Dual effects include antioxidant and pro-oxidation of ascorbic acid on the redox properties of bovine hemoglobin

Abstract

The oxidation reactions have become the main obstacle of development of bovine hemoglobin-derivates products. Herein, the effects of vitamin C (Vc), a easily available natural antioxidant reagent, on the redox reaction of bovine hemoglobin were systematically investigated through methemoglobin (MetHb) formation and spectrophotometric analysis and oxygen affinity monitoring of hemoglobin. The results showed that Vc presented antioxidant effects in the initial stage of reaction and then could accelerated the MetHb content increasing by production of hydrogen peroxide, which can be indirectly characterized by the formation of choleglobin in the following side reactions. The dual effects of Vc include antioxidant and pro-oxidant effects could be confirmed by the spectrophotometric spectrums analysis in this research. The results of this research supplied the novel insight into understanding of redox properties of bovine hemoglobin and also revealed the main obstacle in exploration of Vc application in the future development of bovine hemoglobin-derivates products.

Keywords:

• Vitamin C
• bovine hemoglobin
• antioxidant
• pro-oxidant
• spectrophotometric spectrum

Introduction

Hemoglobin, a kind of heme protein, was extensively investigated during the past decades mainly due to its physiological function of oxygen binding and releasing in red blood cells. Previous research had shown that the hemoglobin could play critical effects in pathophysiology of some diseases or tissue dysfunctions [1]. Besides, another great potential application of hemoglobin is that the hemoglobin-derivates could be used for oxygen carriers, which were considered to be the most promising candidate of red blood cell substitutes and thus had been attracting more and more attentions [2,3]. The hemoglobin-based oxygen carriers (HBOCs) had been intensively studied as the candidates of blood substitutes and were proven to be effective in the treatment of ischemic diseases such as acute anemia [4], hemorrhagic shock [5], microcirculation dysfunctions [6] and so on.

However, the exploration of HBOCs as blood substitutes was blocked by the side effects presented in the clinical trials. Compared to the control groups, the side effects incidence of HBOCs was nearly three times higher, although its curative effects reached over 91% [7]. After extensive research, the oxidation has been proven to be one of the main reasons to the toxicity of hemoglobin [8]. Usually, the hemoglobin could be easily oxidized to methemoglobin (MetHb) by the uncontrolled auto-oxidation reaction due to the absence of protection from the anti-oxidant enzymes systems include superoxide dismutase (SOD), catalase (CAT) and so on [9]. As oxidation products of hemoglobin, MetHb formation could lead to the disability of oxygen delivery of normal hemoglobin and trigger a series of free radical related reactions, causing inflammatory responses and tissue injuries after infusion [10]. In addition, prohibition of oxidative reaction of hemoglobin is also rather important for the long-term preservation of hemoglobin [11]. It was reported that effective oxygen delivery to hypoxic tissues could only be confirmed when MetHb content was less than 10% in HBOCs solution [12]. Therefore, blocking the oxidative reaction pathway of hemoglobin will be of great significance in the development of HBOCs.

To alleviate the side effects of HBOCs after infusion, a wide range of antioxidants were investigated include glutathione [13], phenols [14] and anti-oxidative enzymes [15–17]. Vitamin C (Vc, Figure 1) is an essential nutrient closely related with the functions of tissue repairing, and the antioxidant function of Vc has been well known for a long time. Numerous research reports have showed that Vc could be used in the treatment of variety of diseases with its physiological functions of free radical scavenging and antioxidant effects [18]. In our previous work, Vc presented a powerful antioxidant protection on HBOCs derived from human cord blood, which primarily established the potential applications of Vc on the development of HBOCs [11]. To this day, series of HBOCs products have been developed derived from human blood, bovine blood, porcine blood and recombinant hemoglobin, among which the bovine hemoglobin-derived HBOCs were most intensively studied.

Figure 1. The molecular structure of Vitamin C.

As a cheap and easily available natural product, Vc has been widely used in the industrial and medical area according to its effectiveness and safety. In this study, with the aim of deepening and extension of Vc application in the development of HBOCs, the effects of Vc on oxidation reactions of bovine hemoglobin were investigated. Through spectroscopic analysis, the UV/Vis spectrums of HBOCs solution were monitored during the Vc reaction with bovine hemoglobin, based on which the mechanisms of the reactions would be explored. The results in this study will provide the theoretical basis and experimental reference for the Vc application in the future development of bovine HBOCs products.

