Purification and Viral Inactivation of Hemoglobin from Human Placenta Blood

Abstract

A new procedure was developed to obtain high-quality polymerized human hemoglobin by modifying purified hemoglobin with PLP and polymerized with GDA. Comparing polymerized hemoglobin products obtained from different methods, the product from the new procedure has similar physical, chemical, and biological properties in the molecular distribution, methemoglobin concentration, oxygen carrier capacity, P₅₀ and spectral analysis. Furthermore, the new procedure of modification after polymerization can save PLP greatly, and significantly reduce the cost. So the procedure of modification after polymerization is a better way in research and production.

• Placental hemoglobin
• modification
• polymerization
• P50
• spectral analysis

Stablizing hemoglobin structure, prolonging the circulation half-life, and lowering the O₂ affinity is very important in the research of hemoglobin-based oxygen carriers [1], [2]. American Northfield Corporation modified human hemoglobin with Pyridoxalated-5-phosphate (PLP) and polymerized hemoglobin with Glutaraldehyde (GA). The product which is on its phase III clinical trials is perspective. Chengmin Yang et al. reported the modification and polymerization of human placental hemoglobin and made great progress [3]. This paper investigated a number of polymerization methods and further optimized the process of the placental hemoglobin modification.

MATERIALS

Blood Source

The normal human placental blood was supplied freshly by TianJin Union Stem Cell Gene Engineering Corp.

Chemicals and Reagents

5-Pyridoxalated was purchased from Sigma; all other chemicals used in this study were at analytical reagent grade.

Instrument

CARY50 ultraviolet-visible spectrophotometer (VARIAN); Superdex^TM 75Gel column (Amersham Biosciences); HPLC (VARIAN); Stat PHOX blood gas analyzer (NOVA).

METHODS

Preparation of Placental Hemoglobin

The placental hemoglobin was isolated and purified by established methods in our laboratory and the methods were described in reference [4].

Study of the Process on Polymerization and Modification (All Steps were Carried out in 4°C Water Bath)

The procedure of PLP modification before GDA polymerization

The stroma-free hemoglobin solution obtained from step 2.1 is completely deoxygenated by flushing with nitrogen and vacuumizing. The solution is pyridoxylated using pyridoxa-5’-phosphate, which was dissolved in tris-hydrochloride buffer on about 4:1 molar ratio to hemoglobin. After 3-4h a reducing agent sodium borohydride, which was dissolved in sodium hydroxide, is added to the solution, and the PLP-HbF is obtained after incubation for 4h. Excess reagents were removed by dialysis against 0.9% sodium chloride. Subsequently, the PLP-HbF is polymerized using 1% glutaraldehyde. After 1-2h a reducing agent sodium borohydride, which was dissolved in sodium hydroxide, is added to the solution; the Poly-PLP-HbF is obtained after incubation for 4h. Excess reagents may be removed by dialysis against 0.9% sodium chloride.

The procedure of PLP modification after GDA polymerization

The stroma-free hemoglobin solution is completely deoxygenated by flushing with nitrogen and vacuumizing. The solution is polymerized using 1% glutaraldehyde. After 2h incubation a reducing agent sodium borohydride, which was dissolved in sodium hydroxide, is added to the solution; the Poly-HbF is obtained after incubation for 4h. Excess reagents may be removed by dialysis against 0.9% sodium chloride. Subsequently, the Poly-HbF is pyridoxylated using pyridoxa-5’-phosphate, which was dissolved in tris-hydrochloride buffer on about 4:1 molar ratio to hemoglobin. After 3-4h a reducing agent sodium borohydride, which was dissolved in sodium hydroxide, is added to the solution; the Poly-PLP-HbF is obtained after incubation for 4h. Excess reagents may be removed by dialysis against 0.9% sodium chloride.

Detection Methods

The concentration of Hb was determined using a standard method by converting Hb into cyanmethemoglobin and detecting the absorption at 540nm as described in reference [5].The methemoglobin concentration was determined using standard spectral analysis methods described in the reference [5].

The polymerization rate was detected by SDS-PAGE using 12.5% gel, and HPLC assay using SuperdecTM 75 Column (10mm×300mm) with 1/15M PBS (pH7.3) at flow rate of 0.5ml/min. The HPLC assay was conducted at 25°C, and monitered at wavelength 280nm.

Oxygen carry capacity assay and P₅₀ determination were performed according to refrence [7]. Testing samples were prepared by dialysis against saline solution (pH 7.2 – 7.4), and then adjust Hb concentration to 6.5%∼7%.

Spectral assay was conducted at wavelength 220∼1000 nm with Hb concentration at 5mg/100ml.

RESULTS

The principal Hb in adults is HbA and in placental is HbF. Normal Hb consists of four chains (α₂β₂, α₂γ₂). The α chain and β (γ) chain combine tightly into a dimmer, and two dimmers combine loosely into a tetramer. The affinity of Hb for O₂ is regulated by 2,3-diphosphoglyerate (2,3-DPG). However, lots of 2,3-DPG was lost during the purification procedure. Due to the depleting of 2,3-DPG, isolated Hb therefore cannot readily release O₂ until the tissue O₂ tension is much lower (low P₅₀). PLP is an analogue of 2,3-DPG, which could bind to the 2,3-DPG pocket within Hb. PLP-modified Hb would have a lower oxygen affinity and a higher P₅₀. The reported paper is mainly on the procedure of modification with PLP before polymerization with GDA. The paper studied the physical, chemical, and biological properties of Hb products from different procedures between modification before polymerization and modification after polymerization.

