Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to modified hemoglobins
and uses thereof. More specifically, it relates to
covalently modified and polymerized hemoglobins, processes
for their preparation, and processes of preserving biologi-
cal organs therewith.
Human inkernal organs for transplantation are
normally preserved in cold saline or buffer solutions. ~he
normal viability periods of these organs are from a few
hours to vne day. This viability period can be critical,
especially in the case of liver transplantation, since a
removed liver can keep its viability for only a very short
period of time (4-6 hours)~
One suggested method for increasing the preserva-
tion time of organs is a perfusion of the organ with
hemoglobin (Hb) - based oxygen carriers. These materials
can also be used for effective cardioplegia in open-heart
surgery.
However, whilst a wide variety of Hb-based oxygen
carriers are known, they all have excessive oxygen affin-
ities and inadequate oxygen delivering capacities at the
hypothermic conditions needed for organ perfusion (10-
15 C). Workers in the field have tended to concentratetheir efforts ~n developing Hb-~ased oxygen carriers for
tran~fusion purposes, which have oxygen a~finity resembling
that of whole blood at 37 C, in ~heir search for an
adequate Hb-based blood substitute.
The oxygen affinity of Hb-based oxygen carriers
is commonly and conveniently expressed as its P50 ~ the
partial pressure of oxygen at which 50% of the hemoglobin
siteæ are saturated with oxygen. The higher the P50 value,
the lower the oxygen affinity. The P50 value is highly
dependent on temperature, the oxygen affinity increasing as
the temperature decreases. For example, red blood cells at
15 C have a P50 value of 2 torr, compared with 26.5 torr at
37C. Accordingly, for successful use in hypothermic organ
. .
,
' '' :',"
-- 2 --
perfusion, acellular oxygen~delivering red cell substitutes
should have a P~O value much higher than 27 torr at 37 C,
and ideally between 55 and 100 torr at 37~C~
~ a~ NoO 4 061r~36 Morris et al., dis-
closes a pharmaceutically acceptable, intramolecularly
crosslinked, stromal free hemoglobin or use as a blood
substitute and blood plasma expander. A wide variety of
difunctional crosslinking reagents are suggested in Morris
lQ et al., including divinylsulfon~. In examples VI-IX,
Morris st al. describe reacting hemoglobin with divinylsul-
fone. In each case, an intramolecularly crosslinked
hemoglobin is obtained, with P50 values at 37 C of from 7-55
torr. Morris et al. also suggest the use of their cross-
linked hemo~lobin or the storage and preservation of
viable isolatad perfused mammalian organs for their event-
ual transplant into ~ recipient.
The present invention provides novel divinylsul-
~one-treated hemoglobins which possess properties desired
in oxygen carriers. One of these products is a non-cross-
linked, intr~molecularly modified hemoglobin, which has a
near normal oxygen-delivery capacity at 15C, a relatively
low oxygen af~inity, a normal physioloyical half~lifet a
low methemoglobin content, and relatively high Hill coefPi-
cient. This combination of properties renders it suitable
not only as a component of a potential oxygen carrier ~or
trans~usional purposes, but also as an oxygen carrier in
acellular perfusional fluids. Another of the products is
divinylsulfone-modified polymerized hemoglobin. This has
a long retention time, low oxygen affinity and lower
methemoglob:Ln content. Itq combination of properties
renders it primarily suitable as an oxygen carrier for
transfusional purposes. The invention also provides
mixture~ of these two materials in controlled relative
proportions, in which the combination of their respective
properties can be optimized. It also provides a process
"` 207~2
whereby the individual products, or mixtures thereof in
controlled, predetermined relative proportions can be
obtained, in a single synthetic step using a single
reagent, divinylsulfone (DVS).
Divinyl.sulfone reacts w.ikh primary groups on the
peptide chains of hemoglobin, to form secondary amine
linkages. In a first reaction step, a modified hemoglobin
is formed:
O O
11 11
CH2ac~--s--CH=CH2 + H2N--Hb ~ CH2=CH--7--CH--CH--NH~Hb
~5 ~ 0
DVS Hb modified Hb
~Hh-~VS)
In a second reaction step/ a crosslinked hemoglo-
bin is formed:
O O
ll ll
Hb-NH2 + CH2 = CH-S-CH.CH.NH.Hb ~ Hb.NH.CH.CH~S.CH.CH.NH.Hb
Il 1~
C O
(Hb-DVS) poly (Hb-DVS)
The crosslinking can theoretically take place
between two globin chaine of the same hemoglobin tetrameric
units, in which case the product is referr0d to as intramo-
lecularly crosslinked, or between two globin chains of
different hemoglobin tetrameric units, in which case the
product is intermolecularly crosslinked, effectively
polymeriz~d. However, in contradiction to the predicted
theory, and contrary to the teachings of the aforementioned
Mor~is et al. patent, it is found in accordance with the
present invention that, under selected conditions, divinyl-
sulfone reacts with hemoglobin to produce a product which
. ...
