A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Ribonucleic acid is a biologically important type of molecule that consists of a long chain of nucleotide units. The unfolding of ribonuclease, lysozyme, a-chymotrypsin, and goat P-lactoglobulin by urea and guanidine hydrochloride (GmHCl) has been followed with the use of optical rotation measurements. Urea denaturation leads to a more negative rotation for each protein than does GmHCl denaturation, but the concentration dependence is such that the rotations are almost identical in the absence of denaturant. This indicates that both urea and GmHCl denaturation lead to a similar, randomly~coiled conformations. By far the simplest method of following the denaturation process is the absorbance at 287 nm. There are six tyrosyl residues in ribonuclease which become exposed to the solvent upon denaturation. The equilibrium constant for the denaturation reaction is: KD = [D]/[N] = (YN-Y)/(Y-YD). YD and YN must be determined by linear extrapolation. The basic data then is transformed into values of KD at denaturant concentrations where both [N] and [D] are finitely large. The simplest way of obtaining KºD for a laboratory exercise is to assume that ∆G for denaturation is a linear function of the concentration of denaturant. Thus a plot of ln KD versus [GdmCl] gives as the intercept ln KºD, or KºD in the absence of denaturant.
1. We need 4 ml of 8 M guanidinium chloride, 3-4 ml of 5mg/ml ribonuclease, and about 5-10 ml of 1% NH4HCO3 buffer. Nine solutions are prepared as shown in the table.
Tube Number | Volume Rnase (ml) | Volume 1% NH4HCO3 (ml) | Volume8M GdmCl (ml) | 1 | 0.2 | 0.80 | 0 | 2 | 0.2 | 0.70 | 0.10 | 3 | 0.2 | 0.60 | 0.20 | 4 | 0.2 | 0.50 | 0.30 | 5 | 0.2 | 0.40 | 0.40 | 6 | 0.2 | 0.30 | 0.50 | 7 | 0.2 | 0.20 | 0.60 | 8 | 0.2 | 0.10 | 0.70 | 9 | 0.2 | 0 | 0.80 | 10 | 0.8 | 0.40 | 0.80 | 11 | | (1ml of solution 10 + 1 ml buffer) | | 2. Ribonuclease is added first first, followed by buffer, followed by denaturant. 3. Tubes should be gently vortexed and allowed to stand for a least 15 min before absorbances are read. 4. The A287 for tube…