Download Rapid purification of heart muscle enzymes using dye affinity

Biochemical Society Transactions ( 1 996) 24
Rapid purification of heart muscle enzymes using dye
affinity aqueous two-phase systems.
RAYMOND SLOAN and ROBERT J. ELLIOT.
.%hod of Bdogy and Bochemistw, The Queen's University of Belfast,
Belfast BT9 7BL. Northern Irelend.
Aqueous two-phase systems can be prepared by mixing two,
water soluble, polymers at certain concentrations in water [I]. The
mixture will separate into two aqueous phases (containing > 75 %
water); each phase being rich in one of the two phase-forming polymers.
The highly aqueous nature, of the phases, provides a mild environment
for the rapid fraaionation of labile molecules and cell particles.
Aqueous two-phase systems can be prepared using, the polymers
polyethyleneglycol (PEG) and dexwan, or PEG and certain salts (e.g.
ammonium sulphate). Protein added to an aqueous two-phase systems
will partition between the two phases, generally favouring the lower
phase of a PEG and dextran system. The behaviour can be described by
the partition cocffcient, K, defined as (conmeation in upper phase /
concentration in lower phase). The inclusion of a ligand, covalently
attached to PEG. in an aqueous two-phase system causes the specific
extraction of proteins (capable of binding to the ligand) into the upper
PEG-rich phase.
The ligand-bound protein is purified from
contaminating proteins, that remain in the lower phase [I].
The present work examines the use of reactive dyes, covalently
attached to PEG, 8s ligands for the purification of two medically
significant nucleotide-dependent enzymes (lactate dehydrogenase and
pyruvate b a s e ) within an aqueous two-phase system.
A protein extract was prepared from porcine heart muscle by
homogenisation of the muscle tissue in 10 mM phosphate (pH 7.5)
buffer, containing ImM EDTA, ImM PMSF and ImM 2mercaptoethanol. The homogenate was clarified by centrifugation
(5000 x g for 30 minutes) and the resultant Supernatant subjected to
ammonium sulphate fractionation. The enzymes LDH and PK were
found enriched in the 5 1 60 96 (saturated) ammonium sulphate
fraction, and were separated from malate dehydogenase (a potential
assay contaminant) which was concentrated in the 4 1 - 50% fraction.
The aqueous two-phase systems were prepared from
concentrated polymer and buffer stock solutions. Hydoxypropyl starch
(Reppal PES 200, supplied by Reppe Glykos AB) was used in place of
dextran. The total composition of the systems was 8 % (w/w) PEG
8000. 16 % (wlw) Reppal PES 200, 1 mM EDTA and 1 mM 2mercaptoethanol, buffered using 10 mM hiethanolamine-HCI (pH 7.0).
The PEG-dye ligands were prepared using a modification of the method
of Johansson 121. The PEG-dye ligand. where included, accounted for
0.08 Yo (w/w) of the system. Phase separahon was accelerated by low
speed centnfugation and was completed in under 2 minutes.
The reactive dyes Procion Red HE-7R and Procion Green HE4BD (gifts from ICI Dyestuff Divsion) were selected as ligands for
LDH and PK respectively, following a study on thc specificity of
thirteen reactive dyes with unfractionated extract proteins. The selection
was based on the specificity and binding strength, of the dye ligands, for
the target enzymes.
The selected PEG-ligands were employed as l'ollows. D i a l y d
aliquots of the 5 1 - 60 O/O ammonium sulphate fraction, accounting for
10 YO(w/w) of the aqueous two-phase systems. were extracted using the
PEG-dye as a ligand. The upper PEG-nch phase, containing the PEGdye and the ligand-bound enzyme, was collccted after phase separation.
