title: Characterization of the truncated hemoglobin THB1 from protein extracts of Chlamydomonas reinhardtii.
abstract: 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 in vitro using 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 Chlamydomonas reinhardtii. The recombinant THB1 (rTHB1) has favorable solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the in vivo protein is a critical step in 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 b heme and revealed that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage.
authors: Department of Biophysics, Johns Hopkins University, Baltimore, MD, 21218, USA.
authors: Eric A
authors: EA
authors: Johnson
authors: Department of Biophysics, Johns Hopkins University, Baltimore, MD, 21218, USA.
authors: Juliette T J
authors: JT
authors: Lecomte
keywords: Chlamydomonas reinhardtii; Hemoglobins; THB1; oxygen binding
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