P42 Gene expression profiles of H2S and O2-binding hemoglobins of the clam Lucina pectinata

Abstract

Background Hydrogen sulfide (H 2 S) was thought to be a metabolic poison for years. Nevertheless, there is growing evidence of its involvement in many physiological and pathological processes. Despite these recent findings, there are a variety of invertebrate organisms that can tolerate high hydrogen sulfide levels since they live in sulfide-rich environments, such as hydrothermal vents, cold seeps, and muds. It is believed that these organisms have Hemoglobins (Hbs) capable of binding H 2 S for detoxification and/or transporting it to internal bacteria symbionts. However, these mechanisms are not well understood. It has been demonstrated that human hemoglobin/myoglobin can form sulf-hemoglobin species at high H 2 S and O 2 concentrations, thus inactivating the protein. On the other hand, the three Hbs from the clam Lucina pectinata , which lives in sulfide-rich environments, does not form the sulfhemoglobin complex, which has been proposed to be due to the presence of Gln in position E7. The L. pectinata’s Hbs differ in function since hemoglobin I (HbI) is a sulfide-reactive protein and Hemoglobin II (HbII) and Hemoglobin III (HbIII) are oxygen-reactive proteins. Methods In order to understand the physiological role of these hemoproteins, we evaluated their gene expression at the mRNA level by real time PCR. The Hb mRNAs expression levels were compared to that of the histone H3 mRNA and the 18S rRNA. Total RNA was isolated by the TRIzol method in order to obtain higher RNA yields than that obtained using column-based methods. RNAs were further purified by n -butanol and diethyl ether extractions in order to achieve better A 260 /A 230 ratios and higher amplification efficiencies during one step real time RT-PCR. The histone H3 gene was expressed in similar levels in most tissues except in visceral mass, while the 18S rRNA gene levels were more uniform throughout all tissues. Thus, we compared the three hemoglobin mRNA levels to that of the 18S rRNA using the clam’s foot tissue as calibrator. Results Ctenidia was the tissue of highest expression for all the Hb mRNAs, whereas foot and muscle had lower levels of expression. In ctenidia, oxygen-reactive Hb mRNAs were expressed at higher levels than HbI mRNA, as reflected at the protein level in previous studies. In contrast, the visceral mass mRNA expression of HbI was higher than for HbII and HbIII. Conclusion The presence of HbI in different tissues may confirm that HbI, in spite of transporting H 2 S, may also have an alternative role of protecting the tissues from H 2 S damage. In conclusion, understanding the role of these Hbs in the clam tissues can help elucidate how these organisms survive in these extreme environments. HbI is presently being evaluated as a potential biomolecule to be used to capture or detect H 2 S.