The lower phase, containing residual protein, can be re-extracted with a
fresh upper phase contaming the same PEG-dye ligand. to improve the
enzyme yield. After completion of the extrachon process, the like
upper phases were pooled. Each combined upper phase was washed
with a protein-free lower phase and the upper phase re-collected after
phase separation. This step acted as a 'washing' stage, and removed
non-specific and weak binding proteins. The punfied, and ligand
-
bound, enzymes were eluted, from the washed upper phase by the
addition of solid ammonium sulphate (added as 15 % (w/w) of the
upper phase). In the new two-phase system formed, the purified
enzymes were present in the salt-rich lower phase. The ionic strength of
the new upper PEG-rich phase (- 0.5 M) weakened any ionic
interactions between the enzyme and ligand, and allowed the enzyme to
partition to the lower salt-rich phase for collection [2].
The ammonium sulphate fractionation of the heart muscle proteins
allowed the separation of the two target enzymes (LDH and PK) from
other enzymes that could be bound by the reactive dyes. The recovery
and purification of LDH and PK from the ammonium sulphate fraction,
using the aqueous two-phase system, are shown in tables 1 and 2.
Table 1. Porcine heart muscle: uurification of LDH using Procion Red
HE-7B as an a f f i t v ligand in an aqueous two-ohase svstem.
Table 2. Porcine heart muscle; ourification of PK usha Procion Green
HE-4BD as an affinitv liaand in an aaueous two-ohase system.
I
PEG
ammonium sulphate fraction
upper phase (ligand-bound PK)
'washed' upper phase
PK
PMSF
I
%
yield
100
96
89
I
specificactivity
(IJ/mg)
11.4
I
_-_--
I
purif
factor
1
1
__-_
___
Both enzymes were recovered in greater than 85% yield from
the ammonium sulphate fractionation. The affinity aqueous two-phase
systems resulted in good recovery of both LDH and PK from the
ammonium sulphate fraction, with purification factors that are
consistent with conventional dye affinity column chromatography [3].
Previous workers have demonstrated differences between the use of
reactive dye ligands immobilised to solid mahices and in aqueous twophase systems [4]. It has been proposed that the proteidmahix
interactions may also be in,olved during column chromatography [ 5 ] ,
while aqueous two-phase systems present a sterically unhindered ligand
molecule to the protein's binding site.
Although the aqueous two-phase systems, in the present work,
were performed using small volumes systems, the results can be
extrapolated to large volume preparations. The scaling up would
involve the propornonal increase of all system components, with
identical results obtained at each extractive step. Extractive enzyme
purification, use two-phase systems, has been performed using volumes
from a few miIIiIitres to more than I m3, with scale up factors in excess
of 1 x lo4 being achieved without affecting reeovery or purification
values [6,7]. The present work with heart muscle enzymes, therefore.
would be suitable for large scale preparations.
P.-.4. (1986) "Pamtion of Cell Parhcles and
Macromolecules", 3rd edition. pp8 - 39 and 91-96, Wiley, N.Y.
Johansson, G (1984) Methods Enzymol. 104,356 364.
Konecny, P., Smrz, M., Borak. J. & Slovakova, S. (1987) J.
Chromatogr. 398,387 390.
Naumann, M., Reuter, R., Metz, P. & Kopperschlager, G. (1989) J.
Chromatogr. 466,319 - 329.
Beissner, R.S. & Rudolph, F.B. (1978) J. Chromatogr. 161, 127 135.
Kroner, K.H., Cordes, A,, Schelper, A., Morr, M., Buckmann, A.F.
& Kula, M-R. (1982) in "Affinity chromatography and related
techniques" (Gribnau, T.C.J., Visser, J. & Nivard, R.J.F., eds.), vol.
9, pp491 - 501, Elsevier, Amsterdam.
Sehutte, H., Kroner, K.H., Hummel, W. & Kula, M-R. (1983) Ann.
N. Y. Acad. Sci. 413,270 - 282.
1. Albertsson,
2.
3.
4.
5.
6.
ethylenediminetetra-acetic acid
lactate dehydrogenase
polyethyleneglycol
pyruvate kinase
phenylmethylsulphonylfluonde
I
extraction stage
Abbreviations used
EDTA
LDH
1 1 9s
7.
-
-