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Measuring solution viscosity and its effect on enzyme activity

Biological Procedures Online volume 5, pages 108–115 (2003)Cite this article

Abstract

In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H+-ATPase from the plasma membrane ofKluyveromyces lactis. A direct correlation was found between viscosity (η) and the inhibition of the maximum rate of catalysis (V max). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers’ equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H+-ATPase.

Abbreviations

EGTA:

Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid

PIPES:

Piperazine-N,N′-bis(2-ethanesulfonic acid)

Tris:

Tris(hydroxymethyl)aminomethane

SDS-PAGE:

Sodium-dodecyl-sulphate-Polyacrylamide Gel Electrophoresis

YPD:

Yeast-Peptone-Dextrose

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Authors and Affiliations

  1. Área Académica de Nutrición, Instituto de Ciencias de la Salud ICSA, Universidad Autónoma del Estado de Hidalgo (UAEH), Abasolo 600, C.P 42000. Pachuca, Hidalgo, Mexico

    José G. Sampedro

  2. Departamento de BioquÍmica, Instituto de FisiologÍa Celular, Universidad Nacional Autónoma de México (UNAM), A.P. 70-242, 04510, Mexico City, Mexico

    Salvador Uribe

Authors
  1. Salvador Uribe
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  2. José G. Sampedro
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Correspondence to José G. Sampedro.

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Published: May 1, 2003

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Uribe, S., Sampedro, J.G. Measuring solution viscosity and its effect on enzyme activity. Biol. Proced. Online 5, 108–115 (2003). https://doi.org/10.1251/bpo52

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  • DOI: https://doi.org/10.1251/bpo52

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Biological Procedures Online

ISSN: 1480-9222

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