| Determination of aggregate formation between injector and column inlet
Turbidity measurements provided a means to monitor formation of insoluble aggregates of lysozyme. To investigate the effect of sample application on the aggregate formation between the injector and column inlet, stream before column inlet was directly connected to the detector to monitor the aggregates. That is, the SEC column was temporarily removed and the detector was set at 450 nm for aggregates measurement. The denatured protein sample (1, 5, 10 and 20 g/l) was loaded though a sample loop which had been previously equilibrated with refolding buffer (0.1 M Tris–HCl, pH 8.2, 1.2 mM GSH, 1.2 mM GSSG, 1.5 M sodium chloride, 1 mM EDTA and 2 M urea). The sample loop dimension was divided into two types of standards for sample loading: one is fixed internal diameter (0.25 mm) but variable tubing length (10.2–400 cm). The other is fixed tubing length (25.5 cm) but variable internal diameter (0.17, 0.5, 0.75 and 1.0 mm). The amount of insoluble aggregated protein was determined by integrating chromatograms. It was hypothesized that the integral area is proportional to the amount of aggregate formation.
2.4. Refolding of lysozyme using size-exclusion chromatography
The high performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) was equipped with a dual λ absorbance detector (Waters 2487) and a binary HPLC pump (Waters 1525). All eluents were filtrated by nitrocellulose membrane (0.2 μm) and degassed beforehand. A 20 μl sample of denatured lysozyme (5 g/l) was injected onto a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech, Bjorkgatan, Sweden) previously equilibrated with refolding buffer (0.1 M Tris–HCl, pH 8.2, 1.2 mM GSH, 1.2 mM GSSG, 1.5 M sodium chloride, 1 mM EDTA and 2 M urea) and eluted with 0.5 ml/min mobile phase flow rate at room temperature. Sample fractions were collected and analyzed for the enzyme activities. |