High-Pressure FTIR Study of the Stability of Horseradish Peroxidase. Effect of Heme Substitution, Ligand Binding, Ca⁺⁺ Removal, and Reduction of the Disulfide Bonds

Abstract

The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.

Ultrasensitive ELISA+enhanced by dendritic mesoporous silica nanoparticles

“…S4 pink lines), the peak at 1650 cm -1 is assigned to amide C=O stretching vibration and 1530 cm -1 to N-H vibration. 20,21 In the spectra of HRP loaded DMSNs (DMSNs-H, Fig. S4 observed in free HRP disappears and a broad peak centered at 1640 cm -1 is found, which is explained by the overlap of amide C=O stretching in HRP and Schiff base formed during the HRP conjugation process (Scheme 2).…”

“…The peak at 1448 cm -1 is attributed to the N-H stretching vibration, 19 while 1550 cm -1 to N-H in plane bending. 20 The peak at 2950 cm -1 is assigned to the C-H stretching which belongs to the grafted amino silane. These results suggest the successful modification of amino groups.…”

Influence of high isostatic pressure on structural and functional characteristics of potato protein

“…A pressure of 400 MPa reduced solubility to 96 and 93% at treatment temperatures of 20 and 40°C, respectively. At higher temperatures proteins are more sensitive to a pressure-induced unfolding (Winter, Lopes, Grudzielanek, & Vogtt, 2007) visible in the typical elliptical shape of the protein phase diagram (Smeller, Meersman, Fidy, & Heremans, 2003). Tertiary interactions in patatin are already weakened at temperatures higher than 28°C (Pots, De Jongh, Gruppen, Hamer, & Voragen, 1998) which may sensitize the structure to pressure denaturation.…”

Biology under extreme conditions

“…The data that bacterial life may still be possible at pressure above 1 GPa [7] came therefore as a surprise. Only very few proteins have been found to be resistant to such high pressures [20][21][22].…”

“…Secondly, when the fibres are formed in H 2 O and then transferred to D 2 O, a subsequent compression up to more than 1 GPa does not induce an extensive exchange of H for D. This indicates that the core of the fibrils is extremely resistant to pressure, a phenomenon that is only observed in very limited number of proteins [20][21][22]. Further studies are in progress to see whether this is a general property of fibrils formed from a wide variety of proteins.…”