Materials and methods

Reagents and instruments

Fresh bovine blood was purchased from the Mcdonald Campus Cattle Complex at McGill University (Sainte-Anne-de-Bellevue, Montreal, Canada), and the stroma-free hemoglobin was prepared by the method as described previously [19]. Vc was obtained from Sigma Aldrich (Ontario, Canada). All other chemicals and reagents were purchased in analytical grade and used without any further pretreatment.

Ultrospec 2100 pro UV/Visible Spectrophotometer equipped with SWIFT IIsoftware (Biochrome, Cambridge, England); HEMOX^TM-ANALYZER (TCS Scientific Corp., New Hope, PA); Experimental pH meter (Accumet^® Basic, Fisher Scientific Company, Singapore); Centrifuge A G4580 R (Eppendorf, Germany); Refrigerator (Whirlpool Gold, Trademark of Whirlpool, Benton Harbor, MI) The Amicon Ultra-15 Centrifugal Filter Tube with Ultracel-10 membrane and the Biomax Polyetherdulfone ultrafiltration disc with 300KD cutoff were purchased from Millipore.

Preparation of stroma-free bovine hemoglobin

In this work, the stroma-free bovine hemoglobin (SFHb) was prepared from the fresh bovine blood and the preparation method of SFHb has been described in previously [15,19]. Fresh bovine blood with citrate was firstly centrifuged at 6000 rpm at 4 °C for 60 min to separate the red blood cells from the plasma and other component such as white blood cells and planet. Then the obtained RBC were washed for three to five times with sterile, pre-cold saline followed by suspended in the potassium phosphate buffer solution (12.5 nM and pH 7.4) for 30 min. The extraction was carried out by adding two volumes of ice-cold toluene to the RBC lysis solution to remove stromal lipids and other hydrophobic compounds. The SFHb could be obtained after centrifugation of samples at 16,000 g for 2 h at 4 °C.

Reduction of bovine MetHb by Vc

Firstly, bovine SFHb solution with concentration of about 0.005 mmol/L was prepared in potassium phosphate buffer solution (0.05 mol/l and pH 7.4). Then potassium ferricyanide solution was added into the prepared bovine SFHb solution to oxidize the ferrous (Fe²⁺) bovine hemoglobin into ferric form (Fe³⁺) in the bovine SFHb solution. After that the mixed solution was dialyzed through the membrane (with 10 kD MW cut-off) in PBS solution (0.05 mol/l and pH 7.4) to remove the excessive potassium ferricyanide and then the MetHb solution was obtained. A certain amount of Vc stock solution with concentration of about 0.57 mol/l was added into the MetHb solution to make sure the final concentration of Vc in the mixed solution was 2.84 mmol/L. The reduction reaction was triggered by Vc addition and the MetHb content was termly tested during the reaction time till the MetHb content keep in a stable value.

Effects of different Vc concentration on the redox reaction of bovine hemoglobin

The bovine SFHb stock solution with concentration of about 0.005 mmol/L was prepared previously and divided into five parts with equal volume in polyvinyl chloride centrifuge tubes. Certain amounts of Vc stock solution with concentration of 0.57 mol/l were added into the 0.005 mmol/L bovine SFHb solution and the final Vc concentration in the mixture solution were 0.00 mmol/L, 1.42 mmol/L, 2.84 mmol/L, 4.26 mmol/L and 5.68 mmol/L. The reaction was considered to be started with the Vc addition at the temperature of 4 °C. The solvent of the reaction solution is potassium phosphate buffer solution (0.05 mol/l and pH 7.4 ± 0.1). The whole reaction process was proceed at the temperature of 4 °C and the bovine hemoglobin properties in the reaction solutions were termly monitored during the reaction time include MetHb content, pH values, oxygen affinity and so on.

UV/Vis spectrum analysis of bovine hemoglobin solution during the reaction

The properties, molecular structure and functional moieties changing of hemoglobin could be presented by the UV/Vis spectrums test. To investigate the effects of Vc reaction on the properties of the reaction solution, the UV/Vis spectrums of bovine SFHb solution were detected by the spectrophotometric methods in the presence and absence of Vc during the experiment duration. In this study, the effects of Vc on bovine hemoglobin redox properties were investigated in 0.05 mol/l PBS (pH 7.4) at 4 °C using Ultrospec 2100 pro UV/Visible Spectrophotometer equipped with SWIFT IIsoftware, England. When detecting the UV/Vis spectrums, testing solution was obtained through adding 30 μl bovine SFHb sample solution into 1.5 ml 0.05 mol/l PBS solution with pH 7.4 and then was UV/Vis scanned in the wavelength of 300–750 nm by UV/Visible Spectrophotometer (Ultrpspec 2100^TM, Amersham Bioscience, Sweden). In the process of UV/Vis spectrum detection, the absorbance values of special wavelength points were recorded simultaneously to monitor the changing of some compounds in the reaction solution.

