they unfold the enzyme completely into a polypeptide chain. This
fully unfolded RNase is then allowed to refold after removing the
denaturants by dialysis. If the polypeptide chain successfully refolds
into its native shape, the RNase will regain its catalytic activity.
1. Mix 5 solutions in 5 microfuge tubes, as below:
a 1 ml RNase (0.5mg/ml) + 0.9 ml sodium acetate buffer to
keep the pH unchanged
b 1 ml RNase (0.5mg/ml) + 0.9 ml denaturants (2-ME +
urea) to unfold the enzyme
c dialyzed solution B (dialysis overnight to remove the
denaturants for refolding)
d boiled 0.1 ml RNase + 0.9 ml buffer (as control)
e 1 ml buffer + 0.9 ml denaturants (as control)
2. Incubate the solutions in a 50°C water bath for 5 minutes to
speed up the reaction.
3. Use a cork borer to make five wells in the RNA agar plate
using aseptic techniques.
4. Add 100 μl of each solution into a different well of the agar plate.
5. Cover the agar plate with lid and incubate at 37oC for
6. Overlay the agar plate with 3 ml of 1M perchloric acid for
5 minutes at room temperature. The perchloric acid will
make the RNA agar milky.
7. Pour off the perchloric acid to check whether clear zones are
formed around the wells. Measure and compare the diameter
of the clear zones.
Results are shown in Figure 3.
Discussion and Analysis
The clear zone at A (Figure 3) indicates that RNase had diffused and
broken down the RNA around the well. The breakdown of RNA is
not due to the buffer and denaturants, as shown by the absence of
clear zone at E. At B, no clear zone has formed, showing that RNase
had been denatured by 2-ME and urea by being unfolded into polypeptide chains. At C, when the denaturants had been removed by
dialysis, a smaller clear zone was observed. This shows that some
RNase molecules had regained catalytic activity by refolding back
into its native, three-dimensional shape spontaneously. The clear
zone, however, is smaller than that at A because of two probable reasons: (1) Some RNase molecules may have reacted with urea during
heating by a process called carbamylation, which prevented their
Figure 3. RNA agar plates after incubation and addition of perchloric acid. Clear zones indicate RNase activity.