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. , : : ~
'.~' '.
~07~8~2
-- 4 --
is substantially free from intramolecular crosslinks.
The products according to the invention are thus
covalently modified, non-crosslinked nemoglobin and/or
inter~olecularly crosslinked hemoylobin, and/or mixtures o~
the two modified hemoglobins in controlled relative propor-
tions. The products are mod.i~ied and/or crosslinked with
sulfone-secondary amino covalent bonds, and the products
are substantially free from intramolecular crosslinks.
They show oxygen affinities at 12-15 C which render them
suitable for use in organ perfusion.
Figure 1 is a graph of the ion exchange chroma-
tography puri~`ication of the produ~t prepared according to
Example l below,
Figure 2 is the results of electrophoresis
according to Example 3 below;
Figure 3 is a Hb-GPC analysis curved derived
according to Example 4 below;
Figure 4 is a Hb-GPC of typical poly-hemoglobin
derivatives ~rom the examples below;
Figure 5 is the SDS-PAGE of HbBv and derivatives
from Example 5 below;
Figure 6 is a graphical pres~ntation of the
dependence o~ oxygen affinity on pH o~ the products of the
invention, according to ExamplP 6 below;
Figure 7 is a graphical presentation of the
dependence of oxygen af~inity on temperature o~ the prod-
ucts of the invention, according to Example 7 below;
~7~2
-- 5 --
Figure 8 is a graphical presentation of the
clearance of intravenously injected hemoglobin products,
according to Example 8 below: and
Figure g is a plot of oncotic pressure against
hemoglobin concentration, derived according to Example 9
below.
The products of the present invention are pre-
pared by a process o~ reacting bovine or human deoxygenated
stromal free h~moglobin under anaerobic conditions with
divinylsul~one. When relatively low concentrations of
hemoglobin and DVS are used in anaerobic one step reaction,
one can produce a modified hemoglobin having exactly the
same elution time on gel-permeation HPLC as that of the
native, unmodified Hb, indicating that initially only a
modi~ied, non-crosslinked product is formed. In a two step
reac~ion where low concentrations of DVS are used in the
f irst step and higher concentrations in the second step, a
product comprising 50% modified Hb and 50% modified
polymeriæed Hb tpoly-Eb-DVS) can bQ obtained. Raising the
DVS concentration still higher in the second step yields a
product comprising 15% modified Hb and 85% poly-Hb-DVS.
Thus by varying the concentration o~ DVS in the reaction
mixture, one can vary the relative proportions o~ modified
Hb and poly-Hb produced, in a aontrolled manner to obtain
a product of pre-selected composition. In all cases, the
product produced is substantially free from intramolecular
crosslinking, and has the desirable oxygen carrying prop-
erties described above.
The modification reaction to produce Hb-DVS is
carried out using deoxygenated Hb, under anaerobic condi-
tions. Relatively low concentrations of deoxygenated Hb
and DVS in solution are preferably used, suitably ~rom 5-
10~ of deoxygenated Hb in the reac~ion solution, and 0.5-
1.5 microlitres DVS per ml of solu~ion. The reaction is
~,
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, . :. .
,, :: :
, ' ' ' , " '
," ~, ~
~07~8~2
carried out under slightly basic conditions (pH 7-7.8),
suitably buffered to a pH value in that range with sodium
phosphate buffer. The temperature should be kept within
the range 1-5 C, and t~e reaction takes place for a period
of 18~30 hours.
The second stage reaction, to convert the Hb-DVS
to poly-Hb-DVS is conducted under essentially the same
conditions, except that a higher concentration of Hb-DVS in
solution is used and a higher concentration of DVS is
present. These higher concentrations are, suitably, from
12-20% Hb-DVS in the reaction solution, and from 2-5
microlitres DVS per ml of reaction solution. Other reac-
tion conditions, including pH, temperature, anaerobic
reaction, and time of reaction can be the same in the
second ~ep as in the first step. The reactions are
appropriately quenched after the desired reaction time by
addition of bu~fered lysine-HCL. The unreacted vinyl
groups of DVS are deactivated by lysine-HCL.
Thus according to the present invention, it is
found that under certain conditions the anaerobic reaction
of Hb with the bifunctional reagent DVS can produce an
intramolecularly-modi~ied derivative, Hb-DVS, without the
introduction of an undesired non-speci~ic intramolecular
crosslinkage. In order to direct the reaction to this
path, one uses relatively low concentrations of Hb and low
molar ratios of DVS:Hb-tetramsr. The Hb-DVS so formed is
remarkably homogeneous with respect to its molecular mass
and electrophoretic properties, as demonstrated in the
specific examples below.
