heme concentration from the mixtures. H, heme; Hb, hemoglobin; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.