L-arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA.
L-arginine hydrochloride (L-ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which L-ArgHCl as co-solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co-solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of L-ArgHCl on protein refolding. In this article, we present data, which clearly establish that L-ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S-carboxymethylated rPA (IAA-rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol(-1) under the tested conditions. This finding is in marked contrast to a previously proposed model in which L-ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA-rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation-aggregation scheme, in which L-ArgHCl exerts a strong effect on the pre-equilibria leading to formation of the aggregation seed.
Tischer
Alexander
A
Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany.
Lilie
Hauke
H
Rudolph
Rainer
R
Lange
Christian
C
D001120:Arginine / Q000031:analogs & derivatives*; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D008745:Methylation; D010960:Plasminogen Activators / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry*; D012995:Solubility
D011994:Recombinant Proteins; D001120:Arginine; D010960:Plasminogen Activators; D019791:Guanidine