Oxidation of bovine hemoglobin (MetHb content measurement)

In this research, the oxidation degrees of bovine SFHb sample in reaction solution were expressed by the MetHb contents, which were detected by the multi-wave method of Evelyn and Malloy [20]. In addition, the ratio changing of the absorbance at the wavelength at 630 nm during the reaction was also recorded to monitor the bovine MetHb content. All measurements were repeated at least three times, from which the resultant average values were calculated.

Measurement of oxygen binding affinity of bovine hemoglobin

The oxygen binding affinity of the bovine hemoglobin solution was tested through oxygen equilibrium curves measurement using the HEMOX^TM-ANALYZER (TCS Scientific Corp.) in Tris-HCl suffer solution with pH 7.4 at the temperature of 37 °C. In this study, the oxygen affinity of tested sample was expressed by the P50 value, which was defined as the oxygen partial pressure associated with the half ratio of oxygen saturated hemoglobin.

Results

The reduction of bovine MetHb by Vc

In this study, the MetHb contents were monitored with the co-existence of bovine MetHb (with content about 100%) and Vc of 2.84 mmol/L concentration (w/v) at temperature of 4 ± 1 °C and the results were shown in Figure 2. It could be seen that the MetHb contents decreased from about 99.87 ± 1.44% to 3.86 ± 1.45% with the reaction time increasing in 24 h, indicating the bovine MetHb could be effectively and fast reduced in the presence of Vc. In the initial time of the reaction, the MetHb content was sharply decreased to 44.75 ± 2.76% in one hour, suggesting the powerful reduction ability of Vc on the bovine hemoglobin. Then, as the reaction time increasing, the bovine MetHb reduction rate gradually decreased till the MetHb content kept nearly constant after 12 h. These results confirmed the reduction effect of Vc on bovine MetHb and implied the potential antioxidant effects of Vc on bovine hemoglobin and its derivates.

Figure 2. The time course of bovine MetHb reduction by Vc.

Effects of different Vc concentrations on the redox reaction of bovine SFHb solution

With the presence of Vc concentrations of 1.42, 2.84, 4.26 and 5.68 mmol/L, the redox reaction of bovine SFHb solution was investigated at the temperature of 4 °C within duration of 37 days and the bovine hemoglobin solution with no Vc addition was set as control group. The MetHb contents in sample solution were monitored termly during the reaction and the results were shown in Figure 3(A) and (B). From Figure 3(A), during the reaction process, it could be seen that compared with the slow increasing of MetHb content in control group, the MetHb contents in all sample solutions decreased initially, which was similar with the results in MetHb reduction experiments and proved the reduction effects of Vc on bovine MetHb once again.

Figure 3. The effects of different concentration Vc on redox property of bovine hemoglobin solution. (A) The bovine MetHb contents changing with the presence of different Vc concentrations. (B) The MetHb contents in bovine hemoglobin solution with different Vc concentrations.

It is notable that the MetHb content rise up again at some time points after reduction. The MetHb content firstly increased in sample solution with 5.68 mmol/L Vc concentration, and then followed by that with Vc concentration of 4.26, 2.84 and 1.42 mmol/L in turn. The higher Vc concentrations, the later the MetHb content increasing, indicating that the MetHb content increasing was closely related with the Vc concentration. In addition, Figure 3(B) showed the MetHb content in bovine hemoglobin solutions at the end of investigation duration. It could be seen that the bovine MetHb content in hemoglobin sample solution with higher initial Vc concentration was much higher than in that with lower initial Vc concentrations. The higher the initial Vc concentration, the higher the bovine MetHb contents at the end of the investigated duration. The results in Figure 3(A) and (B) suggested that Vc presented dual effects include both reduction/antioxidant and pro-oxidant on the redox properties of bovine hemoglobin.

