Characterization of the truncated hemoglobin THB1 from protein

Truncated hemoglobins (TrHbs) belong to the hemoglobin superfamily, but unlike their distant vertebrate relatives, little is known about their principal physiologic functions. Several TrHbs have been studied using in vitro engineered recombinant peptides. These efforts have resulted in a wealth of knowledge about the chemical properties of TrHbs and have generated interesting functional leads. However, questions persist as to how closely these engineered proteins mimic their counterparts within the native cell. In this report, we examined THB1, one of several TrHbs from the model organism The recombinant THB1 (rTHB1) has favorable Chlamydomonas reinhardtii. solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the protein is a critical step in in vivo understanding the physiologic function of this protein. Using a simplified three-step purification protocol, 3.5-L batches of algal culture were processed to isolate 50–60 μL fractions enriched in THB1. These fractions of C. reinhardtii proteins were then subjected to physical examination. Using gel mobility, optical absorbance and immunoreactivity, THB1 was identified in these enriched fractions and its presence correlated with that of a heme molecule. Mass spectrometry confirmed this cofactor to be a type heme and revealed b that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage. This article is included in the Oxygen-binding and collection. sensing proteins Referee Status: