Journal of Biochemical and Biophysical Methods, 23 (1991) 53-66 © 1991 Elsevier Science Publishers B.V. 0165-022X/91/$03.50 ADONIS 0165022X9100079R
53
JBBM00888
Preparation of unaltered hemoglobin from human placentas for possible use in blood substitutes G. Fasan a, M. Grandgeorge
2 C. Vigneron
3 and E. Dellacherie 1
CNRS URA 494, ENSIC, Nancy, France, 2 Institut Mdrieux, Charbonni~res-les-Bains, France and 3 Centre Rdgional de Transfusion Sanguine, Vandoeuvre-les-Nancy, France
(Received 9 July 1990) (Revision received 15 December 1990) (Accepted 22 February 1991)
Summary Hemoglobin extracted from human placentas could be used as the basis of blood substitutes provided it could be prepared on a large scale with appropriate oxygen-binding properties. Unfortunately, the industrial conditions under which it is extracted, produce hemoglobin with high oxygen affinity and which is no longer influenced by the classical effectors. These characteristics were shown to be caused by a degradation of the c~-chain brought about by an arginine carboxypeptidase present in the placental tissues and leading to the disappearance of the C-terminal arginine residue. This carboxypeptidase which is released from the tissues during the process of crushing the frozen placentas, degrades the protein during the chromatographic purification procedure. The addition of an inhibitor of this carboxypeptidase (for example, arginine) as soon as the placentas are thawed and during the chromatographic process, makes it possible to obtain placental hemoglobin with oxygen-binding properties quite similar to those of HbA prepared from peripheral venous blood. Keywords: Placental hemoglobin; Placental carboxypeptidase; Large-scale chromatographic purification
Introduction Up to now, most of the hemoglobin-based blood substitutes described in the literature have been synthesized using human hemoglobin (HbA) obtained from Abbreviations: HbA, human adult hemoglobin; HbF, human foetal hemoglobin; HbP, placental hemoglobin purified by the industrial-scale procedure; Hbp, unpurified native placental hemoglobin; HbAp and HbFp, respectively, degraded adult hemoglobin and degraded fetal hemoglobin contained in HbP; IEF, isoelectric focusing; BHC, benzene hexacarboxylic acid; DPG, 2,3-diphosphoglyeerate. Correspondence address." Dr. E. Dellacherie, ENSIC, BP 451, 54001 Nancy Cedex, France.
54 gifts of peripheral venous blood [1]. However, it is also possible to obtain hemoglobin by extracting it from human placentas. Placental blood contains both HbA and H b F (foetal hemoglobin) and after large-scale purification following VOron's method [21 a hemoglobin mixture containing between 25 and 30% of H b F (compared with about 1% for peripheral venous blood) is obtained. Although H b F is known to possess oxygen-binding properties which differ from those of HbA, the use of placental hemoglobin for preparing oxygen-carriers can be considered, as the differences are not very important and as the concentration in H b F is not too high. However, the major proNems posed by the use of placental hemoglobin on a large scale are caused by the conditions under which it is extracted from the placentas and purified. Placentas are kept in frozen storage and to extract hemoglobin they have to be crushed before thawing. Then tissues and fibrinogen are removed by filtration and several steps of chromatography, precipitation and diafiltration are carried out on the resulting filtrate. Finally, a 10% hemoglobin solution is obtained with a yield of 35 1 per 100 kg of placenta. However, hemoglobin obtained in this way has a high oxygen affinity. Moreover, its cooperativity is much less than that of pure HbA and it is no longer sensitive to the classical effectors, making it quite unsuitable for blood substitution purposes. We have analyzed the structure of hemoglobin prepared on a large scale from human placentas and determined the reasons for the alterations observed. This study allowed us to propose some modifications in the large-scale extraction procedure in order to obtain hemoglobin with suitable properties. Materials and Methods Pure HbA was prepared frQm outdated Mood as previously described [3]. Benzene hexacarboxylic acid (BHC) was obtained from Aldrich (Belgium). Carboxypeptidase B, L-arginine and L-tyrosine were purchased from Sigma (U.S.A.). Model oligopeptides were obtained from Bachem (Switzerland). All the other reagents were obtained from Prolabo (France) or Merck (F.R.G.). Total hemoglobin was estimated by Drabkin's procedure [41, concentrations of methemoglobin were measured by the method of Evelyn and Malloy [5] and its reduction by dithionite was performed according to Zygmunt et al. [6]. 2,3-Diphosphoglycerate (DPG) was estimated by a classical method [7]. Placental plasma was prepared by pressing a freshly thawed placenta. Isoelectric focusing was performed in the presence of K C N (100 mg/1). The samples were applied to Phastgel IEF 5-8 and developed with the Phast system (Pharmacia, Sweden). Electrophoresis Of a, fi and ~, chains obtained under strongly dissociating conditions (6.5 M urea, p H 6.0) [8] was carried out with the Sebia system (France). Oxygen equilibrium curves were determined at 37°C in 69 mM Bistris buffer, p H 7.4, 0.14 M NaC1, 44 m M glucose with the Hemox Analyzer B (TCS, Southampton, U.S.A.).
Purification of placental hemoglobin by the industrial scale procedure (HbP) The different steps of this procedure are as follows:
55 ® thawing of crushed frozen placentas at 37°C in a solution containing ethanol (8%) and cellulose, which facilitates pressing • pressing in order to remove tissues and fibrinogen • acidification at p H 5.1 by acetic acid in order to precipitate the lipoproteins • diafiltration in 10 mM phosphate buffer, p H 5.25 (membrane cut-off: 10000) • chromatography on DEAE-Spherodex (IBF. France) in 50 m M phosphate buffer, p H 5.25 in order to remove albumin ® alcoholic precipitation 125% ethanol. 5°C) of immunoglobulins Pyrogenic products and group substances were removed by an additional chromatographic step, followed by diafiltration in 0.1 M NaC1. For large-scale purification of placental Hb in the presence of arginine, 150 kg of placentas were treated as described above but in the presence of arginine which was added in the thawing solution (14 g/l). Temperature was maintained close to 0°C during the whole process. The final chromatography was also carried out in the presence of arginine at 5 g/1.
