Molecular basis for the performance and mechanisms of methylated decolorized bovine hemoglobin flocculants

A slaughterhouse by-product, bovine blood hemoglobin (Hb), has been reported as an adequate flocculant. However, the dark brown/red color originated from the heme prosthetic group of Hb severely limited its widespread adoption. To improve its flocculation efficiency and expand its applications, we removed the heme group by acidified acetone, carboxymethyl cellulose, and hydrogen peroxide oxidation. These heme-depleted Hbs (apo-Hbs) were further modified by methylation, yielding apo-MeHbs with considerably increased size (R-h similar to 70-186 nm) and elevated isoelectric point (pI similar to 10.0) compared to Hb and apo-Hbs (R-h similar to 50-130 nm and pI similar to 7.0). The apo-MeHbs exhibited significantly improved flocculation performance, evidenced by reduced optimal doses (similar to 15 mg/g kaolin) and broadened pH adaptability (pH 4.5-8.5). We demonstrated that charge neutralization was the dominating cause of their exceptional flocculation performance, and soluble aggregates contributed to bridging suspended particles. We further investigated the molecular transformations in Hb caused by these chemical modifications to understand the mechanism and establish a structural indicator for the flocculation improvement. Combined with the increased surface hydrophilicity, the decreased alpha-helix and simultaneously increased beta-sheet and unstructured elements along with the disrupted tertiary contacts greatly facilitated the highly efficient flocculation process observed with the modified Hb.