Heat denaturation of pepsinogen in a water‐ethanol mixture

Abstract

The effect of ethanol and pH on thermodynamic parameters and cooperativity of pepsinogen heat denaturation was studied by scanning microcalorimetry. Addition of 20% ethanol decreases the protein denaturation temperature by 10.7°C at pH 6.4 and 15.8°C at pH 8.0. It also decreases the denaturation heat capacity increment from 5.8 to 4.2 kcal/K, mol. The dependences of calorimetric denaturation enthalpy on denaturation temperature both in water and 20% ethanol are linear and intersect at about 95°C. In 20% ethanol the pH shift from 5.9 to 8.0 results in a decreased number of cooperative domains in pepsinogen. This process causes no changes either in the secondary structure or in the local surroundings of aromatic amino acids. It is concluded that ethanol addition does not affect the cooperativity of pepsinogen denaturation substantially until the pH change provokes redistribution of charges in the protein molecule.

Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs)

“…showed ammonium sulphate acting as the best precipitating agent. Despite the fact that denaturation of enzymes occurs upon precipitation with organic solvent due to hydrophobic interactions between the solvent and the nonpolar groups of the enzyme, 82,83 numerous reports underscore the preparation of active CLEAs using water miscible organic solvents like acetone, ethanol, dimethoxyethane, tert-butyl alcohol, isopropyl alcohol, acetonitrile, etc. CLEAs of lipase, 46,[84][85][86] horseradish peroxidase 87 and poly-3-hydroxybutyrate depolymerase 88 using acetone, papain 89 and lipase 90 using ethanol, (R)oxynitrilase, 91 penicillin acylase, 46 hydroxynitrile lyase 92 and lipase-a-amylase-phospholipase A 2 93 using dimethoxyethane, penicillin G acylase, 34 penicillin acylase 94 and aminoacylase 47 using tert-butyl alcohol, nitrilase 95 using isopropyl alcohol, chloroperoxidase 96 using acetonitrile as precipitant have been prepared.…”

Effect of whey protein denaturation on the physicochemical properties of whey-based products

“…The denaturation temperatures (peak temperature) were 77.5 and 75.0 o C, respectively. Based on calorimetric studies, a similar decrease in the denaturation temperature of several proteins has been reported (Kishore & Ranjana, 2001;Kundu & Kishore, 2004;Makarov, Protasevich, Bazhulina, & Esipova, 1995). Finally, from the thermogram C in Figure 5.5, it becomes clear that ethanol, when present in the protein solution, denatured the proteins almost completely, as indicated by the absence of any significant endothermic peak.…”

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins

“…The increase in initial glutaraldehyde concentration caused a significant increase in the relative activity of Pd HNL‐CLEA and it was obtained as 91.4% at the end of the same crosslinking time (Run 5). Ammonium sulfate has been successfully applied as precipitating agent in various CLEA preparations for the reason that most of the organic solvents used as precipitating agent have been reported to be the cause of denaturation of enzymes . In this study, when the concentration of ammonium sulfate saturation was 50%, the relative activity of Pd HNL‐CLEA was determined as 77.3% at the initial glutaraldehyde concentration of 20% and crosslinking time of 15 h (Run 13).…”

“…Ammonium sulfate has been successfully applied as precipitating agent in various CLEA preparations for the reason that most of the organic solvents used as precipitating agent have been reported to be the cause of denaturation of enzymes. [38][39][40][41][42] In this study, when the concentration of ammonium sulfate saturation was 50%, the relative activity of PdHNL-CLEA was determined as 77.3% at the initial glutaraldehyde concentration of 20% and crosslinking time of 15 h (Run 13). Furthermore, the concentration of the precipitation agent also affects the recovered activity of CLEAs.…”