At the increased concentrations of Hb-DVS and
relatively high molar ratio of DVS:Hb~tetramer, the mod-
i~ied hemoglobin so formed can be anaerobicallypolymerized. From 50-85% of the starting material can be
thus polymerized, to produce a poly Hb-DVS which is not
~7~2
homogeneous with respect to molecular weight, but which, on
analysis, shows the presence of molecular masses of 16 kda
and multiples thereof. No product corresponding to a 32
kda molecular weight is obæerved, demonstrating the lack of
intramolecular crosslinkages, even at the high DVS concen-
tration needed for the polymerization, thereby indicating
that poly-Hb-DVS does not contain intramolecular crosslink-
ages as part of its structure. This conclusion is consist-
ent with the very low oxygen affinity and methemoglobin
content of the material.
The conditions of reaction outlined above are
important for obtaining the products of the present inven~
tion. When too high a temperature is used, very high
quantities of methemoglobin are produced, which is undesir-
able. Similarly, when too high a molar ratio of DVS:Hb-
tetramer i~ used, even at low tempera~ures, the methemoglo-
bin level is too high, implying the possibility of non-
specific intramolecular crosslinkage. The intramolecular
modification obtained with the relatively low molar ratio
of DVS:Hb-tetramer described above appears to change the
molecular conformation in such a way that subsequent non-
speci~ic intramolecular crosslinking can be virtually
avoided.
Both Hb-DVS and poly-Hb-DV~ possess properties
desired in oxygen carriers. The long retention time of
poly-Hb-DVS in the mammalian circulatory system, apparent
from the specific examples below, its low oxygen a~finity
and low methemoglobin content make this protein a potential
oxygen carrier for transfusional purposes. The modified
derivative, Hb-DVS with it6 normal physiological half life,
low oxygen affinity, low methemoglobin content and rela-
tively high Hill coe~ficienk is suitable for use as a
potential oxygen carrier in acellular per~usional fluids.
The findings herein are based upon experimental work
conducted on animal hemoglobin, namely bovine hemoglobin
::
2~7~
-- 8 --
(BvHb), but are helieved equally applicable to other
hemoglobins, including human hemoglobin.
T~e invention is further described and illus-
trated with reference to the following ~pecific examples:
BOVINE NEMOGLOBIN
Stroma~ree bovine hemoglobin (HbBv) was prepared
by t~luene extraction and crystallization~ essentially
accordinq to a procedure previously described for human
hemoglobin b~ De Venuto et al. [1]. The washed crystals
were dissolved in distilled water and dialy~ed twice at 2 C
(1:25, v/v) against water and then three more times (1:15,
v/v) again~t cold 0.0S M sodium phosphate buffer (pH 7.4~.
Sterilization was achieved by passing the final solution
through a sterile 0.22 ~m Millipore Sterivex-GS filter.
Total hemoglobin and methemoglobin concentrations were
~easured on an IL 482 CO-Oxymeter (Instrumentation Labora-
tory) calibrated for Hb v extinction coefficients. Differ-
ent batches of the final solution contained 10-15% (w/v)
hemoglobin and 1.5-3% methemoglobin (~ of total hemoglo-
bin). These were kept at 2-4 C and used within 48 hours
~rom the time they were prepared.
EXAMPLE 1 - P:REPARATION OF HbBv--DVS
A 25 ml aliquot of 8% (w/v~ solution of HbBv in
0.05 ~ sodium phosphate buffer (pH 7.4) (reaction buffer),
containing 0.25 ml of caprylic alcohol to prevent foaming,
was deoxygenated by flushing with nitrogen in a 100 ml
Pyrex reactor for 3 hours. Next, a 25 ~l aliquot of DVS
was added to the reaction vessel, and the reagents were
stirred gently under a continuous flow of nitrogen for 24
hours. ~he unreacted vinyl groups were deactivated by the
addition of 2.0 ml of deoxygenated 2.0 M lysine-HC1 in 0.05
M sodium phosphate buffer (pH 8.0). The quenching reaction
,. . . , : : :
. . .
' , ~: :, , .
207~8~
was carried out under a nitrogen atmosphere for an addi-
tional 18 hours. After quenching, the reaction solution
was clarified by centrifugation at 15000 xg for 30 minutes.
The solution was dialyzed thrice (1:40l v/v) against 0.02
M Tris-HC1 buffer (pH 7.4) in order to remove unbound DVS
and excess lysine. The protein was then converted to the
carbonyl form, and the HbBv-DVS was separated from the
unmodified hemoglobin on a DEAE-Sephadex column (~-50, 2.5
x 30 ~m) which was equilibrated with the same CO saturated
buffer. The column was loaded with 5 ml batches and eluted
with a linear gradient of pH and ionic strength using 0.05
M Tris malaate (pH 6.5) as the second buffer. The second
~ands from all batches were pooled together and concen-
trated to ~15% (w/v) by ultrafiltration (YMT-30 membrane,
Amicon). All work was done at 2-4 C. The final HbBv-DVS
solution was sterilized as before and storecl at the same
temperature. ~hese solutions were used not later than 2
weeks after preparation. Just before use, the carbonyl
derivative of HbBv-DV5 was converted to the oxy form as
previously described by Shih et al. The amount of methemo-
globin in the final oxy derivative was 3-4% of the total
hemoglobin.
