Circular Dichroism Test Chemistry - Lysozyme Refolding
In the presence of 6M guanidine hydrochloride, the protein lysozyme is in an unfolded state. By using the stopped-flow spectrometer configured for asymmetric mixing, the initial 6M guanidine hydrochloride concentration is reduced to 0.55M by the time it reaches the point of observation and this is sufficient to allow the unfolded lysozyme to regain its tertiary structure. The refolding process is accompanied by a biphasic change in CD signal where an initial decrease in CD signal during the first 70ms is then followed by an increase in CD signal during the following 500ms.
Sample Preparation
| Solution 1:- | 10mM phosphate buffer at pH7 |
| Stock solutions A: | 0.2M di-sodium hydrogen orthophosphate (MW = 141·96) |
| Dissolve 14·2g di-sodium hydrogen orthophosphate in 500mL distilled water. | |
| Stock solutions B: | 0.2M sodium dihydrogen orthophosphate (MW = 156·01) |
| Dissolve 15·6g sodium dihydrogen orthophosphate in 500mL distilled water. | |
Preparation of pH7, 10mM buffer:-
Take 12.5mL of stock solution A + 12.5mL of stock solution B and make up to 500mL with water.
| Solution 2:- | 2.2mg/mL lysozyme in 6M guanidine hydrochloride |
| Solute: | Guanidine hydrochloride (GuHCl) |
| Source: | Sigma G-7153 (Sigma Ultra) |
| MW: | 95.53 |
Prepare 6M guanidine hydrochloride by dissolving 57·3g GuHCl in 100mL pH7 phosphate buffer (10mM).
| Solute: | Lysozyme (Muramidase from hen egg white) |
| Source: | Boehringer 1243 004 |
| MW: | 14,400 |
| Conc: | 2.2mg/mL in pH7 phosphate buffer (10mM) |
Prepare 2.2mg/mL lysozyme in 6M guanidine hydrochloride solution
Risk Assessment
Guanidine hydrochoride causes severe irritation, avoid skin contact and contact with mucous membranes in particular. Wash hands after handling.
| Measurement Conditions | |
| Syringe Ratio: | 1:10* (250µL + 2.5mL) i.e. 11x dilution |
| Lysozyme conc. in cell: | 200µg/mL |
| GuHCl conc. in cell: | 0.55M |
| Probe wavelength: | 225nm |
| Acquisition period: | 1-2s (with oversampling) |
| Number of data points | 1000 |
| Light source: | Xenon-Mercury |
* 250µL syringe contains 2.2mg/mL lysozyme in 6M guanidine hydrochloride
2.5mL syringe contains 10mM phosphate buffer at pH7