Structural studies of HbP (placental Hb purified by the industrial-scale procedure) The analysis of H b P was carried out according to the following procedure: ///~ HbP-containing solution
Cation-exchange chromatogr~ HbA e
HbFe
Electrofocusing Electrophoresis Precipitation of H b P and analysis of c~-amino-acids of the supernatant
Precipitation of globins then cation-exchangechromatography
/
/
/ BF
O~p ~/~ Tryptic map
Trypsindigestion reverse-phaseHPLC Collecting of the fragments come out in the void volume
l Cation-exchange H P L C
~/~ Tryptic map
Trypsindigestion reverse-phaseHPLC Collecting of the fragments come out in the void volume
Cation-exchange HPLC
56
The amino-acids of the supernatant were analysed on a Beckman 7300 amino-acid analyser. All the separation steps were carried out following procedures described elsewhere [9]. The first cation-exchange chromatography (separation of H b A p and HbFp, degraded H b A and H b F contained in HbP) was performed according to Schroeder et al. [10] with carboxymethylcellulose (Whatman) with an increasing NaC1 gradient and in the presence of KCN. The c~ and fl globins of H b A e thus separated were prepared by the a c i d / a c e t o n method. They were separated by carboxymethylcellulose chromatography in 8 M urea and in the presence of mercaptoethanol [11]. Digestion with trypsin (treated with tosylphenylalanytchloromethane) was performed in 25 m M a m m o n i u m bicarbonate, p H 8, at ambient temperature using an enzyme/substrate weight ratio of 8 × 1 0 - 4 . The pepfide fragments were separated by reverse phase high-performance chromatography on a n Aquapore RP 300 column (25 × 0.46 cm; Brownlee Labs, U.S:A,) according to Wajcman [12] with an increasing gradient of water-acetonitrile-trifluoroacetic acid (A: 0.05% T F A in water; B: 50/50, acetonitrile/0.1% T F A in water). The last cation-exchange chromatographic step was carried out on a Mono S column (5 × 0.5 cm, Pharmacia) under 2 sets of conditions. In one experiment, the sotutes were eluted at p H 5.8 at a flow rate of 1.5 m l / m i n , with a gradient of A: 10 m M sodium malonate and B: 10 m M sodium malonate, 300 m M LiC1 (Fig. 5). In the second one, the p H was 3.8, the flow rate 1.5 m l / m i n and the gradient was constituted of A: 10 m M sodium formate and B: 10 m M sodium formate, 300 m M LiC1 (Fig. 6).
Results
Influence of placental plasma on the oxygen-binding properties of hemoglobin The oxygen-binding parameters of placental hemoglobin obtained by the largescale purification procedure described in Materials and Methods (HbP) are reported in TaMe 1. The effect on H b P of benzene hexacarboxylic acid (BHC), a strong effector of HbA, was also determined. Isoelectric focusing (IEF) of H b P is given in
TABLE 1 Oxygen-binding parameters of placental Hb purified according to the method described in Materials and Methods (HbP), determined at pH 7.4, 69 mM Bistris buffer, 0.14 M NaC1, 37°C, after reduction of methemog!obin by dithionite
/'5o (Torr) nSob
HbP
HbP + BHC a
HbA
H bA + BHC
2.0 1.1
2.5 0.9
13.5 2.6
42.5 2.6
a BHC = benzene hexacarboxylic acid; B H C / H b molar ratio = 15; b Hill coefficient at Ps0-
a
57
Fig. 1. IEF of a sample of H b P (!) compared to that of H b A (2).
Fig. 1 and shows 2 bands
corresponding
to species with isoelectric points lower than
that of HbA. F i r s t o f all, w e c h e c k e d
that the application
of the same purification
procedure
to
TABLE 2 Influence of placental plasma on the oxygen-binding parameters of H b A Storage medium
Storage time b (h)
DPG content c
Ps0 (Torr)
n 50
Adult blood hemolysate
Adult plasma
0 18 120
0.10
14.0 14.5 14.0
2.55 2.4 2.30
Adult blood hemolysate
Placental plasma a
18 120
0.55
15.0 9.5
2.20 1.40
Pure H b A 100 g / l
Placental plasma a
18 120
0.45
18 10
2.05 1.40
a Placental p l a s m a / H b A solution volume ratio = 1; b at 4°C; c D P G / H b molar ratio measured as described in the Materials and Methods. Precision 10%; other conditions and symbols as in Table 1.
58
H
degraded HbF
-,--/
H
degraded
HbA Fig, 2. IEF of samples of Hbp stored in placental plasma at 37°C. (1): 0 h: (2): 1 h; (5): 2 h; (6): 3 h; (7): 4 h: (8): 6 h; (4): 24 h: (3): pure HbA.
frozen total peripheral venous blood yielded HbA with oxygen-binding properties similar to those of H b A prepared by the classical method. On the other hand. we found that the oxygen-binding properties of a red cell hemolysate or of pure HbA stored for a long time at 4°C in a placenta1 plasma obtained from a freshly-crushed non-frozen placenta were modified as shown in Table 2, i.e. the Ps0 and the Hill coefficient both decreased strongly. Another experiment was carried out with a freshly frozen placenta.This placenta was rapidly crushed in an alcoholic solution (8% ethanol) and the crushed product was rapidly thawed and filtrated. Then the resulting filtrate was kept at 37°C for several hours. Fig. 2 shows the IEF of the sample after various terms of storage, tt can be seen that at r - 0. the hemoglobin contained in the mixture (Hbp) shows 2 bands, one corresponding to H b A and the other to HbF. After 24 h, both bands are shifted towards lower pH, which proves that the species have gained a more negative net electric charge. The oxygen-binding properties of Hbp were determined after 24 h of storage. The results are reported in Table 3 and show that whereas the Hbp contained in a fresh placenta has oxygen-binding parameters similar to those of HbA, its Ps0 at 37°C is drastically decreased after 24 h of storage at 37°C; it is no longer influenced by the presence of an effector, and the cooperativity has almost completely disappeared.
TABLE 3 Oxygen-binding properties of non-purified placental hemoglobin (Hbp) during storage at 37°C, compared to those of H b P
Hbp Hbp HbP
Storage
Ps0 (Tort)
time (h) at 3 7 ° C
N o BHC
In presence of B H C a
ns0
0 24
12.3 2.5 2.0
36.2 2.3 2.5
a 15 mol B H C / m o l Hb; other conditions and symbols as in Table 1.
2.5 1.2 0.9
59
Fig. 3. I E F of Hb A after incubation with carboxypeptidase B at 370C, 50 m M Tris buffer, p H 7.5, for 3 h (3) compared with HbP (1) and pure H b A (2),
Influence of carboxypeptidase B on HbA Pure H b A was incubated with carboxypeptidase B at 37°C, in 50 m M Tris buffer, p H 7.5, for 3 h. The resulting mixture was then examined by I E F and the result of this analysis is shown in Fig. 3 and compared with the I E F of pure H b A and of HbP. The P~0 of H b A after this treatment was 1.85 Torr and the ns0 was 1.2. Fig. 3 shows that, as already described [13,14], H b A is digested by carboxypeptidase B to give des(Arg 141a)-hemoglobin and that the corresponding band appears at the same position as one band of HbP, probably that of H b A p (the degraded H b A contained in the placental large-scale purified Hb), the other one being that of HbFp (degraded HbF). On the other hand, the oxygen affinity of carboxypeptidase-digested H b A (Ps0 = 1.85 Torr) and its Hill coefficient (ns0 = 1.2) are quite similar to those of HbP. These results, together with those shown in Table 2, thus seem to indicate that
TABL E 4 Influence of arginine on the oxygen-binding parameters of an adult blood hemolysate stored under different conditions Storage medium
Storage time c (days)
Ps0 (Torr)
n 50
Adult plasma
0 7 7
12 11.5 9.5
2.55 2.5 1.75
7
11.5
2.55
Placental plasma a Placental plasma a + arginine b
a Placental p l a s m a / h e m o l y s a t e volume ratio = 3; b concentration = 10 g / l ; e at 4°C; other conditions as in Table 1.
