Artificial Cells, Nanomedicine, and Biotechnology

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Extraction of superoxide dismutase, catalase,

and carbonic anhydrase from stroma-free
red blood cell hemolysate for the preparation
of the nanobiotechnological complex of
polyhemoglobin–superoxide dismutase–catalase–
carbonic anhydrase

C. Guo, M. Gynn & T. M. S. Chang

To cite this article: C. Guo, M. Gynn & T. M. S. Chang (2015) Extraction of superoxide
dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate
for the preparation of the nanobiotechnological complex of polyhemoglobin–superoxide
dismutase–catalase–carbonic anhydrase, Artificial Cells, Nanomedicine, and Biotechnology,
43:3, 157-162, DOI: 10.3109/21691401.2015.1035479

To link to this article: https://doi.org/10.3109/21691401.2015.1035479

Published online: 11 May 2015.

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Artificial Cells, Nanomedicine, and Biotechnology, 2015; 43: 157–162
Copyright © 2015 Informa Healthcare USA, Inc.
ISSN: 2169-1401 print / 2169-141X online
DOI: 10.3109/21691401.2015.1035479

Extraction of superoxide dismutase, catalase, and carbonic anhydrase

from stroma-free red blood cell hemolysate for the preparation of the
nanobiotechnological complex of polyhemoglobin–superoxide
dismutase–catalase–carbonic anhydrase
C. Guo, M. Gynn & T. M. S. Chang

Artificial Cells and Organs Research Centre, Departments of Physiology, Medicine and Biomedical Engineering,
Faculty of Medicine, McGill University, Montreal, Quebec, Canada

in the control group. The investigators propose that it has

Abstract
potential for use in treating fatal hemorrhagic shock when
We report a novel method to simultaneously extract superoxide
donor blood is not available (Moore et al. 2009).
dismutase (SOD), catalase (CAT), and carbonic anhydrase
The normal red blood cell (RBC) has three main func-
(CA) from the same sample of red blood cells (RBCs). This
tions: delivery of oxygen to tissues and cells; removal of oxy-
avoids the need to use expensive commercial enzymes, thus
gen radicals; and transport of carbon dioxide from tissues
enabling a cost-effective process for large-scale production of
to the lungs (Chang 2007). Ischemia/reperfusion injuries
a nanobiotechnological polyHb–SOD–CAT–CA complex, with
may occur in severe hemorrhagic shock, due to the produc-
enhancement of all three red blood cell functions. An optimal
tion of oxygen radicals during reperfusion with transfusion
concentration of phosphate buffer for ethanol–chloroform
(D’Agnillo and Chang 1998, Alayash 2004). The original
treatment results in good recovery of CAT, SOD, and CA after
blood substitutes were prepared from a RBC-stroma-free
extraction. Different concentrations of the enzymes can be used
hemolysate (SFHb) that contains all the red blood cell
to enhance the activity of polyHb–SOD–CAT–CA to 2, 4, or 6
enzymes, namely superoxide dismutase (SOD), catalase
times that of RBC.
(CAT), and carbonic anhydrase (CA) (Chang 1964, 1971).
Keywords: antioxidant, blood substitutes, carbonic anhydrase, In order to enhance the antioxidant enzyme activities, we
catalase, enzyme extraction, erythrocytes, nanobiotechnology, have added higher concentrations of the enzymes SOD and
nanomedicine, oxygen carrier, polyhemoglobin, stroma-free CAT, to prepare PolyHb–SOD–CAT (D’Agnillo and Chang
hemolysate, superoxide dismutase 1998). This has prevented ischemia/reperfusion in a murine
model of hemorrhagic shock stroke (Powanda and Chang
2002). In hemorrhagic shock, the mortality and the severity
of myocardial ischemia are related to the elevated intracel-
Introduction
lular pCO2 (Sims et al. 2001). The increase of intracellular
Polyhemoglobin (polyHb) is a first-generation blood sub- carbon dioxide level may lead to acidosis, malfunction of
stitute prepared by the basic method of crosslinking the the central nervous system, and even death (Geers and Gros
hemoglobin molecules with glutaraldehyde (Chang 1971). 2000). The RBC enzyme CA is the most important compo-
This basic principle was later developed independently by nent for the transport of CO2 (Geers and Gros 2000, Arthurs
two U.S. groups and tested in clinical trials (Jahr et al. 2008, and Sudhakar 2005). This fact led this laboratory to prepare
Moore et al. 2009, Kim and Greenburg 2014). Bovine polyHb an even more up-to-date nanobiotechnological complex of
has been approved for use in patients in South Africa and polySFHb–SOD–CAT–CA, with higher concentrations of all
Russia (Kim and Greenburg 2014). U.S. clinical trials on three enzymes than those normally present in RBCs (Bian
ambulance hemorrhagic shock patients have shown that et al. 2012, Bian and Chang 2015). This polySFHb–SOD–
human polyHb can be given on the spot without blood CAT–CA complex with enhanced RBC enzyme activities
typing, and can delay the need for blood transfusion by 12 has been tested in a murine model of 90-min hemorrhagic
hours, while the saline control group requires blood trans- shock with loss of 2/3rds of blood volume. The result shows
fusion within 1 hour (Moore et al. 2009). The percentage of that it is more effective than whole blood in lowering the
non-fatal cardiac side-effects was 3%, compared with 0.6% elevated intracellular pCO2, preventing cardiac side-effects,

