L-Arginine increases the solubility of unfolded species of hen egg white lysozyme

Abstract

L-Arginine (L-Arg) has been widely used as an enhancer of protein renaturation. The mechanism behind its action is still not fully understood. Using hen egg white lysozyme as a model protein, we present data that clearly demonstrate the suppression of the aggregation of denatured protein by L-Arg. By chemical modification of free cysteines, a series of unfolded lysozyme species were obtained that served as models for unfolded and intermediate states during the process of oxidative refolding. An increased equilibrium solubility of unfolded species and intermediates in the presence of L-Arg seems to be its major mechanism of action.

Figure 1.

Refolding of lysozyme. (A) Denatured-reduced lysozyme was refolded at pH 8.2 and room temperature in the presence of a glutathione redox-shuffling system and increasing concentrations of l-ArgHCl as described in the text. The protein concentration was 0.25 mg mL⁻¹. [l-ArgHCl] was 0, 0.2, 0.5, and 0.9 M (bottom to top). The refolding yield was monitored by measuring the enzymatic activity. (B) The final refolding yields were expressed as percentage of the specific activity of native lysozyme and plotted as function of [l-ArgHCl]. (C) The time course of refolding was analyzed as a pseudofirst-order reaction, yielding apparent time constants k_app. Data in B and C represent mean values ± SD from three independent experiments. Lines are meant to guide the eye.

Figure 2.

Denaturant-induced folding/unfolding (A) and thermal stability (B) of lysozyme. (A) The GuHCl-induced folding/unfolding equilibrium of lysozyme was monitored by tryptophan fluorescence at pH 7.0 without l-ArgHCl (filled circles), in the presence of 0.5 M l-ArgHCl (open circles), and of 1.0 M l-ArgHCl (filled triangles). Protein concentration was 20 μg mL⁻¹. Data were normalized assuming a linear dependency of the intrinsic fluorescence for both the native and the denatured state. Lines represent fits to a two-state unfolding model. (B) The thermal unfolding of lysozyme was monitored by DSC at pH 6.0 without l-ArgHCl (filled circles), in the presence of 0.5 M l-ArgHCl (open circles), and of 1.0 M l-ArgHCl (filled triangles). Protein concentrations were 0.5 mg mL⁻¹. Lines represent fits of the data to a two-state unfolding model.

Figure 3.

Kinetics of aggregation during the refolding of lysozyme. (A) Time-dependent formation of aggregates at increasing concentrations of denatured lysozyme in the presence of 50 mM l-ArgHCl. Lysozyme concentration was 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.5, and 0.6 mg mL⁻¹ (bottom to top). (B) Time-dependent formation of aggregates in the presence of increasing concentrations of l-ArgHCl at a protein concentration of 0.3 mg mL⁻¹. [l-ArgHCl] was 0, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.4 M (top to bottom). The aggregation was monitored by light scattering at 600 nm.

Figure 4.

Solubility of iodoacetamide-modified (A) and iodoacetic acid-modified (B) denatured lysozyme. Preparations of the modified forms of denatured lysozyme (protein concentration ∼5 mg mL⁻¹) were dialyzed overnight at room temperature against buffer at pH 8.5, containing increasing amounts of l-ArgHCl (filled circles) or NaCl (open circles). The concentrations of soluble protein in the supernatant were determined after centrifugation, as described in the text. Data represent mean values ± SD from four independent experiments. Lines are meant to guide the eye.