60
the placentas contain a type of B-carboxypeptidase which is responsible for the degradation observed in HbP. As arginine is known to be an inhibitor of this type of carboxypeptidase, this amino acid was added to a mixture of adult blood hemolysate and placental plasma, and the oxygen-binding parameters of the resulting sohition were measured after 7 days of storage at 4°C. The results are given in Table 4 and show that arginine impedes the deterioration of HbA, and specially that of its cooperativity, when the protein is stored in placental plasma.
_b ~HbA
5HbA Ta
40
20
20
U
40 Elutlon t l ~
Elutlon t i m
12
i l i
0
20
40
Elution i I ~
na;n
0 - -
--
--2~
40 ElvtJ~ tlme
Fig. 4. Tryptic maps of a and fl chains of pure HbA (a, b) and of H b A p (c, d) on a C s reverse phase (see Materials and Methods for the chromatograpNc conditions; the broken lines represent the variation of B concentration in A).
61
Analysis of HbP The structure of H b P obtained after the large-scale purification procedure was analysed by the method described in Materials and Methods. Fig. 4 shows the chromatograms of tryptic digests of a and fl subunits of H b A v obtained after separation of H b P into H b A p and HbFp. When compared to the tryptic maps of pure HbA, no difference was observed. Therefore, as it was assumed that the
2.3
0.2
t4
-~ o~
0.1 ..J
vl
4
8
12
Y
rain 0
4
8
EIut 1on tlme
12
rain
E]utlon tllne
~--[5
0,3
b 2
02
BHbAp
g
~HbAp
0,1
o~
j
T7
0
4
"8
12
rain .0
ElutJon tltne
4
8 Elutlon tlrre
12
min
Fig. 5. Cation-exchange chromatography at p H 5.8 of the non-hydrophobic tryptic fragments isolated by C 8 reverse-phase chromatography. (a, c): tryptic fragments corresponding to the fl and a chains of pure H b A respectively and (b, d): to HbAp. Elution pH: 5.8. For the other conditions, see Materials and Methods.
62
HbAp
ca O
_b
"
'
I
-
03
0
L_
6
/~ 8 12 mn E[uUontime Fig. 6. Cation-exchangechromatographyat pH 3.8 of the non-hydrophobictrypticfragmentsof ~-chains, isolated by C8 reverse-phasechromatography. (a): ~-chain of HbAp; (b): c~-chainof HbA. For the other conditions, see Materials and Methods.
modification in H b A P was caused by a carboxypeptidase, necessarily located on the C-terminal end of globins--i.e, on the T14 fragment for the c~-chains and on the T15 for the fl-chains--and as, on the reverse-phase column, these non-hydrophobic peptides were eluted in the void volume together with other fragments, we pooled all of them (i.e. theoretically T14 , T10 , Ts, Tv, T 2 for the c~-chains and Tls , Ts, T7, T 6 for the/?-chains) and eluted them on a Mono S cation-exchange column at p H 5.8° The elution profiles thus obtained are given in Fig. 5 and are compared with those obtained with pure HbA. All the peaks in both chromatograms were assigned by separately injecting the peptides corresponding to R(T14 , ~['10, Ts) and fi(Tls, Ta, T6). Figs. 5a and 5b show that the elution profiles corresponding to the fi-chains of H b A and H b A p are quite similar (T8 cannot be seen because it is eluted with the injection solvent, acetonitrile). On the other hand, Figs. 5c and 5d, corresponding to the c~-chains of H b A and HbAp, prove that in the elution profile related to H b A p , the W14 fragment (H-Tyr-Arg-OH) has disappeared. In order to check whether only Arg a141 was removed from HbP, or whether Tyr ~ 4 0 was also removed, the same peptide mixtures were eluted on the same cation-exchange column but at a lower p H (3.8). The corresponding elution profiles are shown in Fig. 6. Under these conditions, when injected alone as a model, tyrosine is eluted in 1.7 rain. In Fig. 6b (corresponding to HbA) no peak is found at this retention time, whereas Fig. 6a (corresponding to H b A p ) shows the peak, proving that the c~-chain of H b A P has lost the C-terminal residue, Arg 141, but still contains the amino-acid Tyr 140.
63
a degraded a
P Y
Fig. 7. Electrophoresis under dissociating conditions on cellulose acetate of HbP (2) compared to HbA (3,4) and to a mixture of HbA/HbF (1). The bands correspond to the UL,p and y separated globins, native and degraded.
Moreover, the amino-acids contained in the supernatant obtained after precipitation and removal of the proteins from the initial solution of HbP Were also analysed. The only amino-acid discovered was ornithine together with urea, probably resulting from the transformation of arginine by a placental enzyme, arginase. Finally, Fig. 7, which shows the electrophoretic bands of the different globins obtained in dissociating conditions [8] from HbP, HbA and a mixture of HbF/HbA, confirms that in HbP only the a-chains are altered. Influence of arginine on the storage of Hbp A solution of Hbp (prepared by rapidly crushing and thawing a fresh placenta, and not purified) was stored for 20 h at 37°C and at pH 6.9 in the absence of
HbF
degraded mL /--degraded HIbA
Fig. 8. IEF of Hbp after incubation in a placental plasma at 37°C for 20 h, pH 6.9; (1, 2, 3, 4): respectively, with 0, 5, 10 and 20 g/l of arginine. The bands correspond to HbA and HbF, native and degraded.
64
HbF
1
2
3
4
5
6
7
8
9
degraded HbA Fig. 9. IEF of placental Hb a~ each step of purification in the presence of arginine, (1): after pressing; (2): after acid precipitation of lipoproteins; (3 ~: after diafiltration in a phosphate buffer: (4): HbP: (5): before chromatography in a phosphate buffer: (6): after storage m a phosphate buffer; (7): after the final chromatography; (8): HbP; (9): fimshed product.
argihine and in the presence of various concentrations of this amino-acid. The IEF of these samples (Fig. 8) shows that, at least on a qualitative scale, the amount of degraded H b A and H b F was much smaller when argihine was added to the solutions, which proves that arginine inhibits the action of the carboxypeptidase.
Large-scale purification of placental Hb in the presence of arginine The mixture obtained after the large-scale purification in the presence of arginine was analysed by ~EF at each purification step and the results of these analyses are shown in Fig. 9. Only a slight degradation was observed after diafiltration in a phosphate buffer (samples 3, 5 and following), as this step removes the arginine. After this purification procedure 5.20 kg of H b were obtained, containing about 16% of methemoglobin. The Ph0 and nh0 values of this sample were 11.75 Torr and 1.95 respectively (after reduction of methemoglobin by dithionite), to be compared with 13.5 Torr and 2.6 for H b A and 2.0 Torr and 1.1 for H b P (Table 1).