Correspondence: Professor T. M. S. Chang, Artificial Cells and Organs Research Centre, Departments of Physiology, Medicine and Biomedical Engineering,
Faculty of Medicine, McGill University, Montreal, Quebec, Canada. E-mail: artcell.med@mcgill.ca
(Received 20 January 2015; accepted 6 March 2015)

157
158 C. Guo et al.

and preventing ischemia/reperfusion injuries in the liver, ultrafiltration disc with 300 kDa cut-off were purchased from
intestine, and heart (Bian and Chang 2015). Millipore. All other experimental and analytical reagents
Stroma-free hemolysate (SFHb) consists of the RBC con- were purchased from Sigma.
tent with the membrane removed. This contains hemoglo-
bin and RBC enzymes including CAT, SOD, and CA. SFHb Preparation of SFHb
containing these enzymes has been used to prepare the The method was based on that of Chang (2007). Briefly, fresh
earlier blood substitutes (Chang 1964, 1971). However, even heparinized bovine blood was centrifuged at 4,500 g for 30
RBCs with normal CAT, SOD, and CA enzyme activities are min at 4°C. The plasma supernatant containing white blood
not sufficient to prevent irreversible shock in serious sus- cells and platelets was aspirated carefully on ice, leaving the
tained hemorrhagic shock (Kim and Greenburg 2014, Bian red blood cells (RBCs). The RBCs were washed with 0.9 M
and Chang 2015). We therefore successfully prepared the sodium chloride three times. They were then lysed by adding
polySFHb–SOD–CAT–CA complex by crosslinking SFHb with 2 times the volume of hypotonic sodium phosphate buffer
additional CAT, SOD, and CA (Bian, Rong and Chang 2012, (12.5 mM, pH 7.4) to 1 volume of RBCs. The solution was
Bian and Chang 2015). However, commercial CAT, SOD, mixed by repeated inversion and swirling, then left standing
and CA are too expensive to scale-up production towards for 30 min at 4°C. A volume of 0.5 ml of cold toluene was
potential clinical use. Gu and Chang (2009) have extracted added to the mixture. After shaking vigorously, the superna-
SOD and CAT from SFHb. Ethanol–chloroform was used to tant was allowed to stand for 3 hours at 4°C. The upper layer
denature the hemoglobin, followed by acetone precipitation of the mixture, which contained toluene, stromal lipid, and
to purify and precipitate the SOD. CA can also be extracted cellular debris was removed by aspiration. The toluene step
by the method of ethanol–chloroform treatment and ace- was repeated one more time, and the remaining solution
tone precipitation (da Costa Ores et al. 2012). However, this was centrifuged at 16 000 g for 2 hours at 4°C. The superna-
method results in the inactivation of CAT. Thus CAT had to tant was then filtrated by a filter paper. Toluene was removed
be precipitated separately by adding ammonium sulfate by vacuum. The stroma-free hemoglobin solution was mixed
(Gu and Chang 2009). The effect of buffer on the stability of by stirring, and distributed into 15 ml centrifugal tubes and
proteins has been studied by Ugwu and Apte (2004). They stored at ⫺ 80°C.
have shown that a phosphate buffer at a pH of 7.4 promotes
the conformational stability of enzymes. Therefore, we use Quantitative determination of Hb concentration
