348 Maunders3.8. Sulfhydryl Reagents
Sulfhydryl reagents are essential for the action of polynucleotide
kinase (18), whose activity falls to 2% in their absence. Maximum activ-
ity can be obtained in the presence of 5 mM dithiothreitol (DTT) (32).
However, 80% of this maximum activity can be achieved using 10 mM 13-
mercaptoethanol in place of DTT, and 70% with 10 mM glutathione (32).
3.9. Enzyme Assay and Unit Definition
Assaying the activity of T4 polynucleotide kinase entails measure-
ment of the transfer of a radiolabeled phosphate group from T-32p ATP
to an acid insoluble product (2). The phosphate acceptor is normally
a duplex DNA molecule, enzymatically treated to create 5'-hydroxyl
groups. This may be achieved by partial digestion using micrococcal
nuclease, which specifically produces 5'-hydroxyl and Y-phosphate
termini, or using pancreatic DNAse followed by alkaline phosphatase,
which removes the 5'-phosphate groups originally created.
The standard assay conditions are as follows: 70 mM Tris-HCl, pH
7.6; 10 mM MgC12; 5 mM DTT; 66 ~/y-32p-ATP; and 0.26 mM 5'-OH
salmon sperm DNA. Incubate at 37°C.
Commercial suppliers of the enzyme may use slightly different
assays, including variations in the nature of the DNA acceptor, the
concentrations of 5' termini and ATP, and the inclusion of other buffer
components such as spermidine. One unit of activity is defined as the
amount of enzyme that catalyzes the incorporation of 1 nmol of 32p
into an acid insoluble form in 30 min at 37°C.
The 3'-phosphatase activity can be determined by incubation with
AMP (9). The enzyme is incubated with 16 mM 3'-AMP for 60 min at
37°C, and the released inorganic phosphate measured. Preparations
from mutant or recombinant sources that are nominally phosphatase-
free hydrolyze <0.1% of the AMP (23).
- Experimental Procedures
4.1. Uses of Polynucleotide Kinase
The major use ofT4 polynucleotide kinase in the molecular biology
laboratory is for the specific phosphorylation of the 5' termini of DNA
and RNA, either by direct kinasing or by the exchange reaction. This
may be for the purposes of labeling the molecule, or merely to enable
the molecule to be further manipulated.