Discussion Large-scale purification of hemoglobin from frozen placentas yields a mixture of two main hemoglobin species i.e. degraded H b A and degraded HbF, the latter representing about 25% of all the hemoglobin molecules. On the other hand, the solution of placental hemoglobin thus purified (HbP) contains about 30% of methemoglobin, and its oxygen-binding properties are greatly attered even after reduction of methemoglobin: the Ps0 is about 2 Torr (compared with 13.5 for pure HbA), it is not influenced by the presence of an effector such as BHC and cooperativity is nil (Table 1). These important modifications were assumed to be caused by an enzyme which is liberated from the placental tissues as the crushed frozen placentas are thawed. Then, as the industrial purification procedure is relatively long and as a low temperature cannot be maintained at each step, the enzyme can act as soon as the placentas are defrozen. As a proof, when H b A was incubated with a placental plasma, a similar alteration of its oxygen-binding properties was observed (Table 2);
65 on the other hand. when frozen placentas were r a p i d l y crushed a n d thawed, the h e m o g l o b i n c o n t a i n e d in the resulting filtrated solutions ( H b p ) e x h i b i t e d oxygenb i n d i n g p r o p e r t i e s close to those o f native H b A , whereas, after 24 h of storage at 37°C. these p r o p e r t i e s were c o m p l e t e l y altered a n d b e c a m e similar to those of H b P . T h e enzyme involved was a s s u m e d to be an arginine c a r b o x y p e p t i d a s e , as the I E F of H b P ( p l a c e n t a l H b p u r i f i e d b y the industrial-scale procedure) (Fig. 1) and of H b p ( u n p u r i f i e d fresh p l a c e n t a l H b ) after storage for 24 h in p l a c e n t a l p l a s m a (Fig. 2) p r e s e n t e d a b a n d at the same p o s i t i o n as that of H b A after i n c u b a t i o n with c a r b o x y p e p t i d a s e B (Fig. 3). To c o n f i r m this a s s u m p t i o n , the structure of H b A e o b t a i n e d from H b P after s e p a r a t i o n from the other species was d e t e r m i n e d b y the classic c h r o m a t o g r a p h i c p r o c e d u r e [9-12] and the t r y p t i c m a p s of a a n d / ~ chains of H b A p were f o u n d to b e quite similar to those of pure H b A . A n a d d i t i o n a l c h r o m a t o g r a p h i c step on a c a t i o n - e x c h a n g e Column was then p e r f o r m e d , at 2 different p H s . on the m i x t u r e of n o n - h y d r o p h o b i c t r y p t i c f r a g m e n t s theoretically i n c l u d i n g the t e r m i n a l ones. a T 1 4 a n d .BT15. This c h r o m a t o g r a p h i c step p r o v e d that in H b A p , o n l y the a - c h a i n was different to that o f H b A . as the p e a k of the c~T14 f r a g m e n t could not b e seen (Fig. 5d) a n d as a p e a k c o r r e s p o n d i n g to a tyrosyl r e s i d u e a p p e a r e d (Fig. 6b). These results thus p r o v e that the a l t e r a t i o n of h e m o g l o b i n o b s e r v e d after the industrial-scale p u r i f i c a t i o n p r o c e d u r e is caused b y the p r e s e n c e of an arginine c a r b o x y p e p t i d a s e which leads to d e s ( A r g 1 4 1 a ) h e m o g l o b i n . This h y p o t h e s i s is c o n f i r m e d b y Ito et al. [15], who recently evidenced large a m o u n t s of c a r b o x y p e p t i d a s e N (kininase I) in the m i c r o s o m a l fraction of h u m a n p l a c e n t a . T h e final p r o o f was given b y the results of a large-scale p u r i f i c a t i o n p e r f o r m e d in the presence of arginine, an i n h i b i t o r of this t y p e of enzyme, which y i e l d e d h e m o g l o b i n with suitable o x y g e n - b i n d i n g properties. L a r g e quantities of p l a c e n t a l h e m o g l o b i n with o x y g e n - b i n d i n g p a r a m e t e r s quite similar, after r e d u c t i o n of m e t h e m o g l o b i n , to those of pure H b A can thus be p r e p a r e d . M o r e o v e r , as m e t h o d s of r e d u c i n g m e t h e m o g l o b i n on a large scale have b e e n d e v e l o p e d [16,17], the use of this source of h e m o g l o b i n can also b e c o n s i d e r e d for p r e p a r i n g new i n t r a v a s c u l a r oxygen carriers.
References 1 Dellacherie, E., Labrude, P., Vigneron, C. and Riess, J.G. (1987) Synthetic carriers of oxygen. In: Bruck, S.D. (Ed.), CRC Critical Reviews in Therapeutic Drug Carrier Systems Vol. 3, CRC Press, USA, pp. 41-94. 2 V6ron, J.-L. (1983) Valorisation of human placental hemoglobin, Ph.D., Lyon, France. 3 Labrude, P., Mouelle, P., Menu, P., Vigneron, C., Dellacherie, E., L6onard, M. and Tayot, J.L. (1988) Solutions of hemoglobin coupled with polyethylene glycol 1900, Int. J. Artif. Organs 11, 393-402. 4 Van Kampen, E.J. and Zijlstra, W.J. (1960) Standardization of hemoglobinometry, II: the hemiglobincyanide method. Clin. Chim. Acta 6, 538-545. 5 Evelyn, K.A. and Malloy, H.T. (1938) Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J. Biol. Chem. 126, 655-662. 6 Zygmunt, D., Labrude, P.and Vigneron, C. (1987) Structural and functional properties of oxyhemoglobin regenerated from methemoglobin obtained chemically or by freeze-drying. Int. J. Biol. Macromol. 9, 197-204.
66
-
8 9
10 11
12 13 14 15 16 17
Michal. G. (1974~ In: Bergmeyer, H.U. (Ed.), Methods of enzymatic analysis, 2rid edition, Academic Press. Inc.. Vol. 3. p. 1433. Rosa. J., personal communication. Baldouti. F.. Baudin-Chich, V.. Kister. J.. Marden. M.. Teyssier. G.. Poyart, C., Delaunay, J. and Wajcman, H. (1988) Increased oxygen affinity with normai heterotropic effects in hemoglobin Loire [a88(F9) Ala-Ser]. Eur. J. Biochem. 177. 307-312. Schroeder. W.A.. Pace, L.A. and Huisman. T.H.S. (1976) Separation of hemoglobins on CM-cellulose with Bistris and sodium chloride developers. J. Chromatogr. 118. 295-302. Clegg, J.B.. Naughton. M.A. and Weatherall. D.J. (19661 Abnormal human HbS: separation and characterization of the a and the /~ chains by chromatography, and the determination of two new variants. Hb Chesapeake and HbJ (Bangkok). J. Mol. Biol. 19. 91-108. Wajcman, H., personal communication. Zito, R.. Antonini. E. and Wyman, J. (1964) The effect of oxygenation on the rate of digestion of human hemoglobin. J. Biol. Chem. 239. 1804-1808. Kilmartin. J.V.. Hewitt. J.A. and Wootton. I.F. (1975) Alteration of functional properties associated with the change m quaternary structure in unliganded hemoglobin. J. Mol. Biol. 93, 203-218. Ito. Y., Mizutani. S.. Kuraucl'2. O.. Kasugai, M.. Naruta. O. and Tomoda, Y. (1989) Purification and properties of carboxypeptidase N (kininase I) m human placenta. Enzyme 42, 8-14. Durliat. H. and Comtat. M. (1987) Electrochemical reduction of methemoglobin either directly or with r a v i n mononucleotide as mediator. J. Biol. Chem. 262. 11497-11500. Labrune. P. (1990) A bio-electrochemical method for the methemoglobin reduction. Ph.D., Toulouse, France.
