Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Process Biochemistry 42 (2007) 303–309 www.elsevier.com/locate/procbio The role of pH and its control on effective conjugation of bovine hemoglobin and human serum albumin Chunyang Zheng a,b, Guanghui Ma a,b, Zhiguo Su a,b,* a National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China b Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Received 24 May 2006; received in revised form 21 August 2006; accepted 22 August 2006 Abstract Human serum albumin (HSA) and bovine hemoglobin (Hb) conjugate is a promising candidate as a blood substitute. However, preparation of the conjugate is problematic because both proteins tend to conjugate between themselves rather than crosslink each other. In this work, a facile process for conjugation of Hb and HSA was developed through control strategy of the reaction. The reaction was carried out in a buffer containing borax-borate and mannite. The borax-borate was used for pH buffering while mannite was used as a pH switch and a reaction promoter. As a result, self-conjugation of Hb and self-conjugation of HSA were minimized. After the one-step conjugation reaction in aqueous solution, followed by the one-step purification by ion-exchange chromatography, the conjugate of HSA and Hb was obtained with the total yield about 50%. The P50 and the Hill coefficient for the product were 16.1 mmHg and 1.82, respectively. # 2006 Elsevier Ltd. All rights reserved. Keywords: Human serum albumin (HSA); Bovine hemoglobin (Hb); Borax-borate buffer–mannite system; Conjugate; Blood substitute 1. Introduction Blood transfusion is a key method in emergency rescue and in some medical operations such as open-heart surgery. However, the shortage of available blood, the requirement of blood-typing, and the danger of transmitting certain diseases are serious problems. To circumvent these problems, blood substitutes have been proposed to replace real human blood in various medical treatments [1]. Among different blood substitutes, hemoglobin-based blood substitute has been the focus of research. Polymerized bovine hemoglobin has been approved for medical use in South Africa [2]. Polyethylene glycol modified hemoglobin is also under clinical trial [3]. Recently, a human serum albumin (HSA) and bovine hemoglobin (Hb) conjugate was reported to be a good candidate for blood substitute [4]. Considering the case of severe blood loss, especially hemorrhage, HSA is also needed [5], the HSA–Hb conjugate is theoretically assumed to be a * Corresponding author at: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China. E-mail address: [email protected] (Z. Su). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.08.011 better blood substitute than pure hemoglobin. Moreover, as a hydrophilic macromolecule with anionic charge under physiologic conditions, HSA could also render the conjugate a negative surface charge and make it away from the endothelial cells lining the lumen of the circulatory system, which was speculated to further prolong the half-time of the substitute [6]. However, in the trial of Bonhard and Boysen to couple albumin and hemoglobin [7], a single-step conjugation reaction using homobifunctional crosslinker resulted in self-conjugation of HSA and/or Hb. When cross-linking two proteins, the crosslinker may react initially with either one of the proteins, forming an active intermediate. This intermediate further reacted with a second protein through the free end of the crosslinker. Because the reaction was not specific, the chance for reaction with the same protein and with another protein would be the same. To circumvent this problem, the two-step procedure was established. First, one of the proteins to be conjugated is reacted with the homobifunctional reagents, and then excess crosslinker and by-products, e.g. self conjugates or polymers, are removed. The downstream processing of the monomer-activated protein is difficult because the physicochemical properties of the self conjugates or polymers and the activated monomer are very similar. In the second stage, the separated activated protein is mixed with the other protein molecules to be conjugated often in 304 C. Zheng et al. / Process Biochemistry 42 (2007) 303–309 reagent grade. Water used in the experiments was prepared with a RiOs ultrapure water system (Millipore, Bedford, MA). 2.2. Hemoglobin preparations Bovine blood erythrocytes were obtained freshly from a local slaughterhouse, followed by osmotic hemolysis and stroma removal with centrifugation at 30,000 g for 30 min. The stroma-free Hb solutions were purified by selective DEAE cellulose adsorption, essentially as described by Cheung et al. [12]. 2.3. Determination of relationship between mannite content and pH value Fig. 1. Scheme of the coordinating mechanism of the Borax-borate buffer– mannite system. large amount to ensure reaction, and the final conjugation process occurs between two proteins. A post-separation is also required to remove the unreacted proteins. The two-step conjugation is generally time-consuming and at low yield, which limits the application of this method in the scale-up production [8]. Therefore, minimizing the ineffective self-polymerization without engineered properties during the conjugation process is a challenge to the preparation and further investigation of this kind of candidate blood substitute. Borax-borate buffer–mannite system within the pH range 4.0–6.0 has once been applied to create pH gradient to study the micro-heterogeneity of human serum albumin [9]. The original pH value of the borax-borate buffer is about pH 9.0. By adding appropriate mannite to the borax-borate buffer, the pH of the borax-borate buffer system could be switched easily in the large range from 4 to 9. On the other hand, as a polyhydroxy compound, mannite forms easily dissociating complex with boric acid as shown in Fig. 1 [10,11]. The formation of the dissociating complex could influence the pKa of the available groups and then microenvironment of the reactant proteins, which is an important factor to facilitate the conjugation process. In this work, the effect of pH of the reaction system on conjugation process using homobifunctional glutaraldehyde as crosslinker was investigated, and two separate pH ranges were found to be effective to minimize the formation of the self conjugates of HSA and self conjugates of Hb, respectively, which made it possible to control the reaction by changing pH of the reaction buffer. Therefore, Borax-borate buffer–mannite system is utilized presumably to function both as a pH switcher and a reaction promoter. This method offers means to facilely prepare the conjugates of HSA and Hb while minimizing the formation of self conjugates by-products of HSA and/or Hb. The yield is about 50%, and the production circle is about 6 h with only one-step ion exchange chromatography separation involved. 