Fig. 1 o~ the accompanying drawings presents the
ion-exchange chromatography purification of the product,
HbBv-DVS, on a DEAE-Sephadex A-50 column. The figure is
discussed ln the "Results" below.
~A~IE-2 - PREPARATION OF POLY HbBv-DVS
To 25 ml of 15% (w/v) solution of HbBv DVS in
reaction bu~fer, 0.25 ml of caprylic alcohol was added.
The HbBv-DVS solution was then deoxygenated by flushing
with nitrogen for 3 hours. Next, 75 ~l of DVS was added
and the reaction was allowed to proceed, with gentle
stirring, for 24 to 72 hours. Every 24 hours, the pH of a
small aliquot of the reaction solution was detarmined and
;.
,,,~ , ,
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:: ",
. .
21~ 8~
... ,i
-- 10 --
the progress of the reaction was monitored by HP-GPC. If
necessary, the pH was adjustad to 7~4 with 0.5 M NaOH.
Once polymerization had produced the desired molecular
weight distribution for Poly Hb~v DVS, the reaction was
quenched by adding 2.0 ml of deoxygenated 2.0 M lysine-HCl
in reaction bu~fer. The reaction mixture was kept
anaerobîc and stirred for an additional 18 hours, and was
then clarified by centrifugation at 15000 xg for 30 min-
utes. The solution was dialyzed thrice (~:40, v/v) against
a reaction bufPer containing 0.1 M NaCl, an~ the proteln
wa~ converted to the carbonyl form. The reaction solution
was then applied, 5 ml batch at a time, to a 4x50 cm Biogel
P-100 column (Bio-Rad), pre-equilibrated with the same CO
saturated bu~fer. In this gel-filtratlon medium, all
materials with molecular weights higher than ~100,000 were
immediately excluded from the columnl enabling a good
separation of the polymerized material (M~ 2 130,000~ from
the tetrameric HbBv-DVS fraction,. The pooled polymerized
fractions were then concentrated by ultrafiltration to ~10%
(w/v). All work was done at 2-4 C~ The final Poly-HbBv-
DVS s~lution was sterilized as before, and stored at the
same temperature. These solutions were used within 2 weeks
~rom the time o~ preparation. Just before use, the
carbonyl derivative of Poly-HbBv DVS was converted to the
~5 oxy form. The amount of met~emoglobin in the later sol-
ution was less than 6~.
~A~oe~ ELLULOSE ACETATE ELECTROPHORESIS
Electrophoretic follow up of the reaction of the
synthesis of HbBv-DVS was carried out using cellulose
acetate electrophoresis. Super Sepraphore (Gelman) strips
were employed in a Sepratek ~Gelman) cham~er according to
the procedure of Gebott and Peck [3]. The strips were
stained with Ponceau S svlution and destained in 5% acetic
acid. Figs. Z (a), (b) and (c) show the results from,
respectively, HbBv, HbBv-DVS from a 24 hour reaction
:, ' . :, '
' . ; ' ' : '
2~7~
-- 11
mixture (Example 1), and HbBv-DVS Prom a 48 hour reaction
mixture, and are discussed in the "Results" section below.
EXAMPLE 4 - GEL-PERMEATION HPLC
~ P-GPC analyses were per~ormed on a TSK-G 3000 SW
column (7.5 x 600 mm, LKB) and TSK~GWSP pre-column (7.5 x
75 mm, LKB) using a Waters HPLC apparatus equipped with a
model 720 proqrammable system controller, data module, two
pumps and an automatic sample (WISP model 710B). Sample
solutions (100 ~1), wikh a total protein content of 0.5-5
mg, were eluted at 0.8 ml/min with 0.1 M sodium phosphate
buffer (pH 6.8) containing 0.1 M KC1. The column outflow
was monitored at 576 nm, using a variable wavelength
detector (model 450, Waters).
The column was calibrated with highly purified~
well~ch~racterized, non-heme globular protein standards.
The void volume (vO = 11.6 ml, 14.5 min) was d~termined from
the elution of Blue Dextran 20~0. There was a linear
relatiohship between log k~ and the elution time. However,
the apparent molecular weights of the monomeric (M~ =
64,000) and the polymerized h~moglobin components were
shifted with respect to the non-heme protein standards.
These hemoglobin derivatives showed lower apparent molecu-
lar weights than expected. To correct this problem the
las'c three peaks of these components (M~ = 192,000; 128,000
and 64,000) were employed as standards ~or the molecular
weight estimation of the intermolecularly-crosslinked
hemoglobins.