The Molecular Distribution of the Products from Different Procedures

Molecular distribution of Hb products from 2 different procedures is shown in Table 1. Molecular distribution is an excellent indication of the conformation of Hb product. Molecular distribution of modification before polymerization is: 52. 6% (above 64KD), 47.4%(equal or below 64KD); the molecular distribution of modification after polymerization is: 54.1% (above 64KD), 45.9% (equal or below 64KD). So the molecular distribution can be controlled by regulating the reaction condition.

Table 1. Molecular distribution of different procedures

ratio procedure	Mw > 64kD	Mw ≤ 64kD
PLP before GDA	52.6%	47.4%
PLP after GDA	54.1%	45.9%

MetHb Concentration and Oxygen Carrying Capacity

Product from the new procedure has no significant difference on MetHb concentration and oxygen affinity compared to the product from the tradition procedure as indicated by data from five separate experiments (Table 2). Products from PLP modification before GDA polymerization contain an average of 4.74% MetHb and the oxygen affinity capacity is 1.02mL/g, while product from modification after polymerization has 4.95% of MetHb and oxygen affinity capacity is 1.07mL/g, respectively. There is no marked difference between the two processes and oxygen affinity capacity maintained in the adult blood level.

Table 2. Comparation of MetHb concentration and oxygen capacity volume

sample (n = 5)	MetHb concentration (%)	O2 affinity capacity (mL/g)
PLP before GDA	4.74±0.41	1.02±0.001
PLP after GDA	4.95±0.57	1.07±0.002

P₅₀ of Products from Different Processes

Table 3 summarizes the product P₅₀, an important functional property, from different processes. The Hb products from two different processes of PLP modification and GDA polymerization have similar P₅₀ values (29.6 mmHg and 27.5 mmHg, respectively) and P₅₀ values are higher than that of purified Hb. P₅₀ increased from 11.3 mmHg (purified) to 29.6 mmHg in process of modification before polymerization, while P₅₀ increased from 11.3 mmHg (purified) to 27.5 mmHg in process of modification after polymerization. There is no marked difference in P₅₀ between the two processes.

Table 3. Comparison of P₅₀ from of different modification processes

sample (n = 5)		P50 (mmHg)
different modification process	Purified	11.3
	PLP before GDA	29.6±2.63
	PLP after GDA	27.5±0.77

Biological Activities

The change of biological activities of a protein is highly related to changes of its structural conformation. Using spectral analysis, we demonstrated that polymerized Hb from different procedures have almost identical absorption spectrums with purified Hb on a maximum absorption 415nm (Figure 1), which indicates no significant structural changes. So we could infer that a series of modification and polymerization procedures have no adverse effects on the structure of Hb that was the premise of biological activity. Additionally, the purified and modified Hb showed absorption peaks at 540nm and 577nm, respectively, which were the symbols of carrying O₂. The absorption peaks suggest the O₂ carrying capacity might be reserved in the modified Hb, and the procedure has no effects on the oxygen carrying capacity.

Figure 1.  The spectral analysis of different products.

DISCUSSION

Hemoglobin based oxygen carrier has to obey the physical and chemical index as well as retain the biological activity of carrying and releasing oxygen. First of all, depleting 2,3-DPG during purified procedure leads to an increased oxygen affinity, decreased P_50, and then lost the biological functions. In addition, after separation from red blood cells, hemoglobin tetramer divides into the dimmer easily, and the dimers could not be used as blood substitutes because dimmers discrete from kidneys, which damage kidney function. Appropriate modification and polymerization processes must be introduced to the isolated Hb to keep Hb in polymer or oligomer formation and to maintain its basic biological function, such as O₂ transporting. GDA has been a widely used polymerization agent and the polymerization mechanism has been well established. The aldehyde of GDA mainly reacted with lysine -NH₂ residues as well as the αterminal -NH₂ and the sulfhydryl group of cysteine in the polymerization process. The reaction is random and the product is Schiff based. And in the modified process, PLP (which is the analog of 2,3-DPG) reacts with the site of βstrain. Therefore GDA and PLP react with different sites of Hb, respectively, without interference, and thus it must be concluded that they do not compete with each other for the same amino acid –NH₂ groups within the Hb chains. So the physical-chemical and biological properties haven't a marked difference between products from two processes.

In summary, data from this report demonstrated that the molecular distribution, MetHb concentration, oxygen carrying capacity, and spectral analysis are not markedly different between the products from the two procedures. Furthermore, the new procedure of modification after polymerization can save a great amount of PLP compared to that of traditional procedure and subsequently significantly reduced the total production cost of Hb-based blood substitutes. We believe that the procedure of modification after polymerization has great potential both in research and industrial-scale production.