Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers.

Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important parameter for efficient in vitro folding is the composition of the redox buffer, a mixture of a small molecule thiol and small molecule disulfide. The effects of different redox buffers on protein folding, however, have received limited attention. The oxidative folding of denatured reduced lysozyme was followed in the presence of redox buffers containing varying concentrations of five different aromatic thiols or the traditional aliphatic thiol glutathione (GSH). Aromatic thiols eliminated the lag phase at low disulfide concentrations, increased the folding rate constant up to 11-fold, and improved the yield of active protein relative to GSH. The yield of active protein was similar for four of the five aromatic thiols and for glutathione at pH 7 as well as for glutathione at pH 8.2. At pH 6 the positively charged aromatic thiol provided a higher yield than the negatively charged thiols.

Thermal Stability and Photochromic Properties of a Fluorinated Indolylfulgimide in a Protic and Aprotic Solvent.

The photochromic properties and thermal stability of a newly synthesized fluorinated N-ethoxycarbonylmethyl indolylfulgimide substituted on the imide nitrogen were examined in a protic and aprotic environment. The absorption spectra and extinction coefficients of the Z- and C-forms of the fluorinated indolylfulgimide (open and closed, respectively) were measured in a binary 70/30 ethanol/water system and in toluene. The results demonstrated a favorable bathochromic shift of the absorption maxima for both the open and closed forms of the fulgimide when the solvent was changed from aprotic toluene to protic aqueous ethanol. In addition, the photochemical stability of the new fulgimide was found to be high (0.056% and 0.020% degradation each time the fulgimide is cycled between the open and closed form in 70/30 ethanol/water and in toluene, respectively). The thermal stability of both forms of the fulgimide in 70/30 ethanol/water at 50 degrees C, toluene at 80 degrees C, and polymer film (PMMA) at 80 degrees C was measured using UV-Vis and/or (1)H NMR spectroscopy. Both forms of indolylfulgimide display high hydrolytic stability in 70/30 ethanol/water at 50 degrees C, with the Z- and C-forms degrading 1.3%/day and 1.2%/day respectively based on (1)H NMR data. At 80 degrees C in toluene the less stable Z-form lost about 20%/day.

ortho- and meta-substituted aromatic thiols are efficient redox buffers that increase the folding rate of a disulfide-containing protein.

Thiol based redox buffers are used to enhance the folding rates of disulfide-containing proteins in vitro. Traditionally, small molecule aliphatic thiols such as glutathione are employed. Recently, we have demonstrated that aromatic thiols can further enhance protein-folding rates. In the presence of para-substituted aromatic thiols the folding rate of a disulfide-containing protein was increased by 4-23 times over that measured for glutathione. However, several important practical issues remain to be addressed. Aromatic thiols have never been tested in the presence of denaturants such as guanidine hydrochloride. Only two of the para-substituted aromatic thiols previously examined are commercially available. To expand the number of aromatic thiols for protein folding, several commercially available meta- and ortho-substituted aromatic thiols were studied. Furthermore, an ortho-substituted aromatic thiol, easily obtained from inexpensive starting materials, was investigated. Folding rates of scrambled ribonuclease A at pH 6.0, 7.0 and 7.7, with ortho- and meta-substituted aromatic thiols, were up to 10 times greater than those with glutathione. In the presence of the common denaturant guanidine hydrochloride (0.5M) aromatic thiols provided 100% yield of active protein while maintaining equivalent folding rates.