Figs. 3 (a) and (b) are the analysis curves from/
respectively, HbBv, HbBv-DVS isolatPd ~rom a 20 hour
reaction mixture by ionic exchange chromatography (solid
line) and HbBv-DVS 48 hour reaction mixture (dotted line).
Figure 4 show~ the HP-GPC of typical poly HbBv-DVS prepara-
tions (Example 2) namely: (a) poly-HbBv-DVS, 24 hour
: .. :: ,..
.,: .
;,
~7~
-- 12 --
reaction mixture (dotted line) and isolated poly-HbBv-DVS
obtained from the same reaction mixture (æolid line); (b)
poly HbBv-DVS 72 hour reaction mixture (dotted line) and
isolated poly HbBv~DVS obtained from the same reaction
mixture (solid line). They are further discussed in the
"Result~ section below.
EXAMPLE 5 - POLYACRYLAMIDE GEL ELECTROPHORESIS
SDS-PAGE was carried out in slabs (1.5 mm thick),
according to the method of Weber and co-workers ~4], using
polyacrylamide concentration of 12% (w/v). Protein samples
originating from the HP-GPC procedure were concentrated and
desalted (Centriprep-10, Amicon) with 0.01 M sodium phos-
phate buffer (pH 7.0), as part of their preparation for the
SDS-PAGE. This step was necessary in order to obtain the
small sample volumes needed for the continuous buffer
system utilized here and to eliminate SDS precipitation by
the potassium ions present in the HP-GPC elution buffer.
Gels were stained with Coomassie blue R-250 and destained
by di~usion in 12.5% isopropanol and 10% acetic acid.
Relative mobilities, calculated as ratios of distances of
migration of protein bands and dye, were used in conjunc-
tion with a calibration curve obtained from five protein
markers in order to estimate molecular masses. the markers
were bovine serum albumin (66 kDa), bovine erythrocyte
carbonic anhydrase ~29 kDa), bovine pancrea~ trypsinogen
(24 kDa), soybean trypsin inhibitor (20.1 kDa) and bovine
hemoglobin ~16 kDa).
The results are presented in Fig. 5 which shows
SDS PAGE of HbBv and derivatives obtained from its
anaerobic reaction with DVS: (a) mixture of protein
marker~; (b) HbBv-DVS 48 hour reaction mixture7 (c) pure
HbBv-DVS; (d) Poly-HbBv-DVS isolated from a 24 hour reac-
tion mixture; (e) the slowest Hp-GPC peak isolated from
poly-HbBv-DVS 24 hour reaction mixture (dotted peak of E'ig.
2~4~2
- 13 -
4a); (f) 5 ~g of pure HbBv: (g) 5 ~g of pure HbBv-DVS. The
figures are further discussed in the "Results" section
below.
EX~MPLE 6 - oxyGEN EOUILIBRIUM DETE~MINATIoNs
Oxygen equilibrium curves were determined using
a TCS modql B Hemox~analyzer at 37 C. In these experi-
ments, HbBv, HbBv-DVS and Poly-HbBv~DVS (isolated from a 24
hour reaction mixture) were employed at a protein concen
tration of 2 mg/ml. The pH of the samples was measured at
37 C with a Fisher Accumet 750 instrument. If not other-
wise statedl buffers used were 0.15 M Tris-HC1 or Bistris-
HC1 containing 0.15 M C1- ions. ~hese buffer~ were prepared
by titrating the reagents with HC1 to pH 5.5 b~fore read-
justing with NaOH to the desired pH. This was done so that
the molarity of the buffer would also represent the concen-
tration of C1- in solution. The sample6 were equilibrated
with pure nitrogen (les~ than 5 ppm oxygen) and reoxygen-
a~ed wikh 35.2% oxygen in nitrogen. In some cases, whenthe effect of C02 on the oxygenation properties had to be
evaluated, gas mixtures containing 5.0% C0z were employed.
Methemoglobin content after oxygen equilibrium measurements
was 3-8%. oxygenation data were ~ormulated in terms of the
Hill plot (log Y/(1-Y) versus log P) where Y is fractional
saturation of t~e hemoglobin with oxygen and P is the
oxygen pressure in mm Hg. Hill plots were derived using
digitized data points from the oxygen equilibrium curves.
Overall oxygen af~inity and cooperativity were character-
ized in terms of oxygen pressure at half-saturation (P50)
and the Hill coefficient (n50) given by the Hill plot at
half-eaturation.
Data in the present specification were based on
at lea~t two independent determination~ for each point.
Values for individual PgO determinations were within + 1.0
mm Hg of the mean value.
.': '
: ' ., ' ~, ' :,.
~7~8~2
- ~4 ~-
Fig. 6 illustrates the dependence of the oxyyen
affinity on pH for HbBv ~o), HbBv-DVS (-), and poly HbBv-
DVS (~). This figure is further discussed in the "Results"
seçtion below.
