Estimation of the Denaturation Equilibrium Constant for Ribonuclease A Biochemistry Laboratory Exercise Leslie A. Holladay Louisiana Tech University, Ruston. LA 71272
Described below is a biophysical chemistry experiment that allows students to estimiti the denaturation equilihrium constant for hovine pancreatic rilx,nuclease. The experiment requires an ultraviolet spectrophotometer, a few dollars' worth of reagents, and about 3112 hours. Bovine pancreatic ribonuclease is a globular, 13,686-dalton enzyme with 124 amino acid residues in the polypeptide chain and four disulfide crosslinks. The thermodynamics of the denaturation by acid, beat, and guanidinum chloride (GdmC1) have been well characterized (1-5). The denaturant guanidinium chloride appears to cause a two-state transition t o occur in which the great majority of molecules exist in either the native (N) or completely unfolded (D)conformations. By far the simplest method of following the denaturation process is the absorbance a t 287 nm. There are six tyrosyl residues in ribonuclease which become exposed to the solvent upon denaturation. The combined shift in absorbance maximum and drop in extinction coefficient result in a difference spectrum whose near-ultraviolet maximum is a t 287 nm. Let YD = AZa7for the completely denatured form, and Y = tiZ8% YN = A287 for the native form. Then K D = [D]/[N] = (Y, Y)/(Y - YD). Since there are solvent perturbation effects both pre- and post-denaturation YD and YN must he determined by linear extrapolation. The basic data then is transformed into values of K D a t denaturant concentrations where both [N] and [Dl are finitely large. The simplest way of obtaining K k for a laboratorv exercise is to assume that AG for denaturation is a linear function of the concentration of denaturant 15).Thusa lot of In K n versus IGdmCI1 gives as the intercept in the absence of denaturant. l n . ~ ; , or Experimental Procedure An ultraviolet spectrophotometer with a bandpass 2 nm or less will be required. Each group needs 4 ml of 8 M guanidinium chloride (Pierce Chemical Co. #24115), 3-4 ml of 5 mg/ml ribonuclease (dilute Sigma Chemical Co. #R-5250 with 1% NH4HC03, and about 5-10 ml of 1%NH4HC03 buffer. The cost of reagents is currently about $5 per group. Nine solutions are prepared as shown in the table. Ribonuclease is added first, followed by buffer, followed by denaturant. Tubes should he eentlv. vortexed and allowed to stand for at least 15 min hefore absorbance3 are read. The for tuhe 9 should he divided hv two. Solution 10 is nrennred and allowed t o stand for 30 k i n before its absordanie is read. Comparison of A287 for tubes 9 and 10 directly demonstrates the rapid reversal of the denaturation upon dilution of the denaturant.
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1026
Journal of Chemical Education
Plot of absmlmnce al287 nm -US 9; 0 . data for tube 10.
denaturam concentration. 0 ,data fatube
Solution Composition Tube Number
1 2 3 4 5 6
7 8 9 10
Volume RNase (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.8
Volume 1% NHIHCOS (ml) 0.80 0.70 0.55 0.50 0.45 0.40 0.35 0 0.40 (1 m! of solution 9 1 ml buffer)
+
Volume 8 M GdmCl
(ml) 0 0.10 0.25 0.30 0.35 0.40 0.45 0.80 0.80
Calculations A plot of A287 versus denaturant concentration is made. Points 1,2, and 3 are for tubes in which [Dl is too small t o be accurately measured, but a line through these points can be extrapolated to estimate YN a t higher denaturant concentrations. Conversely, points 7 and 8 are for tubes in which [N] is too small; these can likewise be used to estimate YDa t low denaturant concentrations. Tubes 4,5,6, and 9 are for solu-
tions in which ID1 and [N] are easily determined: thevertical distance from the point to the extrapolated YN line is proportional to [Dl, and the vertical distance from the extrapolated Y Dline to the point is proportional to [N]. The ratio of these distances is an estimate of KD. If a point falls outside the area hounded by the two lines, it will provide a negative value for the estimate of KD.This, of course, cannot be used in the next step. The second plot is a plot of In K D against denaturant concentration. The least squares slope and intercept should be calculated. The anti-natural logarithm of the intercept at 0 M for rmanidinium chloride is the estimate for the denaturant equili6rium constant in buffer alone (K;). Studen( Results Eleven groups provided test data. The mean f standard error for A 2 8 1 are shown in the figure. Individual group estimates for KL ranged from 0.28 to 3 X 10-12, with 5 groups obtaining values within the range of literature values (1X to 1 X lo+).
Dlscusslon This experiment exposes undergraduate biochemistry students to several important concepts of protein structure. The abilitv of the denaturant to unfold the nrotein rests largely upon its being a better solvent for the "hydrophobic" residues, removing a principal driving force for folding. The direct demonstration that denaturation is rapidly reversible upon dilution of denaturant demonstrates the flexibility of the secondary and tertiary structure of ribonuclease. The use of A m as a robe for the denatured state illustrates environn&tal effects on the absorption spectrum of a chromonh,..m
Literature Clted 11) (2) (3) (4) 15)
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