53
JBBM00888
Preparation of unaltered hemoglobin from human placentas for possible use in blood substitutes G. Fasan a, M. Grandgeorge
2 C. Vigneron
3 and E. Dellacherie 1
CNRS URA 494, ENSIC, Nancy, France, 2 Institut Mdrieux, Charbonni~res-les-Bains, France and 3 Centre Rdgional de Transfusion Sanguine, Vandoeuvre-les-Nancy, France
(Received 9 July 1990) (Revision received 15 December 1990) (Accepted 22 February 1991)
Summary Hemoglobin extracted from human placentas could be used as the basis of blood substitutes provided it could be prepared on a large scale with appropriate oxygen-binding properties. Unfortunately, the industrial conditions under which it is extracted, produce hemoglobin with high oxygen affinity and which is no longer influenced by the classical effectors. These characteristics were shown to be caused by a degradation of the c~-chain brought about by an arginine carboxypeptidase present in the placental tissues and leading to the disappearance of the C-terminal arginine residue. This carboxypeptidase which is released from the tissues during the process of crushing the frozen placentas, degrades the protein during the chromatographic purification procedure. The addition of an inhibitor of this carboxypeptidase (for example, arginine) as soon as the placentas are thawed and during the chromatographic process, makes it possible to obtain placental hemoglobin with oxygen-binding properties quite similar to those of HbA prepared from peripheral venous blood. Keywords: Placental hemoglobin; Placental carboxypeptidase; Large-scale chromatographic purification
Introduction Up to now, most of the hemoglobin-based blood substitutes described in the literature have been synthesized using human hemoglobin (HbA) obtained from Abbreviations: HbA, human adult hemoglobin; HbF, human foetal hemoglobin; HbP, placental hemoglobin purified by the industrial-scale procedure; Hbp, unpurified native placental hemoglobin; HbAp and HbFp, respectively, degraded adult hemoglobin and degraded fetal hemoglobin contained in HbP; IEF, isoelectric focusing; BHC, benzene hexacarboxylic acid; DPG, 2,3-diphosphoglyeerate. Correspondence address." Dr. E. Dellacherie, ENSIC, BP 451, 54001 Nancy Cedex, France.
54 gifts of peripheral venous blood [1]. However, it is also possible to obtain hemoglobin by extracting it from human placentas. Placental blood contains both HbA and H b F (foetal hemoglobin) and after large-scale purification following VOron's method [21 a hemoglobin mixture containing between 25 and 30% of H b F (compared with about 1% for peripheral venous blood) is obtained. Although H b F is known to possess oxygen-binding properties which differ from those of HbA, the use of placental hemoglobin for preparing oxygen-carriers can be considered, as the differences are not very important and as the concentration in H b F is not too high. However, the major proNems posed by the use of placental hemoglobin on a large scale are caused by the conditions under which it is extracted from the placentas and purified. Placentas are kept in frozen storage and to extract hemoglobin they have to be crushed before thawing. Then tissues and fibrinogen are removed by filtration and several steps of chromatography, precipitation and diafiltration are carried out on the resulting filtrate. Finally, a 10% hemoglobin solution is obtained with a yield of 35 1 per 100 kg of placenta. However, hemoglobin obtained in this way has a high oxygen affinity. Moreover, its cooperativity is much less than that of pure HbA and it is no longer sensitive to the classical effectors, making it quite unsuitable for blood substitution purposes. We have analyzed the structure of hemoglobin prepared on a large scale from human placentas and determined the reasons for the alterations observed. This study allowed us to propose some modifications in the large-scale extraction procedure in order to obtain hemoglobin with suitable properties. Materials and Methods Pure HbA was prepared frQm outdated Mood as previously described [3]. Benzene hexacarboxylic acid (BHC) was obtained from Aldrich (Belgium). Carboxypeptidase B, L-arginine and L-tyrosine were purchased from Sigma (U.S.A.). Model oligopeptides were obtained from Bachem (Switzerland). All the other reagents were obtained from Prolabo (France) or Merck (F.R.G.). Total hemoglobin was estimated by Drabkin's procedure [41, concentrations of methemoglobin were measured by the method of Evelyn and Malloy [5] and its reduction by dithionite was performed according to Zygmunt et al. [6]. 2,3-Diphosphoglycerate (DPG) was estimated by a classical method [7]. Placental plasma was prepared by pressing a freshly thawed placenta. Isoelectric focusing was performed in the presence of K C N (100 mg/1). The samples were applied to Phastgel IEF 5-8 and developed with the Phast system (Pharmacia, Sweden). Electrophoresis Of a, fi and ~, chains obtained under strongly dissociating conditions (6.5 M urea, p H 6.0) [8] was carried out with the Sebia system (France). Oxygen equilibrium curves were determined at 37°C in 69 mM Bistris buffer, p H 7.4, 0.14 M NaC1, 44 m M glucose with the Hemox Analyzer B (TCS, Southampton, U.S.A.).
Purification of placental hemoglobin by the industrial scale procedure (HbP) The different steps of this procedure are as follows:
55 ® thawing of crushed frozen placentas at 37°C in a solution containing ethanol (8%) and cellulose, which facilitates pressing • pressing in order to remove tissues and fibrinogen • acidification at p H 5.1 by acetic acid in order to precipitate the lipoproteins • diafiltration in 10 mM phosphate buffer, p H 5.25 (membrane cut-off: 10000) • chromatography on DEAE-Spherodex (IBF. France) in 50 m M phosphate buffer, p H 5.25 in order to remove albumin ® alcoholic precipitation 125% ethanol. 5°C) of immunoglobulins Pyrogenic products and group substances were removed by an additional chromatographic step, followed by diafiltration in 0.1 M NaC1. For large-scale purification of placental Hb in the presence of arginine, 150 kg of placentas were treated as described above but in the presence of arginine which was added in the thawing solution (14 g/l). Temperature was maintained close to 0°C during the whole process. The final chromatography was also carried out in the presence of arginine at 5 g/1.