~X~MPLE 7 ~ EFFECT ON TEMPERATURE ON THE OXYGEN-
~1 DING P~OPERTIES
The effect of temperature on the oxygen affinity
of HbBv, HbBv-DVS and Poly HbBv-DVS is shown in Fig. 7. A
range of temperatures extending from 15 to 37~C was
e~plored. In this range, at pH 7.4 and in the presence of
0.15 M Cl- ions linear plots of Log P50 VS. 1/T were
obtained, indicating constant heats of oxygenation. one
can see that ~or all three oxygen carriers the P50 values
are decreasing at lowsr temperatures. At any one tempera-
; ture, however, the PgO values of HbBv-DVS and Poly HhBv-DVS
are much high~r than that of HbBv.
The amounts of oxygen that would be unloaded at
37C and 15C by these five human and ~ovine hemoglobin-
based oxygen carriers upon decreasing the PO2 from an
arterial value o~ 100 to a venous level of 40 mm Hg were
calculated from the origina] oxygenation curves used for
the generation of Fig. 7. ~hese values are compared in
Table I, îmmediately below. The Hill coefficient values
for H~Bv-~VS and Poly HbBv-DVS, at all temperatures, were
n50 ~2.0 and n50 - 1.6~ respectively.
2~7~5~
~ 15 -
TABLE I
OXYGEN UNLOADING BY SOM~ HUMAN AND BOVINE
HEMOGLOBIN-BASED OXYGEN CARR~ERS AT 15 AND 37 C
oxygenTemp. P50 ~ % oxyHb
Carrier t ~)(mm Hg)(P02100-49 mm Hg)
Human blood 37 26.5 25
-- __
Human Hb 37 14 6
HbBv 37 27 15
7 6
HbBv-DVS 37 52 37
17 20
Poly HbBv-DVS 37 61 32
22 22
As in the oxygen equilibrium curves of Fig. 6,
the data points in Fig. 7 are for HbBv (o), HbBv~DVS ~-)
and Poly HbBv-DVS (~). This figure and Table I are ~urther
discuæ~ed in the "Results" section bælow.
ÆXA~PLE 8 - INTRA~A$CULAR R~T~ ION ~IME IN ~
Plasma retention pro~iles of HbB~, HbBv-DVS and
Poly HbBv-DVS (isola~ed from a 24 hour reaction mixture)
were obtained from experiments performed on Sprague-Dawley
male rats (300-350 g). Hemoglobin derivatives were
exchanged by difiltration into phosphate-buffered saline
(pH 7.4) (0.01 M sodium phosphate in 0.15 M NaCl) and their
concentrations regulated to 6-8~. These solutions were
~iltered through sterile 0.22 ~m filters just before
injection. ~he rats were anaesthetized with Somnotol. The
right ~emor~l vein was cannulated and a sample of blo~ was
' : ,; ,, :
:: . ,, : ,
~.,:, ~ ..,
.. . ... .
20748~2
- 16 -
withdrawn, ag a control, prior to the bolus injection of
2.0 ml of the tested material. Blood samples (250 ~.l) were
drawn via the vein at 2, 15 and 30 min after the injection~
Subsequently, the surgery site was closed and samples were
continued to be taken at 1, 2, 3, 4 and 6 hours following
the injection time by cutting a minor slice at the end of
khe tail. The samples were centrifllged and analy~ed for
the hemoglobin content o~ their plasma.
Figure 8 graphically presents the clearance of
intravenously injected HbBv (O), HbBv-DVS (-~ (upper panel)
and two different initial plasma levels of poly HbBv-DV5
(lower panel). The dashed lines indicate values obtained
for pla~ma half life. The ~igure is further discussed in
the l'Resultsll section below.
EXAMP~E 9 - COLLOID OSMOTIC PRESSURE
The colloid osmotic (oncotic) pressure for HbBv,
HbBv-DVS and Poly HbBv-DVS was determined on solutions of
the products at various concentrations, at 20 C, with a
W~SCOR 4400 oncometer calibrated with WESCO~ AC-010 Waker
Manometer and 5% (w/v) albumin solution. The ef~ect of the
hemoglobin concentration on the oncotic pressure is pres-
ented in Fig. 9. Virtually the sam2 behaviour is o~tainedfor HbBv and HbBv-DVS. To attain physiological oncotic
pressures, (COP approximately 25 mm Hg3 both the~e deriva-
tives had to be diluted to 7 g/dl, compared to the inter-
molecularly-crosslinked Poly HbBv-DVS which was iso-oncotic
with plasma at 14 g/dl. The relationship between oncotic
pressure and hemoglobin concentration was non linear and
COP values for HbBv and HbBv-DVS were increased approxi-
mately three-fold with respeck to those of Poly HbBv-~VS at
similar concentration~.