potassium phosphate buffer (PPB) to protect CAT during The concentration of Hb was colorimetrically determined by
ethanol–chloroform treatment in the present study. Extract- allowing the samples to react with Drabkin’s reagent (Sigma-
ing individual enzymes from different batches of SFHb is Aldrich). After 12 μl of sample was added to 3 ml of Drabkin’s
neither efficient nor cost effective. In the present study, we reagent in a cuvette, the solution was allowed to stand for
have designed an optimal method for extracting all three 15 min at room temperature, avoiding light. The concentra-
enzymes simultaneously from the same sample of SFHb. tion of cyanmethemoglobin in the solution was measured by
In this study, we have successfully established the method spectrophotometry at 540 nm.
to extract CAT, SOD, and CA from blood and crosslink them
with SFHb to prepare PolySFHb–SOD–CAT–CA. The use of Determination of CAT activity
different concentrations of PPB for the extraction was ana- The established method has been described in the study
lyzed in detail. As a result, we have been able to establish paper by Zhu and Chang [21]. The SFHb and polySFHb
the optimal concentration of PPB for the extraction of CAT samples were diluted at a ratio of 1:200 with PPB (50 mM,
together with SOD and CA. When adding 3 M PPB to SFHb, pH 7.0) to a total volume of 5 ml; while the SFHb mixed with
CAT activity can be retained in the Hb-free supernatant extracted enzymes and polySFHb–SOD–CAT–CA samples
after ethanol–chloroform treatment; SOD and CA can also was diluted at a ratio of 1:1000. The H2O2 solution was pre-
be obtained simultaneously. Finally, the extracted enzymes pared by adding 40.6 μl of 30% H2O2 solution to 9 ml of PPB
would be purified and concentrated, followed by crosslinking (50 mM, pH 7.0). A UV spectrophotometer at 240 nm was
them with SFHb to generate polySFHb–SOD–CAT–CA. Our used to measure the rate of elimination of H2O2 within 15
results show that our modified ethanol–chloroform treat- seconds. For the blank, 1 ml of diluted sample and 0.5 ml of
ment can maintain CAT, SOD, and CA activities with high buffer were mixed. Then, 1 ml of diluted sample mixed with
recovery yields. The method we have established is expected 0.5 ml of H2O2 solution was applied, to determine the CAT
to cut down the costs of scale-up production compared with activity in the sample.
the costs incurred using commercial enzymes.
Determination of SOD activity
The SOD activity assay measures the decrease of cytochrome
Materials and methods
c by superoxide, observed using a UV spectrophotometer at
Materials 550 nm, within 50 seconds [11]. The SFHb and polySFHb
Stroma-free hemoglobin (SFHb) was prepared from bovine samples were diluted 20-fold with PPB (0.1 M, pH 7.4), while
blood purchased from the McDonald Campus Cattle Com- the SFHb mixed with extracted enzymes and polySFHb–
plex at McGill University (Sainte-Anne-de-Bellevue, Canada). SOD–CAT–CA samples were diluted 100-fold. The reaction
Both the Amicon Ultra-15 Centrifugal Filter Unit with buffer prepared consists of potassium phosphate (50 mM,
Ultracel-10 membrane, and the Biomax polyethersulfone pH 7.8), EDTA (10–4 M), xanthine (5 ⫻ 10–5 M), cytochrome
 Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate 159

