Extended Data Fig. 9: Investigating the effects of sequence heterogeneity in heme-binding RHPs by SEC and solvent fractionation.

a, SEC elution profile of heme-bound RHP-H1. RHP and heme were mixed at 1:1 molar ratio to obtain heme-bound RHPs. The mixture was then subjected to SEC. The elution profile was tracked at different wavelengths to distinguish the whole RHP population (monitored at 260 nm) versus the heme-bound RHP fraction (monitored at 380 nm, and RHP doesn’t absorb at 380 nm). Left: raw data. Right: normalized absorbance to account for the differences in absorption coefficients between species. The nearly identical elution profiles suggest that heme binding is consistent across RHP chains in RHP-H1. SEC was performed with an instrument equipped with an isocratic pump (1200 Infinity II, Agilent) and a UV detector. Agilent Bio SEC-5 column was used (300 Å, 7.8 ×300 mm) in this experiment. The elution profile is noisy at elution time t > 10 min due to the buffer and trace DMF elution. b, Comparison of SEC elution profiles of mixed heme/RHP-H1, RHP-H1 alone and heme alone. The elution peak of buffer and solvent was at around 10.5 min. c, NMR spectra of the organic and water phase residues of RHP-H1 after liquid-liquid extraction (500 MHz, CDCl₃). To perform the extraction, 0.6 ml RHP solution (D2O, 50 mg/ml) was mixed with 0.6 ml organic solvent (CHCl₃, DCM, or ethyl acetate). The mixtures were shaken vigorously, and the opaque solutions were centrifuged (8000 rpm, 5–10 min) to obtain well-separated water and organic phases. Each phase was dried to remove solvent and re-dissolved in CDCl₃ for NMR measurements and comparison. In all three trials, the residues recovered from organic phase are below 1 mg. The results show that minimal RHP chains were partitioned into organic phases.