Methods in Molecular Biology • 16 Enzymes of Molecular Biology

RNase A 265

0~I--OH

CH 2

0 OH

O~l '--OH <~=ffi=~

C 2

Base

H

f

I

?

0 OH

\/

//P\

O OH

CH20H

'1 'r

I

Fig. 1. Cleavage of RNA.

~==~

0 OH

O~t--OH

OH

defined a unit of ribonuclease activity as the amount of enzyme capable
of causing a decrease of 100%/min in the absorbance at 300 nm of a
solution of 0.05% yeast nucleic acid in 0.05M acetate buffer at pH 5.0.
Afinsen et al. (9) introduced a stopped assay where the activity is
measured at a temperature of 25°C and a wavelength of 260 nm with
a 0.8% solution of yeast nucleic acid and the undigested RNA is preci-
pitated with 0.75% uranium acetate in 25% perchloric acid. Several other
methods of precipitating the undigested RNA have been used such as
glacial acetic acid (2) or perchlorate alone (10). In addition synthetic
polymers are often used in place of extracted RNA as substrate (11).

2.3. Substrate
RNase A has its highest activity with single-stranded RNA. How-
ever, if the concentration of RNase exceeds 2.2 Kunitz U/mL and the
RNA concentration is of the order of 30 ~g/mL or greater, then double-
stranded RNA or poly A RNA will be cleaved (12). Thus, at low