c (10–5 M), and catalase (500 U/ml). Xanthine oxidase was SFHb. The concentration of phosphate buffer had no effect
diluted with 0.154 M NaCl to 6 U/ml. The mixture of 1.45 ml on the recovery yields of SOD and CA.
of buffer and 25 μl of diluted sample or buffer was applied Based on these results, we used 3M for the final procedure
as blank, and 25 μl of xanthine oxidase (6 U/ml) was then of enzyme extraction, as described in the next section.
added to initiate the reaction.
Final method established for the extraction and
Determination of CA activity concentration of enzymes from stroma-free hemolysate
The procedure for CA measurement was based on the A volume of 20 ml of SFHb is added to a beaker on ice, fol-
method established by Bian in 2012 [17]. Tris buffer (20 mM, lowed by 4 ml of 3M PPB (pH 7.4), to obtain a final concen-
pH 8.3) and diluted samples were prepared, as above. The tration of 9 g/dL SFHb. The mixture is then stirred using a
substrate of the reaction was prepared by bubbling CO2 magnetic stirrer at medium speed for 5 mins, on ice. Next,
through distilled water. Then, 10 μl of diluted sample or the 8 ml of cold ethanol–chloroform solution is added drop-
same amount of buffer as control was added into 3 ml of Tris wise at a speed of 1 drop/sec, and then stirred for 30 min at
buffer, while monitoring pH changes. To start the test, 2 ml of medium speed on ice. The solution is centrifuged for10mins
saturated CO2 solution was added. The time needed for the at 5000 rpm at 4°C. The supernatant layer (usually around
pH of the mixture to drop from 8.3 to 6.3 was recorded. 20 ml) is collected, which contains the recovered SOD, CA,
and CAT, with little or no Hb. The supernatant is transferred
Molecular weight distribution to a new centrifugal tube, and centrifuged at 5000 rpm for 10
A Sephacryl-300 HR column at a flow rate of 130 ml/hour mins at 4°C to remove any remaining precipitated Hb. Next,
was used for the analysis of molecular weight distribution. 12 ml of the supernatant is transferred to the Amicon Ultra-
The column was equilibrated with 0.9% NaCl buffer. After 15 Centrifugal 10 kDa filter unit and centrifuged for 10 min
200 μl of sample with Hb at a concentration of 9 g/dL was at 6000 rpm at 4°C. The remaining 8–10 ml of supernatant
loaded into the column, 0.9% NaCl buffer was used to elute is then added to the same filter unit containing the enzyme
the sample. The molecular weight distribution was recorded solution, and centrifuged at 6000 rpm for 15 mins at 4°C.
by a 280 nm UV detector at a velocity of 5 mm/min. The ethanol remaining in the extracted enzyme solution is
washed out by adding 5 ml of 0.2M Na•PO4 buffer (pH 7.4)
to the centrifugal unit, mixing the solution, and then cen-
Results
trifuging at 6000 rpm at 4°C for 15 min. The washing steps
Effects of Potassium Phosphate Buffer Concentrations on are repeated two more times. Finally, to minimize the final
Enzyme Extraction volume of concentrated enzymes, the enzyme solution is
For the study, we followed the procedure of enzyme extrac- washed with 2 ml of Na•PO4 buffer (pH 7.4) and centri-
tion from hemolysate, as described in the next section, except fuged at 6000 rpm at 4°C for 15 mins. The final volume of
that we used different concentrations of PPB (pH 7.4): concentrated enzymes should be approximately 300 μl.
1. SFHb 3ml ⫹ 4M PPB 0.6ml ⫹ ethanol–chloroform 1.2ml
Final method established for the preparation of
2. SFHb 3ml ⫹ 3M PPB 0.6ml ⫹ ethanol–chloroform 1.2ml
PolySFHb–SOD–CAT–CA using extracted enzymes
3. SFHb 3ml ⫹ 2M PPB 0.6ml ⫹ ethanol–chloroform 1.2ml
A volume of 4 ml of 9 g/dl SFHb is added to different amounts
4. SFHb 3ml ⫹ 1M PPB 0.6ml ⫹ ethanol–chloroform 1.2ml
of the concentrated enzymes, prepared as described above.
5. SFHb 3ml ⫹ 0.1M PPB 0.6ml ⫹ ethanol–chloroform 1.2ml
Then 102 μl of 4M NaCl is added to the solution and shaken
6. SFHb 3ml ⫹ 0.05M PPB 0.6ml ⫹ ethanol–chloroform 1.2ml
at 160 rpm at 4°C for 5 mins. Frozen glutaraldehyde (0.5M)
7. SFHb 3ml ⫹ water 0.6ml ⫹ ethanol–chloroform 1.2ml
is thawed on an ice-water mixture using ultrasonication for
8. SFHb 3ml ⫹ ethanol–chloroform 1ml
30–40 mins. The glutaraldehyde must be mixed evenly before
Table I summarizes the results obtained. The enzyme use. Without stopping the shaker, 162.3 μl of glutaraldehyde
activity in the SFHb was considered as 100%. For CAT, there is added dropwise in 4 equal aliquots at intervals of 15 mins,
was little or no enzyme recovered at the lower phosphate and shaken at 160 rpm for 24 hrs at 4°C. Next, 641.4 μl of 2M
buffer concentrations. Increase in the concentration of lysine (200:1 lysine: protein molar ratio) is added in 2 equal
phosphate ions resulted in increased recovery of CAT, with aliquots over 10 mins and shaken at 160 rpm at 4°C for 1
the highest yield at 3M PPB. The reasons for the more than hour, to quench. The solution is centrifuged at 8000 rpm at
100% apparent recovery of CAT activity will be explained in 4°C for 1 hour to remove any precipitate.
the Discussion. The recovery yields of SOD and CA were both The polySFHb was prepared following the same proce-
around 70–100%, compared with the activity recovered in dure as for the preparation of polySFHb–SOD–CAT–CA, but

