Rapid modification of hemoglobin heme to form enhanced peroxidase-like activity for colorimetric assay

Abstract

Modification of proteins is widespread throughout nature responsible for the vast biodiversity. In this study, we developed a facile method to rapid and selectively modify heme of hemoglobin (Hb) with tartaric acid to form complex (TA-Hb), which exhibits strong peroxidase-like activity comparable to the native horseradish peroxidase (HRP). The structural change of Hb accompanied by the formation of TA-Hb complex was validated by UV-vis absorption, fluorescence, fluorescence lifetime and circular dichroism data. The oxygen atom of the carboxyl group of tartaric acid can easily coordinate with heme and become the axial ligand of porphyrin, and this specific structure facilitates to form stable intermediate, significantly enhancing Hb peroxidase-like activity. This study is expected to provide important insight into the interactions of the physiologically important protein Hb with various ligands. In addition, the unique property of TA-Hb was employed to develop a rapid colorimetric assay for detecting concentration of Hb in human serum samples, regardless of whether the sample is in the form of fresh or long-term storage, liquid or dried blood. This colorimetric assay can detect Hb over the concentration range of 0.01 ~ 0.1 mg/mL and the limit of detection (LOD) is 0.1 μg/mL. Moreover, cascade reactions can be realized based on the peroxidase-like TA-Hb complex formed in situ from the serum samples by combining with the glucose oxidase, which enable this colorimetric assay to detect not only Hb but also blood glucose in the same sample.