Effects of different Vc concentrations on the redox reaction of bovine hemoglobin

In this study, the spectrophotometric methods were used to monitor and analyze the bovine hemoglobin transition in the existence of Vc. The spectrophotometric spectrum of bovine hemoglobin solution with presence of different Vc concentration were investigated and the results were shown in Figure 4, which presented the Soret band and Q band curves, respectively. From Figure 4(A), the Soret band peak intensity of the investigated curves was gradually decreased as the Vc concentration increasing, indicating the heme structure loss during the reaction with Vc [21]. Secondly, the continuous red-shift of the Soret band maximum from 406 nm to 417 nm could be shown as the evidences of ferryl hemoglobin formation. The more Vc concentrations, the more red-shifting of Soret band peaks, implying the correlation between Vc concentrations and ferryl hemoglobin formation [22].

Figure 4. Spectrophotometric spectrums of bovine hemoglobin with presence of different Vc concentrations. (A) The Soret band region curves. (B) Q band region curves.

The Q band curves also showed the effects of different Vc concentrations on the redox properties of bovine hemoglobin. The intensity of the characteristic absorbance maxima around 541 nm and 576 nm of Q band continuously decreased as the Vc concentration increasing, which was indicative for the formation of MetHb [22]. In addition, the formation of MetHb could be simultaneously evidenced by the appearance of the absorbance values at the wavelength of 630 nm and the results were shown in Figure 5, which presented that the final MetHb contents in bovine hemoglobin solutions increased with the Vc concentration increasing, implying the potential closely association between MetHb formation and Vc concentrations in samples solution.

Figure 5. The absorbance values at 630 nm of bovine hemoglobin with different Vc concentrations.

Effects of different Vc concentrations on the redox reaction process of bovine hemoglobin

Besides the effects of different Vc concentrations on the redox of bovine hemoglobin, the Vc effects on the redox reaction process were also investigated through the spectrophotometric spectrums in the bovine hemoglobin solution with 5.68 mmol/L Vc concentration. The results of this investigation were shown in Figure 6 and the Soret band curves and Q band curves were present in Figure 6(A) and (B), respectively. In Figure 6(A), the intensity of absorbance maximum of wavelength of Soret band region decreased continuously as the reaction time increasing, indicating the loss of heme structure of the bovine hemoglobin molecule [21]. On the other hand, the Soret band maximum peaks presented slight red-shift with the reaction went on, suggesting the accumulation of ferryl hemoglobin formation with the reaction time increasing [22].

Figure 6. Spectrophotometric spectrums of bovine hemoglobin with presence of 5.68 mmol/L Vc concentrations during the reaction process. (A) The Soret band region curves. (B) Q band region curves.

As for the Q band curves shown in Figure 6(B), it could be seen that the intensity of the absorbance maximum peak around 541 nm and 576 nm decreased as the reaction time increasing, indicating the increasing formation of MetHb with the reaction went on. Besides, the MetHb content in the bovine hemoglobin sample solutions gradually increased with the reaction time increasing and the results were shown in Figure 7, which presented the MetHb content in the form of the absorbance values at the wavelength of 630 nm.

Figure 7. The absorbance values at 630 nm of bovine hemoglobin during reaction with Vc concentration of 5.68.

Effects of Vc reaction on the oxygen affinity of bovine SFHb

The effects of Vc on the oxygen affinity of bovine hemoglobin were also investigated in this study. The results of the research on oxygen affinity of hemoglobin after the redox reaction with different Vc concentrations and final oxygen affinity of bovine hemoglobin during the redox reaction with 5.68 mmol/L Vc were presented in Tables 1 and 2, respectively. The oxygen affinity was expressed in the form of P50 values and Hill coefficients. In Table 1, both of the results of P50 values and Hill coefficients values were totally decreased as the Vc concentration increasing, indicating the increasing of the oxygen binding and decreasing of the synergistic effect of oxygen binding about bovine hemoglobin. Furthermore, as for the oxygen affinity changing during the bovine hemoglobin reaction with 5.68 mmol/L concentration of Vc, both the P50 values and Hill coefficients presented the overall decline trend with the reaction time increasing, implying the oxygen affinity increasing and synergistic effect decreasing of oxygen binding. In a word, the results of investigation on oxygen affinity showed that the existence of Vc could lower down the values of P50 and Hill coefficients of bovine hemoglobin.

Table 1. The oxygen affinity of bovine hemoglobin with presence of different Vc concentration.

Vc concentration (mmol/L)	0.00	1.42	2.84	4.26	5.68
P50 (mmHg)	31.79 ± 0.25	31.31 ± 0.45	31.28 ± 0.36	29.17 ± 0.42	26.51 ± 0.66
Hill (–)	2.54 ± 0.03	2.48 ± 0.05	2.42 ± 0.03	2.39 ± 0.04	2.32 ± 0.03

Table 2. The oxygen affinity of bovine hemoglobin in the reaction process with 5.68 mmol/L Vc.

