ISSN 1070-4272, Russian Journal of Applied Chemistry, 2007, Vol. 80, No. 9, pp. 1598 !1603. + Pleiades Publishing, Ltd., 2007.
Original Russian Text + N.P. Kuznetsova, R.N. Mishaeva, L.R. Gudkin, 2007, published in Zhurnal Prikladnoi Khimii, 2007, Vol. 80, No. 9, pp. 1566 !
AND POLYMERIC MATERIALS
Sorption Purification of Concentrated Hemoglobin Solutions
N. P. Kuznetsova, R. N. Mishaeva, and L. R. Gudkin
Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia
Received October 26, 2005; in final form, March 2007
Abstract-A procedure was developed for purification of concentrated hemoglobin solutions from hemolyzate
of human erythrocytes to remove soluble cellular impurities using a frontal chromatographic process on
an anion exchanger under conditions of selective absorption of impurities and the absence of sorption of
the target product, hemoglobin.
Hemoglobin (Hb), the main respiratory blood pro-
tein, is contained in specific cells, erythrocytes, serv-
ing the most important function of blood, reversible
transfer of oxygen from lungs to tissues .
An Hb solution is obtained by breakdown of eryth-
rocytes using the osmotic hemolysis with the subse-
quent removal of insoluble cellular fragments (stroma)
by centrifugation. The resulting solution of hemo-
lyzate contains Hb in a high concentration. Along
with Hb, soluble components of erythrocyte mem-
branes, the products of cellular metabolism, having no
definite functional and molecular characteristics, pass
into the solution. Typically, these are phospholipids
and other phosphorus-containing components of a cell
and also intracellular proteins and enzymes, the resi-
dues of plasma and membrane proteins.
One of serious problems arising in using Hb solu-
tions as a basis for the development of blood substi-
tutes, oxygen carriers , is the possibility of toxic
reactions caused by the impurities, soluble stroma
components. In this connection, it becomes necessary
to develop procedures for hemolyzate purification to
remove side cellular components. There are published
data on procedures of Hb purification to remove im-
purities by affinity chromatography , displacement
preparative HPLC , and ion-exchange chroma-
tography using cationic and anionic systems .
The chromatographic methods suggested usually in-
volved presorption of Hb from the hemolyzate solu-
tion with subsequent desorption in a gradient of pH or
salt concentration in the eluting solution.
To be suitable as a base for a blood substitute
(oxygen carrier), Hb (MW 64 500) should practically
fully preserve the physiological activity, which poses
stringent requirements upon operation with its solu-
tions, such as narrow range of ionic strength, pH,
temperature, and the shortest contact time with
the sorbent. In this connection, as a method for purify-
ing Hb from hemolyzate solutions containing Hb
in high concentrations we suggested and studied
the frontal version of anion-exchange liquid chroma-
tography under such conditions when the main pro-
tein, Hb, is not sorbed but foreign soluble components
are selectively absorbed from the hemolyzate.
We used an EDE-10p anion exchanger (Plastics
Plant, Nizhni Tagil, Sverdlovsk oblast, Russia).
The sorption experiments were performed with the
anion exchanger (grain size 0.13 0.2 mm) in the mixed
OH3Cl form, prepared by titration of the OH form of
the anion exchanger with 0.2 N HCl in 0.15 N NaCl
to the corresponding equilibrium pH value.
Removal of impurities from the hemolyzate was
studied in two modes: static (sorption with stirring
for 2 h at 20oC) and dynamic (column size 6.5 0
1.2 cm, anion exchanger volume 6 ml, which corre-
sponds to 2 g of dry sorbent, solution flow velocity
25 ml h
). Approximately 80 ml of concen-
trated Hb solution (hemolyzate) was passed through
the column; 5-ml fractions were collected and ana-
Hemoglobin was prepared from erythrocytes of
human blood by osmotic hemolysis of cells. The
erythrocytes were separated from blood serum by cen-
trifugation (3000 rpm, 4oC, 20 min) and carefully