Table I. Recovery yield of apparent activities of CAT, SOD, and CA after extraction using different concentrations of phosphate ions (see Discussion,
regarding the possible reasons for the higher recovery of CAT activity).
4M PPB 3M PPB 2M PPB 1M PPB 0.1M PPB 0.05M PPB Water Nothing
Final PO42 ⫺ Concentration (mM) 507.8 382.8 257.8 132.8 20.3 14.05 7.8 9.375
CAT recovery yield (%) 194 ⫾ 41 209 ⫾ 65 204 ⫾ 56 118 ⫾ 23 6⫾3 3⫾2 0 11 ⫾ 7
SOD recovery (%) 86 ⫾ 7 88 ⫾ 5 70 ⫾ 10 70 ⫾ 3 77 ⫾ 9 80 ⫾ 8 98 ⫾ 9 80 ⫾ 12
CA recovery (%) 80 ⫾ 10 102 ⫾ 5 74 ⫾ 7 76 ⫾ 5 84 ⫾ 2 84 ⫾ 7 95 ⫾ 4 93 ⫾ 10
160 C. Guo et al.

CATActivity
400000
Before crosslinking
350000
After crosslinking
300000
250000

U/gHb
200000
150000
100000
50000
0

2x

4x

6x
H
SF

A
+C

+C

+C
D

D
O

O
+S

+S

+S
AT

AT

AT
C

C
b+

b+

b+
H

H
SF

SF

SF
Figure 2. Comparison of CAT activities in different samples. Left to
right: SFHb before and after crosslinking; SFHb ⫹ CAT⫹ SOD ⫹
CA 2x (SFHb plus 2x extracted SOD, CAT, and CA) before and after
crosslinking; SFHb ⫹ CAT⫹ SOD ⫹ CA 4x (SFHb plus 4x extracted
SOD, CAT, and CA) before and after crosslinking; SFHb ⫹ CAT⫹ SOD
⫹ CA 6x (SFHb plus 6x extracted SOD, CAT, and CA) before and after
crosslinking.

products polySFHb–SOD–CAT–CA or polySFHb, most of

the molecules were in the ⬎ 669 KDa range (Figure 1A). For
SFHb, most of the molecules were in the 64KD hemoglobin
range (Figure 1B).

Determination of Enzyme Activities and Recovery of Yield

The CAT, SOD, and CA activities were measured for the fol-
lowing samples before and after crosslinking: SFHb, and
SFHb with concentrated enzymes added at 2x, 4x and 6x the
original SFHb enzyme concentrations. After purification of
the polymerized molecules with the 300 KDa ultrafiltration
disc, the enzyme activities of the complexes were measured
(Figures 2–4).
CAT activity in SFHb was found to be 53234 ⫾ 2945 U/g
Hb. After crosslinking into polySFHb, the catalase activity
was 20124 ⫾ 1053 U/g Hb). SFHb plus different amounts of
concentrated extracted enzymes before crosslinking and
after crosslinking also showed good recovery of CAT activi-
ties (Figure 2). For example, the CAT activity for SFHb with
2x extracted SOD, CAT, and CA was 153046 ⫾ 2207 U/g Hb
before crosslinking, and 93034 ⫾ 3537 U/g Hb after cross-
linking. With 4x extracted SOD, CAT, and CA, the activ-
ity was 160320 ⫾ 2205 U/g Hb) before crosslinking, and
Figure 1. (A) The molecular weight distribution of polySFHb–SOD– 135909 ⫾ 3918 U/g Hb after crosslinking. With 6x extracted
CAT–CA, or polySFHb (B) The molecular weight distribution of SFHb. SOD, CAT, and CA, the activity was 326989 ⫾ 7385 U/g
Hb) before crosslinking, and 273919 ⫾ 7379 U/g Hb after
without adding the extracted enzymes. The supernatant of crosslinking.
the crosslinked sample was around 4–5 ml. The sample was SOD activity in SFHb was found to be 1642 ⫾ 48 U/ml. After
purified using a 300 KDa ultrafiltration disc with positive air crosslinking into polySFHb, the SOD activity was 1114 ⫾ 33
pressure. After purification, the Hb concentrations of the U/ml (Figure 3). SFHb plus different amounts of concen-
crosslinked samples were tested and adjusted to 9g/dL with trated extracted enzymes before crosslinking and after
0.1M PPB (pH 7.4). crosslinking also showed good recovery of SOD activities
(Figure 4). For example, the SOD activity for SFHb with 2x
Molecular Weight Distribution extracted SOD, CAT, and CA was 3567 ⫾ 100 U/g Hb) before
The molecular weight distributions of polySFHb–SOD–CAT– crosslinking, and 2846 ⫾ 68 U/g Hb after crosslinking. For 4x
CA, polySFHb, and SFHb were analyzed with Sephacryl extracted SOD, CAT and CA, the activity was 4526 ⫾ 65 U/g
S-300 gel column chromatography. A volume of 200 μl of Hb) before crosslinking and 4149 ⫾ 69 U/g Hb after cross-
supernatant was loaded on the column. In the polymerized linking. For 6x extracted SOD, CAT, and CA, the activity was
 Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate 161