Reaction time (Day)	0	7th	15th	23th	37th
P50 (mmHg)	31.91 ± 1.03	30.77 ± 1.45	30.33 ± 1.55	29.57 ± 2.03	26.51 ± 1.88
Hill (–)	2.54 ± 0.04	2.45 ± 0.03	2.41 ± 0.08	2.33 ± 0.06	2.32 ± 0.05

Effect of Vc on the pH value of the hemoglobin solution

In this research, the pH values of bovine hemoglobin solution were monitored with existence of Vc. Figure 8(A) and (B) showed the results of pH values changing after redox reactions with different Vc concentration and during the reaction process in bovine hemoglobin solution, respectively. It could be seen that, with the Vc concentration increased from 0 to 5.68 mmol/L, the pH value of bovine hemoglobin solution remarkably decreased from more than 7.60 ± 0.05 to about 7.00 ± 0.05. In addition, during the reaction process, the pH values of Hb solution gradually lower down. The higher Vc concentrations, the more pH values decreased, indicating the pH value decreasing was closely associated with the Vc content and its reactions process.

Figure 8. Effects of Vc on pH values of bovine hemoglobin. (A) Effects of different Vc concentrations. (B) pH values changing during redox reactions.

Discussion

Hemoglobin is a very important functional protein in the physiological reaction and plays an indispensable role in the life reaction process such as oxygen delivery, tissue energy conversion, metabolisms and so on [23]. Among the potential applications of hemoglobin, the most concerning aspect is the HBOCs, which has been considered to be the most promising candidates for red blood cells substitutes and nanotherapeutic medicine for ischemic diseases [3]. However, due to the spontaneous uncontrolled oxidation reaction with the absence of enzymatic protection and other oxidizing conditions such as higher temperature, ultraviolet radiation, oxidative reagents and so on, the hemoglobin may be subjected to be oxidized from ferrous form to ferric or ferryl forms, which were disabled in oxygen delivery and may cause protein and tissue damages [12]. Therefore, the oxidation of hemoglobin has been considered to be a main obstacle in the application and development of hemoglobin-derivates.

Up to now, prohibition of oxidative reaction process has been attempted through administration or addition of diversity of antioxidants, enzymes and iron chelators. A series of research works have proved that the oxidation of hemoglobin could be hindered by a wide range of antioxidants such as glutathione, acetylcysteine and plant phenols include catechin, epigallocatechin and so on [14]. Nevertheless, the antioxidant reaction mechanisms of these antioxidants were rather different with each other according to their own properties and molecular structure. In addition, antioxidant ability cannot be used as the feasibility and reliability assessment of antioxidant reagents to be applicable in hemoglobin derivates protection.

Vc is a kind of antioxidant compounds widely existed in natural plants and other resources and also associated with many physiological effects including antioxidant protection, free radical scavenging, anti-inflammatory effects and so on. Vc has been widely used in industrial and medical areas because of its confirmed safety, reliability and effectiveness. Numerous research works have revealed that Vc was effective in prohibition the human-origin hemoglobin derivates and have potential application in the development of HBOCs derived from human cord blood [11,18,24]. There were many kinds of hemoglobin derivates made from variety of sources include bovine, procine and human origin, among which the bovine hemoglobin has been considered to be one of the important raw resource for the preparation of hemoglobin-derivate products.

In the present study, for the future development of bovine hemoglobin-derivate products, the effects of Vc on the bovine hemoglobin were systematically investigated. During the experiments, the MetHb contents always originally decreased and then re-arise with the presence of Vc under physiological conditions, which indicated the dual effects include both antioxidant and pro-oxidant of Vc on the redox reaction of bovine hemoglobin. There were several explanations about these dual effects of Vc on bovine hemoglobin. The antioxidant effect can be of course explained by the Vc ability of reduction and free radical scavenging, which is similar with the Vc antioxidant effect on polyhemoglobin derived from human cord blood. As for the pro-oxidant effects, its mechanism could be described from the reaction between the Vc and bovine hemoglobin with the existence of oxygen (or oxygenated hemoglobin). According to previous work from Gardikas and Kaziro, the ascorbate could reacted with oxygen to form the hydrogen peroxide, which could be further reacted with hemoglobin to form MetHb and hemoglobin-based complex, leading to the formation of choleglobin [25,26]. During these reactions process, the hydrogen peroxide cannot be directly monitored because of its rapid quenching through the reaction with some reactive compounds in reaction system. However, the accumulated formation of choleglobin could be monitored through the spectrophotometric analysis at wavelength of 700 nm [27], which can indirectly reflect the formation of hydrogen peroxide and the results were shown in Figure 9.

