Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Methods for Studying Enzymatic Reactions 53
ment in a high pressure cell. Animal cells can be readily broken by homogenisation,
but plant cells sometimes require rapid freeze/thaw methods in order to
break their tough cell walls.
Using the enzyme assay mentioned above, the enzyme activity can then be
puriWed from the crude extract using precipitation methods such as ammonium
sulphate precipitation, and chromatographic methods such as ion exchange
chromatography, gel Wltration chromatography, hydrophobic interaction chromatography,
etc. PuriWcation can be monitored at each stage by measuring
the enzyme activity, in units per ml, where 1 unit conventionally means the
activity required to convert 1 mmole of substrate per minute. Protein concentration
can also be measured using colorimetric assays, in mg protein per
ml. The ratio of enzyme activity to protein concentration (i.e. units per mg of
protein) is known as the speciWc activity of the enzyme, and is a measure of the
purity of the enzyme. As the enzyme is puriWed the speciWc activity of
the enzyme should increase until the protein is homogeneous and pure, which
can be demonstrated using sodium dodecyl sulphate (SDS)-polyacrylamide gel
electrophoresis. A puriWcation scheme for a hydratase enzyme from the
author’s laboratory is shown in Table 4.1. The puriWcation of the enzyme can
be seen from the increase in speciWc activity at each stage of the puriWcation. An
SDS-polyacrylamide gel containing samples of protein at each stage of the
puriWcation is shown in Figure 4.3. You can see that in the crude extract
there are hundreds of protein bands, but that as the puriWcation proceeds the
28-kDa protein becomes more and more predominant in the gel.
Why do we need pure enzyme If we can see enzyme activity in the original
extract, why not use that The problem with using unpuriWed enzyme for
kinetic or mechanistic studies is that there may be interference from other
enzymes in the extract that use the same substrate or cofactor. There may
also be enzymes that give rise to UV absorbance changes which might interfere
with a UV-based assay. If the enzyme can be puriWed then the turnover number
of the enzyme can be measured, which is the number of mmoles of substrate
converted per mmole of enzyme per second. The turnover number can be simply
Table 4.1 Enzyme puriWcation table (see Figure 4.3). DEAE, diethylaminoethyl.
Volume
(ml)
Enzyme
activity
(units ml 1 )
Protein
concentration
(mg ml 1 )
SpeciWc
activity
(units mg 1 )
PuriWcation
(-fold)
Crude extract 14 13.1 62.0 0.212 1.0
DEAE sephadex pool 19 11.6 17.0 0.684 3.2
Phenyl agarose pool 11 11.8 0.085 140 662
MonoQ anion-exchange pool 6.0 29.2 0.037 787 3710