SOD Activity CA Activity

12000 160000
Before crosslinking Before crosslinking
10000 After crosslinking 140000 After crosslinking
120000
8000
100000
U/gHb

U/gHb
6000 80000
4000 60000
40000
2000
20000
0 0

2x

4x

6x
b

2x

4x

6x

H
H

SF
SF

A
A

+C

+C

+C
+C

+C

+C

D
D

O
O

+S

+S

+S
+S

+S

+S

AT

AT

AT
AT

AT

AT

C
C

b+

b+

b+
b+

b+

b+

H
H

SF

SF

SF
SF

SF

SF

Figure 3. Comparison of SOD activities in different samples. Left to right: Figure 4. Comparison of CA activities in different samples. Left to right:
SFHb before and after crosslinking; SFHb⫹ CAT⫹ SOD⫹ CA 2x (SFHb SFHb before and after crosslinking; SFHb⫹ CAT⫹ SOD⫹ CA 2x (SFHb
plus 2x extracted SOD, CAT, and CA) before and after crosslinking; plus 2x extracted SOD, CAT, and CA) before and after crosslinking;
SFHb⫹ CAT⫹ SOD⫹ CA 4x (SFHb plus 4x extracted SOD, CAT, and CA) SFHb⫹ CAT⫹ SOD⫹ CA 4x (SFHb plus 4x extracted SOD, CAT, and CA)
before and after crosslinking; SFHb⫹ CAT⫹ SOD⫹ CA 6x (SFHb plus before and after crosslinking; SFHb⫹ CAT⫹ SOD⫹ CA 6x (SFHb plus
6x extracted SOD, CAT, and CA) before and after crosslinking. 6x extracted SOD, CAT, and CA) before and after crosslinking.