Structural studies of HbP (placental Hb purified by the industrial-scale procedure) The analysis of H b P was carried out according to the following procedure: ///~ HbP-containing solution
Cation-exchange chromatogr~ HbA e
HbFe
Electrofocusing Electrophoresis Precipitation of H b P and analysis of c~-amino-acids of the supernatant
Precipitation of globins then cation-exchangechromatography
/
/
/ BF
O~p ~/~ Tryptic map
Trypsindigestion reverse-phaseHPLC Collecting of the fragments come out in the void volume
l Cation-exchange H P L C
~/~ Tryptic map
Trypsindigestion reverse-phaseHPLC Collecting of the fragments come out in the void volume
Cation-exchange HPLC
56
The amino-acids of the supernatant were analysed on a Beckman 7300 amino-acid analyser. All the separation steps were carried out following procedures described elsewhere [9]. The first cation-exchange chromatography (separation of H b A p and HbFp, degraded H b A and H b F contained in HbP) was performed according to Schroeder et al. [10] with carboxymethylcellulose (Whatman) with an increasing NaC1 gradient and in the presence of KCN. The c~ and fl globins of H b A e thus separated were prepared by the a c i d / a c e t o n method. They were separated by carboxymethylcellulose chromatography in 8 M urea and in the presence of mercaptoethanol [11]. Digestion with trypsin (treated with tosylphenylalanytchloromethane) was performed in 25 m M a m m o n i u m bicarbonate, p H 8, at ambient temperature using an enzyme/substrate weight ratio of 8 × 1 0 - 4 . The pepfide fragments were separated by reverse phase high-performance chromatography on a n Aquapore RP 300 column (25 × 0.46 cm; Brownlee Labs, U.S:A,) according to Wajcman [12] with an increasing gradient of water-acetonitrile-trifluoroacetic acid (A: 0.05% T F A in water; B: 50/50, acetonitrile/0.1% T F A in water). The last cation-exchange chromatographic step was carried out on a Mono S column (5 × 0.5 cm, Pharmacia) under 2 sets of conditions. In one experiment, the sotutes were eluted at p H 5.8 at a flow rate of 1.5 m l / m i n , with a gradient of A: 10 m M sodium malonate and B: 10 m M sodium malonate, 300 m M LiC1 (Fig. 5). In the second one, the p H was 3.8, the flow rate 1.5 m l / m i n and the gradient was constituted of A: 10 m M sodium formate and B: 10 m M sodium formate, 300 m M LiC1 (Fig. 6).
Results
Influence of placental plasma on the oxygen-binding properties of hemoglobin The oxygen-binding parameters of placental hemoglobin obtained by the largescale purification procedure described in Materials and Methods (HbP) are reported in TaMe 1. The effect on H b P of benzene hexacarboxylic acid (BHC), a strong effector of HbA, was also determined. Isoelectric focusing (IEF) of H b P is given in
TABLE 1 Oxygen-binding parameters of placental Hb purified according to the method described in Materials and Methods (HbP), determined at pH 7.4, 69 mM Bistris buffer, 0.14 M NaC1, 37°C, after reduction of methemog!obin by dithionite
/'5o (Torr) nSob
HbP
HbP + BHC a
HbA
H bA + BHC
2.0 1.1
2.5 0.9
13.5 2.6
42.5 2.6
a BHC = benzene hexacarboxylic acid; B H C / H b molar ratio = 15; b Hill coefficient at Ps0-
a
57
Fig. 1. IEF of a sample of H b P (!) compared to that of H b A (2).
Fig. 1 and shows 2 bands
corresponding
to species with isoelectric points lower than
that of HbA. F i r s t o f all, w e c h e c k e d
that the application
of the same purification
procedure
to
TABLE 2 Influence of placental plasma on the oxygen-binding parameters of H b A Storage medium
Storage time b (h)
DPG content c
Ps0 (Torr)
n 50
Adult blood hemolysate
Adult plasma
0 18 120
0.10
14.0 14.5 14.0
2.55 2.4 2.30
Adult blood hemolysate
Placental plasma a
18 120
0.55
15.0 9.5
2.20 1.40
Pure H b A 100 g / l
Placental plasma a
18 120
0.45
18 10
2.05 1.40
a Placental p l a s m a / H b A solution volume ratio = 1; b at 4°C; c D P G / H b molar ratio measured as described in the Materials and Methods. Precision 10%; other conditions and symbols as in Table 1.
58
H
degraded HbF
-,--/
H
degraded
HbA Fig, 2. IEF of samples of Hbp stored in placental plasma at 37°C. (1): 0 h: (2): 1 h; (5): 2 h; (6): 3 h; (7): 4 h: (8): 6 h; (4): 24 h: (3): pure HbA.
frozen total peripheral venous blood yielded HbA with oxygen-binding properties similar to those of H b A prepared by the classical method. On the other hand. we found that the oxygen-binding properties of a red cell hemolysate or of pure HbA stored for a long time at 4°C in a placenta1 plasma obtained from a freshly-crushed non-frozen placenta were modified as shown in Table 2, i.e. the Ps0 and the Hill coefficient both decreased strongly. Another experiment was carried out with a freshly frozen placenta.This placenta was rapidly crushed in an alcoholic solution (8% ethanol) and the crushed product was rapidly thawed and filtrated. Then the resulting filtrate was kept at 37°C for several hours. Fig. 2 shows the IEF of the sample after various terms of storage, tt can be seen that at r - 0. the hemoglobin contained in the mixture (Hbp) shows 2 bands, one corresponding to H b A and the other to HbF. After 24 h, both bands are shifted towards lower pH, which proves that the species have gained a more negative net electric charge. The oxygen-binding properties of Hbp were determined after 24 h of storage. The results are reported in Table 3 and show that whereas the Hbp contained in a fresh placenta has oxygen-binding parameters similar to those of HbA, its Ps0 at 37°C is drastically decreased after 24 h of storage at 37°C; it is no longer influenced by the presence of an effector, and the cooperativity has almost completely disappeared.
TABLE 3 Oxygen-binding properties of non-purified placental hemoglobin (Hbp) during storage at 37°C, compared to those of H b P
Hbp Hbp HbP
Storage
Ps0 (Tort)
time (h) at 3 7 ° C
N o BHC
In presence of B H C a
ns0
0 24
12.3 2.5 2.0
36.2 2.3 2.5
a 15 mol B H C / m o l Hb; other conditions and symbols as in Table 1.
2.5 1.2 0.9
59
Fig. 3. I E F of Hb A after incubation with carboxypeptidase B at 370C, 50 m M Tris buffer, p H 7.5, for 3 h (3) compared with HbP (1) and pure H b A (2),
Influence of carboxypeptidase B on HbA Pure H b A was incubated with carboxypeptidase B at 37°C, in 50 m M Tris buffer, p H 7.5, for 3 h. The resulting mixture was then examined by I E F and the result of this analysis is shown in Fig. 3 and compared with the I E F of pure H b A and of HbP. The P~0 of H b A after this treatment was 1.85 Torr and the ns0 was 1.2. Fig. 3 shows that, as already described [13,14], H b A is digested by carboxypeptidase B to give des(Arg 141a)-hemoglobin and that the corresponding band appears at the same position as one band of HbP, probably that of H b A p (the degraded H b A contained in the placental large-scale purified Hb), the other one being that of HbFp (degraded HbF). On the other hand, the oxygen affinity of carboxypeptidase-digested H b A (Ps0 = 1.85 Torr) and its Hill coefficient (ns0 = 1.2) are quite similar to those of HbP. These results, together with those shown in Table 2, thus seem to indicate that
TABL E 4 Influence of arginine on the oxygen-binding parameters of an adult blood hemolysate stored under different conditions Storage medium
Storage time c (days)
Ps0 (Torr)
n 50
Adult plasma
0 7 7
12 11.5 9.5
2.55 2.5 1.75
7
11.5
2.55
Placental plasma a Placental plasma a + arginine b
a Placental p l a s m a / h e m o l y s a t e volume ratio = 3; b concentration = 10 g / l ; e at 4°C; other conditions as in Table 1.
