Time, refolding parameter

It is apparent that while incubation in solutions of low guanidine hydrochloride concentration may sometimes cause inactivation (carbonic anhydrase), it may also promote pro r refolding (chymotrypsinogen). In addition, replacement of a strong denaturant with a weaker one is an approach which can be used to successfully refold polypeptide chains. These data suggest that the rate of removal of the denaturant agent can influence yield of active protein. In this respect, time is an important refolding parameter. 

NMR samples contained 0.6 ml receptor (0.5-2.0 mM) dissolved in refolding buffer (vide supra) with 10% DjO. One-dimensional F NMR spectra were obtained at 470 mHz on a General Electric GN 500 spectrometer fitted with a 5 mm F probe. Parameters included 16K data points, 3.0 second relaxation delay and 25 Hz linebroadening for processing spectra. T, relaxation times were measured by the inversion recovery method. The two-dimensional F NOESY NMR spectrum was obtained on a Varian Unity Plus 500 using the standard Varian pulse sequence. A total of 128 experiments with a mixing time of 0.3 seconds were performed with collection of 1024 data points. Quadrature detection in the second dimension was obtained through the method of States and Haberkom. C ( H NMR spectra were obtained on a Varian 500 Unity Plus fitted with a 10 mm broadband probe. 

Fig. 7.9 (pp. 368-369). Comparative studies of unfolding-refolding of three carp parv-albumins (from Lin and Brandts, 1978) at pH 5.8, 25°C. Values of the kinetic parameters, relaxation time, and amplitude are given for each protein. Unfolding experiments are represented by filled symbols and refolding experiments by open symbols (A), (B), and (C) correspond to different parvalbumins, only that studied in (B) contained proline residues (courtesy of Brandts). 

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