Low-cost equilibrium unfolding of heme proteins using 2 μl samples

Neuroglobin: From structure to function in health and disease

“…Guanidine-HCl induces the reversible unfolding of murine holo-Ngb, the unfolded globin population increasing with the guanidine-HCl concentration. The unfolding-folding process follows the simple two-state unfolding model (Guca et al, 2013). On the other hand, human holo-Ngb unfolding by urea and guanidine-HCl follows a two-step and a three-step mechanism, respectively (Zhang et al, 2013a).…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…. These values are higher than the ones determined in this work, which could be attributed to the fact that the method used by Guca et al (2013) to fit their denaturation curves does not take into consideration the dependence of unfolding on the denaturant concentration (especially at low and high concentration of denaturant), thereby they might obtain a higher value for…”

“…The Soret band in heme proteins is characterized by a large extinction coefficient and is sensitive to changes in the environment such as chemical changes, hence the changes in absorbance of the Soret band upon addition of chaotropic agents (e.g., urea and guanidinium hydrochloride) or changes in pH has been extensively used in unfolding studies of heme proteins (Guca et al, 2013;. Therefore, the GuHCl-induced unfolding of Cygb was monitored by the change in intensity of the Soret band upon addition of the caotropic agent GuHCl in 50 mM Tris buffer (pH 7.0).…”

“…Unfolding of Mb has been described using a twostate model and it was proposed that the heme group maintains its interaction with the unfolded polypeptide chain through the proximal His (Moczygemba et al, 2000). Guca et al (2013) describes the GuHCl-induced denaturation of hNgb with a two-state model. However, the acid-induced and GuHClinduced denaturation of apoNgb was proposed to occur through a three-state mechanism in which the transition from the folded state to the unfolding state occurs through the formation of a molten globule intermediate, whereas urea-induced denaturation occurs through a two-state transition (Mu et al, 2010;Zhang et al, 2013a).…”

“…Far-UV circular dichroism (CD) spectroscopy provides information about the secondary structure of a protein and is extensively used as a tool to monitor protein folding/unfolding (Kelly and Price, 1997). In case of heme proteins, the sensitivity of the Soret band to changes in the environment (e.g., chemical changes, pH, temperature) allows the use of absorption spectroscopy to characterize unfolding transitions (Guca et al, 2013;Hamdane et al, 2005;.…”