Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability

• We report on the properties of Glossoscolex paulistus hemoglobin (HbGp).
• HbGp structure contains around 14 and 30 ions of Zn2 + and Ca2 +, respectively.
• The ions, Zn2 + and Ca2 +, play an important role in maintaining of HbGp structure.
• The removal of Zn2 + cations weakens the contacts between the linkers and the dodecamer.
• HbGp shows significant antioxidant activity as a peroxidase enzyme with guaiacol.

In this work we investigate the presence of divalent cations bound to the Glossoscolex paulistus (HbGp) hemoglobin and their effect over the protein stability and the peroxidase (POD) activity. Atomic absorption studies show that the HbGp iron content is consistent with the presence of 144 ions per protein. Moreover, using iron as a reference, the content of calcium was estimated as 30 ± 4 ions per protein, independently of the EDTA pre-treatment or not prior to the acidic treatment performed in the protein digestion. The zinc content was 14 ± 2 ions in the absence of EDTA pre-treatment, and 3 ± 1 ions per protein in the presence of EDTA pre-treatment, implying the presence of one zinc ion per protomer (1/12 of the whole molecule). Finally, the copper concentration is negligible. Different from the vertebrate hemoglobins, where the effectors are usually organic anions, the hexagonal bilayer hemoglobins have as effectors inorganic cations that increase the oxygen affinity and stabilize the structure. Previous studies have suggested that the presence of divalent cations, such as copper and zinc, is related to the different types of antioxidant enzymatic activities as the superoxide dismutase (SOD) activity shown by giant hemoglobin from Lumbricus terrestris (HbLt). Recently, studies on HbGp crystal structure have confirmed the presence of Zn2 + and Ca2 + binding sites. The Ca2 + sites are similar as observed in the HbLt crystal structure. Otherwise, the Zn2 + sites have no relation with those observed in Cu/Zn SODs. Our peroxidase assays with guaiacol confirm the POD activity and the effect of the zinc ions for HbGp. Our present results on HbGp metal content and their stability effects is the first step to understand the role of these cations in HbGp function in the future.

The absence and presence of zinc and calcium ions as promoters of Glossoscolex paulistus hemoglobin protein structural stability: oligomerization, aggregation and enzymatic activity.Figure optionsDownload as PowerPoint slide