60
the placentas contain a type of B-carboxypeptidase which is responsible for the degradation observed in HbP. As arginine is known to be an inhibitor of this type of carboxypeptidase, this amino acid was added to a mixture of adult blood hemolysate and placental plasma, and the oxygen-binding parameters of the resulting sohition were measured after 7 days of storage at 4°C. The results are given in Table 4 and show that arginine impedes the deterioration of HbA, and specially that of its cooperativity, when the protein is stored in placental plasma.
_b ~HbA
5HbA Ta
40
20
20
U
40 Elutlon t l ~
Elutlon t i m
12
i l i
0
20
40
Elution i I ~
na;n
0 - -
--
--2~
40 ElvtJ~ tlme
Fig. 4. Tryptic maps of a and fl chains of pure HbA (a, b) and of H b A p (c, d) on a C s reverse phase (see Materials and Methods for the chromatograpNc conditions; the broken lines represent the variation of B concentration in A).
61
Analysis of HbP The structure of H b P obtained after the large-scale purification procedure was analysed by the method described in Materials and Methods. Fig. 4 shows the chromatograms of tryptic digests of a and fl subunits of H b A v obtained after separation of H b P into H b A p and HbFp. When compared to the tryptic maps of pure HbA, no difference was observed. Therefore, as it was assumed that the
2.3
0.2
t4
-~ o~
0.1 ..J
vl
4
8
12
Y
rain 0
4
8
EIut 1on tlme
12
rain
E]utlon tllne
~--[5
0,3
b 2
02
BHbAp
g
~HbAp
0,1
o~
j
T7
0
4
"8
12
rain .0
ElutJon tltne
4
8 Elutlon tlrre
12
min
Fig. 5. Cation-exchange chromatography at p H 5.8 of the non-hydrophobic tryptic fragments isolated by C 8 reverse-phase chromatography. (a, c): tryptic fragments corresponding to the fl and a chains of pure H b A respectively and (b, d): to HbAp. Elution pH: 5.8. For the other conditions, see Materials and Methods.
62
HbAp
ca O
_b
"
'
I
-
03
0
L_
6
/~ 8 12 mn E[uUontime Fig. 6. Cation-exchangechromatographyat pH 3.8 of the non-hydrophobictrypticfragmentsof ~-chains, isolated by C8 reverse-phasechromatography. (a): ~-chain of HbAp; (b): c~-chainof HbA. For the other conditions, see Materials and Methods.
modification in H b A P was caused by a carboxypeptidase, necessarily located on the C-terminal end of globins--i.e, on the T14 fragment for the c~-chains and on the T15 for the fl-chains--and as, on the reverse-phase column, these non-hydrophobic peptides were eluted in the void volume together with other fragments, we pooled all of them (i.e. theoretically T14 , T10 , Ts, Tv, T 2 for the c~-chains and Tls , Ts, T7, T 6 for the/?-chains) and eluted them on a Mono S cation-exchange column at p H 5.8° The elution profiles thus obtained are given in Fig. 5 and are compared with those obtained with pure HbA. All the peaks in both chromatograms were assigned by separately injecting the peptides corresponding to R(T14 , ~['10, Ts) and fi(Tls, Ta, T6). Figs. 5a and 5b show that the elution profiles corresponding to the fi-chains of H b A and H b A p are quite similar (T8 cannot be seen because it is eluted with the injection solvent, acetonitrile). On the other hand, Figs. 5c and 5d, corresponding to the c~-chains of H b A and HbAp, prove that in the elution profile related to H b A p , the W14 fragment (H-Tyr-Arg-OH) has disappeared. In order to check whether only Arg a141 was removed from HbP, or whether Tyr ~ 4 0 was also removed, the same peptide mixtures were eluted on the same cation-exchange column but at a lower p H (3.8). The corresponding elution profiles are shown in Fig. 6. Under these conditions, when injected alone as a model, tyrosine is eluted in 1.7 rain. In Fig. 6b (corresponding to HbA) no peak is found at this retention time, whereas Fig. 6a (corresponding to H b A p ) shows the peak, proving that the c~-chain of H b A P has lost the C-terminal residue, Arg 141, but still contains the amino-acid Tyr 140.
63
a degraded a
P Y
Fig. 7. Electrophoresis under dissociating conditions on cellulose acetate of HbP (2) compared to HbA (3,4) and to a mixture of HbA/HbF (1). The bands correspond to the UL,p and y separated globins, native and degraded.
Moreover, the amino-acids contained in the supernatant obtained after precipitation and removal of the proteins from the initial solution of HbP Were also analysed. The only amino-acid discovered was ornithine together with urea, probably resulting from the transformation of arginine by a placental enzyme, arginase. Finally, Fig. 7, which shows the electrophoretic bands of the different globins obtained in dissociating conditions [8] from HbP, HbA and a mixture of HbF/HbA, confirms that in HbP only the a-chains are altered. Influence of arginine on the storage of Hbp A solution of Hbp (prepared by rapidly crushing and thawing a fresh placenta, and not purified) was stored for 20 h at 37°C and at pH 6.9 in the absence of
HbF
degraded mL /--degraded HIbA
Fig. 8. IEF of Hbp after incubation in a placental plasma at 37°C for 20 h, pH 6.9; (1, 2, 3, 4): respectively, with 0, 5, 10 and 20 g/l of arginine. The bands correspond to HbA and HbF, native and degraded.
64
HbF
1
2
3
4
5
6
7
8
9
degraded HbA Fig. 9. IEF of placental Hb a~ each step of purification in the presence of arginine, (1): after pressing; (2): after acid precipitation of lipoproteins; (3 ~: after diafiltration in a phosphate buffer: (4): HbP: (5): before chromatography in a phosphate buffer: (6): after storage m a phosphate buffer; (7): after the final chromatography; (8): HbP; (9): fimshed product.
argihine and in the presence of various concentrations of this amino-acid. The IEF of these samples (Fig. 8) shows that, at least on a qualitative scale, the amount of degraded H b A and H b F was much smaller when argihine was added to the solutions, which proves that arginine inhibits the action of the carboxypeptidase.
Large-scale purification of placental Hb in the presence of arginine The mixture obtained after the large-scale purification in the presence of arginine was analysed by ~EF at each purification step and the results of these analyses are shown in Fig. 9. Only a slight degradation was observed after diafiltration in a phosphate buffer (samples 3, 5 and following), as this step removes the arginine. After this purification procedure 5.20 kg of H b were obtained, containing about 16% of methemoglobin. The Ph0 and nh0 values of this sample were 11.75 Torr and 1.95 respectively (after reduction of methemoglobin by dithionite), to be compared with 13.5 Torr and 2.6 for H b A and 2.0 Torr and 1.1 for H b P (Table 1).