9124 ⫾ 338 U/ml before crosslinking and 6712 ⫾ 181 U/ml enough ions to form a hydration shield. Interestingly, the
after crosslinking. phosphate buffer was reported to destabilize the hemoglo-
CA activity in SFHb was 16725 ⫾ 411 U/g Hb. After cross- bin by binding to the 2,3-bisphosphoglycerate (2,3-BPG)
linking into polySFHb, the CA activity was 18638 ⫾ 711 U/g site (Ugwu and Apte 2004).Therefore, the phosphate buffer
Hb (Figure 4). SFHb plus different amounts of concentrated at a pH of 7.4 can protect CAT and simultaneously promote
extracted enzymes before crosslinking and after crosslinking hemoglobin precipitation.
also showed good recovery of CA activities (Figure 4). For Why is it that compared with the original enzyme activi-
example, the CA activity for SFHb with 2x extracted SOD, ties assayed in SFHb, the apparent CAT activities increase
CAT, and CA was 25722 ⫾ 758 U/g Hb) before crosslinking in the extracted enzyme solution after ethanol–chloroform
and 24547 ⫾ 574 U/g Hb after crosslinking. For 4x extracted treatment? There are a number of possible explanations.
SOD, CAT, and CA, the activity was 62776 ⫾ 680 U/g Hb Firstly, hemoglobin has peroxidase-like activity (Paco
before crosslinking and 54707 ⫾ 400 U/g Hb after crosslink- et al. 2009). We have done the following test on the effect
ing. For 6x extracted SOD, CAT, and CA, the activity was of hemoglobin on CAT activity. When CAT is added to the
140243 ⫾ 2911 U/g Hb before crosslinking and 101869 ⫾ 4248 SFHb, the total CAT activity of the mixture is lower than the
U/g Hb after crosslinking. summation of CAT activities in the extracted enzymes and
the SFHb (Figure 2). Therefore, it is likely that hemoglobin
may suppress the CAT as a competitive inhibitor. Secondly,
Discussion and conclusion
ethanol has been found to increase the CAT activity (Mag-
PPB at a pH of 7.4 can prevent CAT from aggregation after ner and Klibanov 1995). We compared the activity of pure
ethanol–chloroform treatment. It is likely that the ion CAT and CAT-ethanol mixture. The ethanol concentration in
strength and pH value of the buffer contribute to the aggre- this test is equal to the concentration during the extraction
gation of hemoglobin and the stability of the three enzymes. procedure. The result shows that 12.5% ethanol can increase
The molecular weights (MW) of hemoglobin, CAT, SOD, and CAT activity by 1.4 times. Thirdly, hemoglobin can be con-
CA are 68 KDa, 250 KDa, 32.5 KDa, and 29 KDa respectively. verted to choleglobin by coupling with ascorbic acid in the
The isoelectric points (pI) of hemoglobin, CAT, SOD, and CA presence of oxygen (Miller 1958). Choleglobin formation
are 6.8, 5.4, 4.95, and 5.9 respectively. As we can see, only can even increase when RBCs are hemolyzed (Foulkes and
the pI of hemoglobin is close to the pH of the solution at 7.4; Lemberg 1949). The hemoglobin–ascorbic acid complex is
thus hemoglobin tends to aggregate when adding organic the intermediary substance in choleglobin formation and
solvents that have reduced dielectric strength (Crowell et al. acts as a hydrogen peroxide donor (Miller 1958). The assay
2013). In spite of its low pI value, CAT also aggregates after of CAT is based on the measurement of H2O2 left after the
ethanol–chloroform treatment, possibly due to its large reaction of samples with H2O2 solution.
size and the ion pairing effect. However, when increasing In conclusion, we have devised a novel method to simul-
the final concentration of phosphate ions from 20.3 mM to taneously extract all three enzymes, CAT, SOD, and CA,
257.8 mM, the improvement in CAT recovery yield can be from RBC hemolysate (SFHb). When using bovine RBC, the
observed (Table I). The phosphate buffer may be able to unlimited and inexpensive source of bovine blood would
protect CAT because a large amount of ions migrate to the be much less costly than commercial enzymes to scale-up
charged surface residues to generate a solvent layer, pre- the production of blood substitute. Earlier studies from this
venting the aggregation of CAT; the pI of hemoglobin is close laboratory have shown that bovine PolySFHb containing the
to the pH of solution, therefore hemoglobin cannot attract bovine RBC enzymes did not show immunological reactions
162 C. Guo et al.

