Experimental Methods for Studying Cellular Heme Signaling

Abstract

The study of heme is important to our understanding of cellular bioenergetics, especially in cancer cells. The function of heme as a prosthetic group in proteins such as cytochromes is now well-documented. Less is known, however, about its role as a regulator of metabolic and energetic pathways. This is due in part to some inherent difficulties in studying heme. Due to its slightly amphiphilic nature, heme is a “sticky” molecule which can easily bind non-specifically to proteins. In addition, heme tends to dimerize, oxidize, and aggregate in purely aqueous solutions; therefore, there are constraints on buffer composition and concentrations. Despite these difficulties, our knowledge of heme’s regulatory role continues to grow. This review sums up the latest methods used to study reversible heme binding. Heme-regulated proteins will also be reviewed, as well as a system for imaging the cellular localization of heme.

Rickettsial pathogen uses arthropod tryptophan pathway metabolites to evade reactive oxygen species in tick cells

“…We monitored the hemin titration and binding to XA or KA in separate experiments by UVvisible spectroscopy as reported (Soto et al, 2012). Hemin exhibits absorbance in the UV-Vis spectrum due to a Soret band near 400 nm (Comer & Zhang, 2018). When 5 μM of XA was titrated with incremental concentration of hemin, we observed the Soret band shifting to 364 nm with a significant increase in its amplitude (Figure 3g).…”

HeMoQuest: a webserver for qualitative prediction of transient heme binding to protein motifs

“…Besides its well-known binding to these hemoproteins as a prosthetic group, heme has been established as a biologically available molecule. Human targets of transient heme binding have been reviewed extensively [1][2][3][4][5][6] (Additional Table 1, supplementary data 3). Well-known representatives are δ-aminolevulinic acid synthase 1 (ALAS1) and transcription regulator protein Bach1, which bind heme via CP-containing motifs [1,7,8].…”

Revisiting the interaction of heme with hemopexin: Recommendations for the responsible use of an emerging drug

“…Hemopexin was able to acquire heme from the isolated hemoglobin alpha chain and a K D of <1pM was derived based on the known association constant of heme and globin (0.19 pM) 27 . Today, sophisticated biophysical methods for direct analysis and real-time monitoring of biomolecular interactions are available 28 . Almost 50 years after the introduction of binding constants for the heme-hemopexin interaction, it seems appropriate to inspire and promote reflection and a debate of the former analyses.…”