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~ESUI.~S
PURI~ICATION AND CHEMICAL CHh~ACTERI~TION OF HbBv-DVS
Fig. 1 presents the ion exchange chromatography
on a DEAE-Sephadex column of HbBv reacted anaerobically
with ~VS. The two ~ractions are nearly equal, each repre-
senting ~50% of the total protein. The fast peak, with
identical retention time to that of pure HbBv, represents
the unmodified hemoglobin. The slower peak is pure HbBv-
DVS. These results are consistent with an electrophoretic
analysis of the reaction mixture. The analysis on cellu-
lose acetate strips indicates that one of the fractions
gave a sharp band with a mobility identical to that of
untreated HbBv, while the protein eluting in the other
peak, HbBv-DVS, gave a sharp band with a higher mobility
(Fig. 2ajib~. HP-GPC of the slower ion-exchange chromatog-
raphy peak shows identical elution time with that of native
HbBv proving that HbBv-DVS is an intramolecularly-modified
monomeric (64 kDa) hemoglobin derivatives (Fig~ 3a, b).
Increasing the reaction t.ime from 24 to 48 hour~,
can result in up to 80-gO~ of HbBv-DVS~ A 50% increase in
the mol.ar ratio between DVS and HbBv can also provide
similar yields. The problem, however, is that under these
conditions there is a concomitant synthesis of small
amounts o~ a dimeric (128 kDa) derivative (Fig. 3b). This
situation is also illustrated in the electrophoretic
analysis of a ~8 hour reaction mixture (Fig. 2c). While
the band corresponding to the native HbBv virtually disap-
peared showing that most of the hemoglobin was modified,
the high mobility HbBv-DVS band is quite di~Eused, which is
probably due to the polymerization.
Fig. 5 presents typical results of SDS-PAGE. The
electrophoretic pattern of pure HbBv-DVS shows a single
band with a mobility corresponding to molecular mass of 16
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kDa (FigO 5C)~ For a 48 hour reaction mixture at relative-
ly high protein loading, an additional faink band corre-
sponding to a molecular mass twice that of the major band
component can be observed (Fig. 5b). When loading small
amounts of proteins on the gels it is sometimes possible to
obtain separation of the ~ and B chains. In Fig. 5E there
can be seen the ~ and B chain bands for the native HbBv.
In Fig. 5g there is a similar pattern for the modified
~bBv-DVS.
PURIFICATION AND CHEMICAL CHARACTERIZATION OF POLY HbBv-DVS
HP-GPC o~ typical Poly HbBv~DVS preparations is
pr~sented in Fig. 4. For a 24 hour reaction mixture the
amounts of the modified monomeric hemoglobin (HbBv-DVS),
and the modi~ied polymerized hemoglobin (poly HbBv-DVS),
are nearly equal. The isolated Poly HbBv-DVS produced
under these ~onditions had a molecular mass range ~rom 130
to about 500 k~a with a weight-average molecular weight, M~
~200,000 (Fig. 6a). For a 72 hour reactisn mixture the
yield of Poly Hb~v-DVS in this case is from 130 to well
above 500 kDa (Fig. 4b).
Cellulose acetate electrophoresis of isolated
Poly Hb~v~DVS, obtained from a 24 hour reaction mixture, is
shown in Fig. 2d. There is only one very di~ferent band
with a mean mobility much higher than that of HbBv or even
HbBv-DVS.
Fig. 5d shows the SDS-PAGE pattern of Poly HbBv-
DVS isolated from a 24 hour reaction mixture. One sees
bands corresponding to a molecular mass of 16 kDa and
multiples thereo~, thus indicating the production of a
mixture o~ modi~ied polymerized hemoglobins. llhe slowest
HP-GPC peak isolated ~rom the same reaction mixture
exhibits one band corresponding to a molecular mass of 16
kDa. This ~and is identified with the nonpolymerized HbBv-
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DVS (Fig. 5e and 6a).
OXYGEN-BINDING PROPERTIES
The ~xygen equilibria of Hb~v, HbBv-DVS and Poly
~bBv-DVS were studied in the pH range 5.8-9~0. The effect
of pH on the oxygen a~finity is presented in Fig. 6. one
can see that while near p~ ~.0 the P50 values for all three
materials are similar (~75 mm Hb)~ they vary at higher pH
values. At pH 9.0, for example, the P50 value for native
HbBv is 12 mm Hg while those for HbBv-DVS and Poly HbBv-DVS
are 31 and 41 respectively.
Figure 7 presents the effect of temperature on
the oxygell affinity of HbBv, HbBv-DVS and Poly HbBv-DVS
solutions at pH 7.40 and shows that both HbBv-DV5 and Poly
HbBv-DVS still exhibited P5~ values of about 20 mm Hg at the
low temperature of 15 C, by contrast with only 7 mm Hg for
native ~ovine hemoglobin.
2~
~ comparison of the oxygen unloading properties
at 15 C and 37 C of products acaording to the present
invention to other human and bovine oxygen carriers is
provided in Table I. From this data it i8 seen that at
15 C the oxygen-unloading of HbBv-DVS (~ = 20%) and Poly
HbBv-DVS (~ = 22%) are comparable to that of human blood at
37C (~ = 25%). Thus under hypothermic conditions Hb
derivatives according to the pres~nt invention display very
desirable oxygen-delivering capacities.