Figure 9. The absorbance values at 700 nm of reaction solution with presence of Vc. (A) The absorbance values at 700 nm of reaction solution with presence of different Vc concentrations. (B) The absorbance values at 700 nm of reaction solution with 5.68 mmol/L Vc concentration.

The A700 values of bovine hemoglobin solution with existence of Vc were shown in Figure 9. The absorbance values of A700 under effects of different Vc concentrations and in the reaction process during reaction with 5.68 mmol/L concentration of Vc were presented in Figure 9(A) and (B), respectively. It could be seen from Figure 9(A) that the A700 values increased with the Vc concentration increasing, indicating the relationship between choleglobin formation and Vc content in reaction solution. The results in Figure 9(B) showed that A700 values increasing with the reaction time increasing, reflecting the accumulated choleglobin formation during the reaction process. Combined with the results of MetHb formation, the results in Figure 9 indirectly reflected the formation of hydrogen peroxide, which was closely associated with the production of bovine MetHb, indicating the Vc pro-oxidation effects on bovine hemoglobin.

Based on the confirmation of choleglobin and MetHb formation in this study, the pro-oxidation effects of Vc on bovine hemoglobin could be explained through the reaction process between Vc and oxygenated hemoglobin (or hemoglobin with existence of oxygen), which was shown in Figure 10. In the investigated reaction system, Vc molecules reacted with oxygen to continuously produce hydrogen peroxide, which could oxidize the bovine hemoglobin to be MetHb and hemoglobin-based complex, leading to the formation of choleglobin. During the reactions process, the hydrogen peroxide and choleglobin production was closely associated with the Vc content in reaction system. The higher the Vc content, the more hydrogen peroxide and choleglobin production. Although Vc could scavenge the produced hydrogen peroxide, the hydrogen peroxide accumulation could enhance the oxidation of bovine hemoglobin as Vc consumption, which could be the evidence of MetHb increased earlier in hemoglobin solution with higher Vc concentrations.

Figure 10. Schematic illustration of reactions related to pro-oxidant effects of Vc on bovine hemoglobin.

It was reported that the Vc addition could enhance the acidic environment in polyhemoglobin solution derived from human cord blood due to the degradation and reaction products of Vc [11]. The molecular structure and properties of hemoglobin was proven to be stable in physiological conditions and may be subjected to be oxidized or denaturalized under acidic conditions. Vc is an acid substance of enol molecular structure and its oxidation reaction could produce a series of acid products, leading to the decreasing of pH values in hemoglobin solution and thus have potential negative influences on the stability and functions of bovine hemoglobin. Therefore, the pH decreasing would be considered to be a notable problem in the future exploration of Vc application in bovine hemoglobin-derivates development.

In summary, Vc was proven to be capable of both antioxidant and pro-oxidation effects in bovine hemoglobin solution. Vc presented the prohibition of bovine MetHb formation in the initial stage of reactions and then accelerate the MetHb content increasing through the production of hydrogen peroxide, which could be indirectly characterized by the formation of choleglobin in the synchronous side reactions. The dual effects of Vc on the redox reactions of bovine hemoglobin could be confirmed through the Spectrophotometric spectrum analysis and could provide novel insight into the basic understanding about the redox properties of bovine hemoglobin. The results in this research suggested that, before exploration of Vc application in development of bovine hemoglobin-derivates, some attempts should be made to prohibit the pro-oxidation effects of Vc and the balance between antioxidant and pro-oxidant effects of Vc should be clearly and effectively established.

Acknowledgements

Dr. Gang Chen came to this laboratory as China Scholarship Council Visiting Scholar form the Institute of Blood Transfusion, Chinese Academy of Medical Sciences.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Professor Chang’s research has been supported by a grant from the Canadian Blood Service/Canadian Institutes of Health Research (CBS/CIHR) that requires the following statement: "the opinion expressed is not necessary that of the CBS/CIHR nor that of the government of Canada". The authors are grateful to the financial support from the Sichuan Province Key Research and Development Program (2017SZ0098), Youth Innovation Porject Plan on Medical Research from Sichuan Medical Association (Q16086), CAMS Innovation Fund for Medical Sciences (CIFMS, No. 2017-I2M-3–021). This research support for the current study reported here also comes from the China Scholarship Council.