Introduction Enzymatic activity in solution is largely influenced by environmental factors such as the physical properties of the system. Viscosity is referred to as an index of the mobility of a system since it is related to the translational diffusion by the Stokes relationship. Kramers’ theory [1] predicts that the rate for the dynamics should have an inverse relationship with viscosity. Even if a large body of work indicates that Kramers’ theory does not always predict protein dynamics properly [2], large amplitude (global) protein motions and, in particular, those involving exterior portions of the protein, appear to follow Kramers’ relation at viscosities above ≈ 1 cP [3,4] whilst diffusive motions involving smaller portions of the protein are less likely to follow Kramers’ relation at high viscosities [2]. Deviations from Kramers’ relation for exterior protein motions has been attributed to solvent composition and viscosity in the immediate vicinity of the protein.Independently of viscosity, other physical variables could affect the enzyme kinetics in solutions. In particular, in highly viscous concentrated solutions, water activity, which is a thermodynamical variable extensively used to describe the chemical potential of water in binary and complex systems, could play a significant role [5].The aim of this study was to investigate and compare the effect of viscosity and water activity (aw) on the activity of horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) in buffered solutions. HRP (44,173.9 Da) is an enzyme with an highly accessible active site whilst LPO is a larger enzyme (78,431 Da) with a deeply buried active site with a restrictive substrate access channel which should limit the influence of physical properties of the solution (such as viscosity and water activity) on enzymatic catalysis.ExperimentalConcentrated binary and ternary buffered solutions with were prepared by using 0.1 M potassium phosphate buffer, maltose, trehalose and maltodextrins ( : 30.000).HRP and LPO activity was tested in buffered solutions using the method of Keesey [6]. Viscosity was measured using a falling ball viscometer while water activity was measured using a dew point hygrometer. All measurements were carried out at 25 °C. Differential scanning calorimetry was used for the T’g determination. Thermograms were obtained after annealing at T’g + 10 °C. ResultsIn viscous solutions characterized by different composition, the inhibition of the HRP activity was dependent both on the thermodynamical properties of the solution, as described by water activity, and on the mobility of the system, as described by the inverse of viscosity and T-T’g. Viscosity was the most important factor in the inhibition of enzymatic activity in solutions characterized by the same T’g value, but when T’g was changed, due to changes in the solutes composition, the latter became the key factor in the regulation of the enzyme activity. HRP activity was much more influenced by changes in the water activity than LPO activity which, in turn, was much more influenced by changes in viscosity. The results of this study suggest that for large enzymes the translational diffusivity which, according to the Stokes-Einstein law, is inversely related to viscosity and molecular weight, is a far more critical factor than the accessibility of the active site. On the other hand, the water status of the solution seems to be a far more critical factor for the activity of enzymes with an active site highly accessible and expose to the external environment.

Activity of horseradish peroxidase and bovine lactoperoxidase as affected by physical properties of the solution

SACCHETTI, Giampiero;NERI, LILIA;MASTROCOLA, Dino;PITTIA, Paola
2012

Abstract

Introduction Enzymatic activity in solution is largely influenced by environmental factors such as the physical properties of the system. Viscosity is referred to as an index of the mobility of a system since it is related to the translational diffusion by the Stokes relationship. Kramers’ theory [1] predicts that the rate for the dynamics should have an inverse relationship with viscosity. Even if a large body of work indicates that Kramers’ theory does not always predict protein dynamics properly [2], large amplitude (global) protein motions and, in particular, those involving exterior portions of the protein, appear to follow Kramers’ relation at viscosities above ≈ 1 cP [3,4] whilst diffusive motions involving smaller portions of the protein are less likely to follow Kramers’ relation at high viscosities [2]. Deviations from Kramers’ relation for exterior protein motions has been attributed to solvent composition and viscosity in the immediate vicinity of the protein.Independently of viscosity, other physical variables could affect the enzyme kinetics in solutions. In particular, in highly viscous concentrated solutions, water activity, which is a thermodynamical variable extensively used to describe the chemical potential of water in binary and complex systems, could play a significant role [5].The aim of this study was to investigate and compare the effect of viscosity and water activity (aw) on the activity of horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) in buffered solutions. HRP (44,173.9 Da) is an enzyme with an highly accessible active site whilst LPO is a larger enzyme (78,431 Da) with a deeply buried active site with a restrictive substrate access channel which should limit the influence of physical properties of the solution (such as viscosity and water activity) on enzymatic catalysis.ExperimentalConcentrated binary and ternary buffered solutions with were prepared by using 0.1 M potassium phosphate buffer, maltose, trehalose and maltodextrins ( : 30.000).HRP and LPO activity was tested in buffered solutions using the method of Keesey [6]. Viscosity was measured using a falling ball viscometer while water activity was measured using a dew point hygrometer. All measurements were carried out at 25 °C. Differential scanning calorimetry was used for the T’g determination. Thermograms were obtained after annealing at T’g + 10 °C. ResultsIn viscous solutions characterized by different composition, the inhibition of the HRP activity was dependent both on the thermodynamical properties of the solution, as described by water activity, and on the mobility of the system, as described by the inverse of viscosity and T-T’g. Viscosity was the most important factor in the inhibition of enzymatic activity in solutions characterized by the same T’g value, but when T’g was changed, due to changes in the solutes composition, the latter became the key factor in the regulation of the enzyme activity. HRP activity was much more influenced by changes in the water activity than LPO activity which, in turn, was much more influenced by changes in viscosity. The results of this study suggest that for large enzymes the translational diffusivity which, according to the Stokes-Einstein law, is inversely related to viscosity and molecular weight, is a far more critical factor than the accessibility of the active site. On the other hand, the water status of the solution seems to be a far more critical factor for the activity of enzymes with an active site highly accessible and expose to the external environment.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11575/11034
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