Abstract
Earlier studies have reported that trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0-8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK(a) of 4.66 +/- 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-alpha-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK(a) of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK(a), whereas it stabilizes proteins at pH values above its pK(a). This conclusion was reached by determining the T(m) (midpoint of denaturation), delta H(m) (denaturational enthalpy change at T(m)), delta C(p) (constant pressure heat capacity change), and delta G(D) degrees (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T(m) and delta G(D) degrees depend on TMAO concentration at each pH value and that delta H(m) and the delta C(p) are not significantly changed in presence of TMAO.
Highlights
Many organisms are known to accumulate low molecular weight organic molecules in their tissues in response to harsh environmental stresses
We have asked the question of whether the effect of trimethylamine Noxide (TMAO) on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-␣-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pKa of TMAO
Heat-induced denaturation curves of proteins in the presence of different TMAO concentrations (0, 0.25, 0.50, 0.75, and 1 M) at five different pH values were measured by following the changes in ⌬⑀300 of lysozyme, ⌬⑀287 of RNase A, and ⌬⑀295 of ␣-LA as a function of temperature
Summary
Commercial lyophilized preparations of RNase A (type III-A), hen egg white lysozyme, and bovine ␣-lactalbumin were purchased from Sigma. Concentration of the protein was determined experimentally using molar absorption coefficient (MϪ1 cmϪ1) values of 9800 at 277.5 nm for RNase A [28], 39,000 at 280 nm for lysozyme [29], and 29,210 at 280 nm for ␣-LA [30]. The concentration of GdmCl stock solution was determined by refractive index measurements [31]. All solutions for optical measurements were prepared in the desired degassed buffer containing 0.1 M KCl. For various pH ranges, the buffers used were denaturation; ⌬Hm, enthalpy change of denaturation; ⌬GD, Gibbs energy change; ⌬GD°, Gibbs energy change at 25 oC. For various pH ranges, the buffers used were denaturation; ⌬Hm, enthalpy change of denaturation; ⌬GD, Gibbs energy change; ⌬GD°, Gibbs energy change at 25 oC
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