INTRAVASCULA~ R~T~NTION IN T~
Clearance of both HbBv and HbBv-DVS from the
circulation is presented in the top portion of Fig. 8.
With dos~s yielding initial hemoglobin plasma contents of
~10 mg/ml, HbBv-DVS showed a vascular half-life of 100 ~ 10
min (n=3 rats), which is not significantly dif~erent from
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that of HbBv (~0 + l0 min, n=3 rats). Poly HbBv-DVS, on
the other hand, displayed a much longer half-life of ~4.5
hours. A lower dose of the same material exhibited a
similar curve but resulted in a slightly shorter half-life
of 4.0 hours (Fig. 8/ bottom).
VISCOSITY STUDIES
Absolute viscosities at 15 C and 37 C for concen-
trations of 7.0 and 14.0 g/dl were determined for HbBv,HbBY-D~S and Poly HbBv DVS. The viscosities of HbBv-~VS
solutions were found to be identical to those of HbBv
solutiGns at the same temperatures and concentrations. The
viscosities of HbBv-DVS solutions, at any measured set of
temperatures and concentrations, are much lower than that
of human whole blood at 37 C. At 15 c, the viscosity of a
13 g/dl Poly HbBv-~VS solution i5 lower than that of human
whole blood. The viscosity of a 14.0 g/dl Poly HbBv-~VS
solution at 15C is only slightly higher than that of human
whole blood at 37 C. These viscosity properties indicate
that these hemoglobin derivatives are likely to have
beneficial effects when used to preserve isolated organs -
a situ~tion that requires perfusion of a constricted
microvasculature under hypothermic conditions.
One symmetrical peak in ion-exchange chromatogra-
phy and one sharp band in cellulose acetate electrophoresis
indicate a well-deEined molecule. The absence of any 32
kDa band from the SDS-PAGE pattern proves the lack of
intramolecular crosslinkage, while the single-peak HP-&PC
demonstrates the absence of intermolecular crosslinkage.
The introduction of a molecule with multiple
negative charges into the B-cleft of human or bovine
hemoglobins could generate strong electrostatic interac-
tions, resulting in a pseudo-crosslinkage, that confers
stability to the tetrameric structure of hemoglobin. Such
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tetrameric pseudo-crosslinkage is characterized by half~
life intravascular retention time increased four to five-
fold with respect to that o~ normal hemoglobin [5,6]. The
similar rekention times o~ the native and modified hemoglo-
bins in the pre~ent investigation rule out ~he possi~ilityfor such pseudo-crosslinkage in HbBv-DVS.
At a 15% concentration of HbBv-DVS and relatively
high molar ratio of ~VS/HbBv-tetramer it was possible to
anaerobically polymerize the modified hemoglobin. From HP-
GPC studies it was determined that 50-80% of the starting
material was polymerized and that the product, Poly HbBv-
DVS was not homogeneous with respect to the molecular
weight. Consistent with these ohservations, the SDS-PAGE
analysis gave bands with mobilities corresponding to
molecular masses of 16 kDa and multiples thereof. The
absence of a 32 k~a band in the slowest HP-GPC peak iso-
lated from a 24 hour reaction mixture (Fig. 5e) demon-
strates the lack o~ intramolecular arosslinkage, even at
the high DVS concentration needed for the polymerization,
and strongly suggests that Poly HbBv-DVS does not contain
intramolecular crosslinkages as part of its structure.
This conclusion is con~istent with the very low oXygen
a~inity and methemoglobin content of this material.
REFERENC~S
1. De Venuto, F., Zuck, T.F., Zegna, A.I. and
Moores, W.Y. (1977) J. Lab. Clin. Med. 89, 509-
516.
2. Shih, T.B., Jones, R.T. and Johnson, C.5. (1982)
Hemoglobin 6, 153-167.
3. Gebott, M.D. and Peck, J.M. (1978) Beckman Micro-
zone Electrophoresis Manual, Chapter 8A, Beckman
Instruments Inc., Fullerton, U.5.A.
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~7~2
- 22 -
4. Weber, K., Pringle, J.R. and Oshorn, M. (1972) in
MethQds in Enzymology (Hirs, C.H.W. and
Timashef~, S.N., eds.~, Vol. 26, pp, 3-27, Aca-
demic Press, London.
5. Bucci, Eo~ Razynska, A., Urbaitis, B. ~nd
Fronticelli, C. (1989) J. Biol. Chem. 264, 6191-
61950
~O Fronticellil C., Bucci, E. ~azyn ka, A.,
Sznajder, J., Urbaitis, B. and Gryczynski, Z.
(1990) Eur. J. Biochem. 193, 331-336.
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