|
| |
Applications:
DYE-LIGAND CHROMATOGRAPHY
- CHARACTERIZATION OF IONTOSORB RED AND IONTOSORB BLUE SORBENTS FOR PROTEIN
CHROMATORAPHIC SEPARATION (Iontosorb RED,
Iontosorb BLUE)
- Kminkova M., Proskova A., Kucera J.: Potrav.
Vìdy, 11 (1993) 291-298
Characteristic of sorbents for bead-cellulose based dye-ligand chromatography
(Iontosorb BLUE and Iontosorb RED) are compared with traditional sorbents of this kind
Blue-agarose and Red-agarose). These characteristics were determined: particle mean radius
(Iontosorb BLUE 0.0987 mm, Iontosorb RED 0.205 mm), optimum linear flow rate (Iontosorb
BLUE 0.95 cm/min, Iontosorb RED 0.86 cm/min), height of theoretical plate (Iontosorb BLUE
0.265 mm, Iontosorb RED 0.510 mm) and reduced height of theoretical plate, that means
height of theoretical plate as particle mean multiples (Iontosorb BLUE 2.68,
Iontosorb RED 2.48). All these characteristics were compared with the same parameters of
Blue-agarose and Red-agarose sorbents. Both types of sorbents were demonstrated to be
congruent with each other in these characteristics. Dye concentration in both types of
sorbents were also similar, but there are certain differences between both types in this
parameter. The highest dye concentration was determined in the Iontosorb BLUE sorbent (3.5 µM/ml),
the lowest in the Iontosorb RED sorbent (0,725 µM/ml). Hence the dye concentrations
in agarose sorbents are much more even (Blue-agarose 2.1 µM/ml, Red-agarose
2.4 µM/ml). The dye concentration is also manifested in the sorption
capacity, which was determined for bovine serum albumin as a model protein. The highest
capacity was observed in the Red-agarose sorbent (13.46 mg/ml), the lowest in the
Iontosorb RED (5.76 mg/ml). The sorbents Blue-agarose (9.33 mg/ml) and Iontosorb
BLUE (7.93 mg/ml) have similar sorption capacities. On the other hand, the lower
capacity results in the higher recovery of bovine serum albumin in the elution
process. The highest recovery was determined for Iontosorb RED (84.3 %), while the
recovery of the highest substituted sorbent Red-agarose was only 41.7 %. The recovery
values of the Iontosorb BLUE sorbent (73.2 %) and of the Blue-agarose sorbent (75.2 %)
were actually the same. Model proteins, such as bovine serum albumin, human serum albumin,
acid phosphatase, catalase and peroxidase, were sorbed on all four sorbents and they were
eluted with an ionic strength gradient applying the standard procedure. This comparison
shows that there are not in fact any differences in elution volumes when the Iontosorb
sorbent and the agarose sorbent with the same dye are used. In both case the sorbents with
triazine dye are more efficient, and this is a well-know and practically utilized property
of the agarose sorbent. The results of this comparison document that bead-cellulose based
sorbent (Iontosorb) are equivalent to agarose based sorbents; this finding is of practical
importance because bead cellulose is more resistant mechanically and it can resist to
pressure up to 0.3 MPa while agarose is resistant to pressures of only 40 cm water column.
In addition, unlike agarose, bead cellulose is easy to be sterilized and it changes its
volume very little if ionic strength and pH value are changed. All these qualities
illustrate the fact than bead-cellulose sorbents are more feasible to industrial
applications of sorption techniques, particularly in the food and pharmaceutic industries.
The detection of identical properties suggests an opportunity for the industrial
employment of sorptions on immobilized dyes.
- COMPETITIVE ELUTION OF LACTATE DEHYDROGENASE FROM CIBACRON BLUE-BEAD CELLULOSE
WITH CIBACRON BLUE-DEXTRANS (Iontosorb DEAE)
- Mislovicova D., Gemeiner P., Stratilova E.: J.
Chromatogr., 510 (1990) 197-204
The efficiencies of elution of lactate dehydrogenase (LDH) from Cibacron Blue
(CB)-bead cellulose with elements ensuring competitive (Cibacron Blue-dextran), biomimetic
(NADH) and displacing (KCl) mechanisms were compared. Competitive elution with CB-dextran
T 10 was shown to be the most effective providing a 38 fold purified enzyme in 83 % yield.
As shown by fast protein liquid chromatography and polyacrylamide gel electrophoresis,
this LDH preparation was free from protein contaminants but contained CB-dextran.
CB-dextran was then removed by ion-exchange chromatography and the yield of LDH decreased
to 62 %. When using a longer column, the enzyme was resolved partially in two fractions.
The isoelectric point of the main fraction was 7.3.
|