Discussion Large-scale purification of hemoglobin from frozen placentas yields a mixture of two main hemoglobin species i.e. degraded H b A and degraded HbF, the latter representing about 25% of all the hemoglobin molecules. On the other hand, the solution of placental hemoglobin thus purified (HbP) contains about 30% of methemoglobin, and its oxygen-binding properties are greatly attered even after reduction of methemoglobin: the Ps0 is about 2 Torr (compared with 13.5 for pure HbA), it is not influenced by the presence of an effector such as BHC and cooperativity is nil (Table 1). These important modifications were assumed to be caused by an enzyme which is liberated from the placental tissues as the crushed frozen placentas are thawed. Then, as the industrial purification procedure is relatively long and as a low temperature cannot be maintained at each step, the enzyme can act as soon as the placentas are defrozen. As a proof, when H b A was incubated with a placental plasma, a similar alteration of its oxygen-binding properties was observed (Table 2);
65 on the other hand. when frozen placentas were r a p i d l y crushed a n d thawed, the h e m o g l o b i n c o n t a i n e d in the resulting filtrated solutions ( H b p ) e x h i b i t e d oxygenb i n d i n g p r o p e r t i e s close to those o f native H b A , whereas, after 24 h of storage at 37°C. these p r o p e r t i e s were c o m p l e t e l y altered a n d b e c a m e similar to those of H b P . T h e enzyme involved was a s s u m e d to be an arginine c a r b o x y p e p t i d a s e , as the I E F of H b P ( p l a c e n t a l H b p u r i f i e d b y the industrial-scale procedure) (Fig. 1) and of H b p ( u n p u r i f i e d fresh p l a c e n t a l H b ) after storage for 24 h in p l a c e n t a l p l a s m a (Fig. 2) p r e s e n t e d a b a n d at the same p o s i t i o n as that of H b A after i n c u b a t i o n with c a r b o x y p e p t i d a s e B (Fig. 3). To c o n f i r m this a s s u m p t i o n , the structure of H b A e o b t a i n e d from H b P after s e p a r a t i o n from the other species was d e t e r m i n e d b y the classic c h r o m a t o g r a p h i c p r o c e d u r e [9-12] and the t r y p t i c m a p s of a a n d / ~ chains of H b A p were f o u n d to b e quite similar to those of pure H b A . A n a d d i t i o n a l c h r o m a t o g r a p h i c step on a c a t i o n - e x c h a n g e Column was then p e r f o r m e d , at 2 different p H s . on the m i x t u r e of n o n - h y d r o p h o b i c t r y p t i c f r a g m e n t s theoretically i n c l u d i n g the t e r m i n a l ones. a T 1 4 a n d .BT15. This c h r o m a t o g r a p h i c step p r o v e d that in H b A p , o n l y the a - c h a i n was different to that o f H b A . as the p e a k of the c~T14 f r a g m e n t could not b e seen (Fig. 5d) a n d as a p e a k c o r r e s p o n d i n g to a tyrosyl r e s i d u e a p p e a r e d (Fig. 6b). These results thus p r o v e that the a l t e r a t i o n of h e m o g l o b i n o b s e r v e d after the industrial-scale p u r i f i c a t i o n p r o c e d u r e is caused b y the p r e s e n c e of an arginine c a r b o x y p e p t i d a s e which leads to d e s ( A r g 1 4 1 a ) h e m o g l o b i n . This h y p o t h e s i s is c o n f i r m e d b y Ito et al. [15], who recently evidenced large a m o u n t s of c a r b o x y p e p t i d a s e N (kininase I) in the m i c r o s o m a l fraction of h u m a n p l a c e n t a . T h e final p r o o f was given b y the results of a large-scale p u r i f i c a t i o n p e r f o r m e d in the presence of arginine, an i n h i b i t o r of this t y p e of enzyme, which y i e l d e d h e m o g l o b i n with suitable o x y g e n - b i n d i n g properties. L a r g e quantities of p l a c e n t a l h e m o g l o b i n with o x y g e n - b i n d i n g p a r a m e t e r s quite similar, after r e d u c t i o n of m e t h e m o g l o b i n , to those of pure H b A can thus be p r e p a r e d . M o r e o v e r , as m e t h o d s of r e d u c i n g m e t h e m o g l o b i n on a large scale have b e e n d e v e l o p e d [16,17], the use of this source of h e m o g l o b i n can also b e c o n s i d e r e d for p r e p a r i n g new i n t r a v a s c u l a r oxygen carriers.
References 1 Dellacherie, E., Labrude, P., Vigneron, C. and Riess, J.G. (1987) Synthetic carriers of oxygen. In: Bruck, S.D. (Ed.), CRC Critical Reviews in Therapeutic Drug Carrier Systems Vol. 3, CRC Press, USA, pp. 41-94. 2 V6ron, J.-L. (1983) Valorisation of human placental hemoglobin, Ph.D., Lyon, France. 3 Labrude, P., Mouelle, P., Menu, P., Vigneron, C., Dellacherie, E., L6onard, M. and Tayot, J.L. (1988) Solutions of hemoglobin coupled with polyethylene glycol 1900, Int. J. Artif. Organs 11, 393-402. 4 Van Kampen, E.J. and Zijlstra, W.J. (1960) Standardization of hemoglobinometry, II: the hemiglobincyanide method. Clin. Chim. Acta 6, 538-545. 5 Evelyn, K.A. and Malloy, H.T. (1938) Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J. Biol. Chem. 126, 655-662. 6 Zygmunt, D., Labrude, P.and Vigneron, C. (1987) Structural and functional properties of oxyhemoglobin regenerated from methemoglobin obtained chemically or by freeze-drying. Int. J. Biol. Macromol. 9, 197-204.
66
-
8 9
10 11
12 13 14 15 16 17
Michal. G. (1974~ In: Bergmeyer, H.U. (Ed.), Methods of enzymatic analysis, 2rid edition, Academic Press. Inc.. Vol. 3. p. 1433. Rosa. J., personal communication. Baldouti. F.. Baudin-Chich, V.. Kister. J.. Marden. M.. Teyssier. G.. Poyart, C., Delaunay, J. and Wajcman, H. (1988) Increased oxygen affinity with normai heterotropic effects in hemoglobin Loire [a88(F9) Ala-Ser]. Eur. J. Biochem. 177. 307-312. Schroeder. W.A.. Pace, L.A. and Huisman. T.H.S. (1976) Separation of hemoglobins on CM-cellulose with Bistris and sodium chloride developers. J. Chromatogr. 118. 295-302. Clegg, J.B.. Naughton. M.A. and Weatherall. D.J. (19661 Abnormal human HbS: separation and characterization of the a and the /~ chains by chromatography, and the determination of two new variants. Hb Chesapeake and HbJ (Bangkok). J. Mol. Biol. 19. 91-108. Wajcman, H., personal communication. Zito, R.. Antonini. E. and Wyman, J. (1964) The effect of oxygenation on the rate of digestion of human hemoglobin. J. Biol. Chem. 239. 1804-1808. Kilmartin. J.V.. Hewitt. J.A. and Wootton. I.F. (1975) Alteration of functional properties associated with the change m quaternary structure in unliganded hemoglobin. J. Mol. Biol. 93, 203-218. Ito. Y., Mizutani. S.. Kuraucl'2. O.. Kasugai, M.. Naruta. O. and Tomoda, Y. (1989) Purification and properties of carboxypeptidase N (kininase I) m human placenta. Enzyme 42, 8-14. Durliat. H. and Comtat. M. (1987) Electrochemical reduction of methemoglobin either directly or with r a v i n mononucleotide as mediator. J. Biol. Chem. 262. 11497-11500. Labrune. P. (1990) A bio-electrochemical method for the methemoglobin reduction. Ph.D., Toulouse, France.