when tested in rats (Zhu et al. 2010). We shall investigate Chang TM. 2012. From artificial red blood cells, oxygen carriers, and
oxygen therapeutics to artificial cells, nanomedicine, and beyond.
the effects of using higher concentrations of enzymes. If
Artif Cells Blood Substit Immobil Biotechnol. 40:197–199.
there is an immunological problem, we can also use our Chang TM. 2013. Selected topics in nanomedicine. In: Chang TMS, Ed.
novel method for the extraction of human hemoglobin and Artificial cells that started nanomedicine. World Science Publisher/
Imperial College Press, pp. 1–21.
enzymes from human placental RBCs (cord RBCs) for the
Crowell AM, Wall MJ, Doucette AA . 2013. Maximizing recovery of
preparation of polySFHb–SOD–CAT–CA. water-soluble proteins through acetone precipitation. Anal Chim
Acta. 796:48–54.
da Costa Ores J, Sala L , Cerveira GP, Kalil SJ. 2012. Purification of
Acknowledgements carbonic anhydrase from bovine erythrocytes and its applica-
tion in the enzymic capture of carbon dioxide. Chemosphere.
This laboratory is not connected with any commercial orga- 88:255–259.
D’Agnillo F, Chang TM. 1998. Polyhemoglobin-superoxide dismutase-
nizations. The research support for the study reported here
catalase as a blood substitute with antioxidant properties. Nat
comes from the joint program of the Canadian Blood Ser- Biotechnol. 16:667–671.
vice/Canadian Institutes of Health Research that requires Foulkes E, Lemberg R. 1949. The formation of choleglobin and the role
of catalase in the erythrocyte. Proceedings of the Royal Society of
the authors to state that: “The opnions expressed in this
London. Series B-Biological Sciences. 136:435–448.
article are the opnions of the investigators of this paper and Geers C, Gros G. 2000. Carbon dioxide transport and carbonic anhy-
not neccssarily those of the Canadian Government nor of the drase in blood and muscle. Physiol Rev. 80:681–715.
Gu J, Chang TM. 2009. Extraction of erythrocyte enzymes for the prep-
Canada Blood Service”.
aration of polyhemoglobin-catalase-superoxide dismutase. Artif
Cells Blood Substit Immobil Biotechnol. 37:69–77.
Jahr JS, Mackenzie C, Pearce LB, Pitman A , Greenburg AG. 2008.
Declaration of interest HBOC-201 as an alternative to blood transfusion: efficacy and safety
evaluation in a multicenter phase III trial in elective orthopedic sur-
The authors report no declarations of interest. The authors gery. J Trauma. 64:1484–1497.
alone are responsible for the content and writing of the Kim HW, Greenburg AG (Eds.). 2014. Hemoglobin-based oxygen carri-
ers as red cell substitutes and oxygen therapeutics. Springer.
paper. Magner E, Klibanov AM. 1995. The oxidation of chiral alcohols
catalyzed by catalase in organic solvents. Biotechnology Bioeng.
46:175–179.
References Miller H. 1958. The relationship between catalase and haemoglobin in
human blood. Biochem J. 68:275–282.
Alayash AI. 2004. Redox biology of blood. Antioxid Redox Signal. Moore EE, Moore FA , Fabian TC, Bernard AC, Fulda GJ, Hoyt DB. 2009.
6:941–943. Human polymerized hemoglobin for the treatment of hemorrhagic
Arthurs G, Sudhakar M. 2005. Carbon dioxide transport. Continuing shock when blood is unavailable: the USA multicenter trial. J Am
Education in Anaesthesia. Critical Care & Pain. 5:207–210. Coll Surg. 208:1–13.
Bian Y, Rong Z, Chang TM. 2012. Polyhemoglobin-superoxide dis- Paco L, Galarneau A , Drone J, Fajula F, Bailly C, Pulvin S, Thomas D.
mutase-catalase-carbonic anhydrase: a novel biotechnology-based 2009. Catalase‐like activity of bovine met‐hemoglobin: interac-
blood substitute that transports both oxygen and carbon dioxide tion with the pseudo‐catalytic peroxidation of anthracene traces in
and also acts as an antioxidant. Artif Cells Blood Substit Immobil aqueous medium. Biotechnol J. 4:1460–1470.
Biotechnol. 40:28–37. Powanda DD, Chang TM. 2002. Cross-linked polyhemoglobin-super-
Bian Y, Chang TMS. 2015. A novel nanobiotherapeutic poly- oxide dismutase-catalase supplies oxygen without causing blood-
[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] brain barrier disruption or brain edema in a rat model of transient
with no cardiac toxicity for the resuscitation of a rat model with global brain ischemia-reperfusion. Artif Cells Blood Substit Immo-
90 minutes of sustained severe hemorrhagic shock with loss of 2/3 bil Biotechnol. 30:23–37.
blood volume. Artif Cells Nanomed Biotechnol. 43:1–9. Sims C, Seigne P, Menconi M, Monarca J, Barlow C, Pettit J, Puyana JC.
Chang TM. 1964. Semipermeable microcapsules. Science. 146:524–525. 2001. Skeletal muscle acidosis correlates with the severity of blood
Chang TM. 1971. Stabilisation of enzymes by microencapsulation with volume loss during shock and resuscitation. J Trauma. 51:1137–
a concentrated protein solution or by microencapsulation followed 1145; discussion 1145–1146.
by cross-linking with glutaraldehyde. Biochem Biophys Res Com- Ugwu SO, Apte SP. 2004. The effect of buffers on protein conforma-
mun. 44:1531–1536. tional stability. Pharm Technol. 28:86–109.
Chang TM. 2007. Artificial cells: Biotechnology, nanotechnology, blood Zhu H, Du Q, Chen C, Chang TM. 2010. The immunological properties
substitutes, regenerative medicine, bioencapsulation, cell/stem of stroma-free polyhemolysate containing catalase and superox-
cell therapy. World Scientific Publisher/Imperial College Press, p. ide dismutase activities prepared by polymerized bovine stroma-
455. Available at http://www.medicine.mcgill.ca/artcell/2007%20 free hemolysate. Artif Cells Blood Substit Immobil Biotechnol.
ebook%20artcell%20web.pdf. Accessed on April 2 2015. 38:57–63.