2. Materials and methods 2.1. Chemicals and reagents Human serum albumin (HSA, fraction V) and glutaraldehyde (50%, v/v, grade I) were from Sigma. All other chemicals obtained were of analytical The relationship between mannite content (w/v) and pH value of the boraxborate system with original pH 9 was investigated. After mannite was fully dissolved, the pH of the Borax-borate buffer–mannite system was determined and averaged of three repeated measurements. 2.4. Preparation of the conjugates One hundred milligrams HSA was dissolved in the 10 ml of 20 mM, pH 9, borax-borate buffer. Then, 480 ml of 5% (v/v) glutaraldehyde was added to the activated HSA molecules. The activation reaction was carried out for 30 min at 4 8C, followed by the addition of mannite till the pH fell to 5.7. One milliliter of 100 mg ml 1 Hb solution was added, and the reaction was allowed to continue for another 4 h at 4 8C, after which excessive Lysine was added to block further reaction. 2.5. Purification of the conjugate The reaction mixture was subjected to the ion-exchange chromatography DEAE Sepharose Fast Flow. The column (10 cm 1.6 cm) was equilibrated by 10 mM PBS buffer, pH 7.0 (buffer A), and eluted with 0–0.1 M NaCl gradient in buffer A. The flow rate was 0.5 ml min 1. The fractions containing the conjugates were pooled and subjected to further analysis. 2.6. SDS-PAGE SDS-PAGE was operated as the method of Laemmli [13]. The samples were diluted with one volume of SDS sample buffer containing 0.1 M dithiothreitol and boiled for 5 min, followed by electrophoresis for 1 h at 200 V. The gel was stained in Coomassie Blue R-250 and then destained. 2.7. Oxygen equilibrium measurements O2 equilibrium curves were measured on a Hemox Analyzer (TCS, PA) at 37 8C, using the Hemox buffer (pH 7.4). The analyzer measures the O2 pressure with a Clark O2 electrode (Yellow Springs Instrument, OH) and simultaneously calculates the Hb saturation via a dual-wavelength spectrophotometer. Values for P50 (the O2 pressure at which Hb is half saturated) are obtained from the O2 equilibrium curves. 3. Results 3.1. Conventional single-step conjugation Glutaraldehyde is a common crosslinker widely used in the preparation of conjugates; however, as a result of high activity, the self conjugates by-products plague its further precise application, especially in the therapeutic field. Fig. 2 shows the GF-HPLC profile of the resultant mixture prepared at pH 8 followed by the conventional single-step conjugation method. The product was indeterminate. The reactant proteins were C. Zheng et al. / Process Biochemistry 42 (2007) 303–309 Fig. 2. Characterization of the conjugation preparation using glutaraldehyde as crosslinker by the conventional single-step method. The samples were loaded on a Shodex gel filtration column, followed by elution at a flow rate of 0.5 ml min 1. 305 of the reactant proteins, and therefore impact interaction of them and the following reaction. As shown in Fig. 3, while the pH of the reaction mixture was more than 9, the selfconjugation of HSA was inhibited, and the activated HSA was in the monomeric formation (Fig. 4a), with trace HSA dimmer that originally existed in the native HSA material; while the pH of the reaction mixture was less than 6, the selfconjugation of Hb was inhibited, and the reaction of Hb and glutaraldehyde was limited to the intra-crosslinking of Hb (Fig. 4b). Hb has molecular weight of 64 kDa, and during SDS-PAGE the four subunits dissociated and presented a band with about 16 kDa, and after intra-molecularly crosslinked two additional bands appeared with the molecular weight of about 32 and 48 kDa as shown in the inset (Fig. 4b). The results demonstrated that whatever pH value was selected as the working pH to couple HSA and Hb, the resultant mixture was sure to include at least the ineffective byproducts of HSA polymer or Hb polymer. highly crosslinked, and even insoluble precipitate was formed. Therefore, proper control strategy of the reaction was needed to minimize the formation of the self conjugate by-products and ensure the production of the conjugates with engineered merits. 3.2. The effect of pH on the conjugation process 3.2.1. The effect of pH on the ineffective self conjugation of HSA or Hb The pH value plays an important role on the cross-linking reaction of proteins. It is clear that high pH value could make the reactive amino group less protonated. The de-protonated amino group is the nucleophilic species and thus can facilitate the reaction between the crosslinker and the functional groups on the protein. On the other hand, the pH of the reaction buffer also has an influence on the charge condition Fig. 3. The effect of pH on the formation of polyhaemoglobin and polyalbumin. The reaction samples under various pH values were determined using GFHPLC. The percentage of the uncoupled monomer was represented by the area of the peak of monomer on the chromatogram. The content of polymer was calculated by the formula of (100% monomer%). The results were obtained from the average of three repeated measurements. Fig. 4. The determination of the reaction samples of HSA (a) (detected by the Shodex column) and Hb (b) (detected by the TSK3000sw column for the slight change of elution behavior) under the selected pH condition using GF-HPLC. After the modification of glutaraldehyde, HSA still remained as monomer, and the subunits of Hb were crosslinked. The inset shows the characterization of the intracrosslinked Hb using SDS-PAGE. Lane (1), native Hb; lane (2), standard marker; lane (3), intracrosslinked Hb. The stacking and running gels were 5% and 15% in acrylamide, respectively. 306 C. Zheng et al. / Process Biochemistry 42 (2007) 303–309 Fig. 5. The conjugate and by-product yield as the function of pH. The conjugation reaction was carried out at 4 8C for 1 h under different pH values, and terminated by addition of excessive lysine solution. The components were determined by TSK3000sw column. The yields of the by-products (crosslinked products except effective conjugate of HSA and Hb) and conjugate were estimated by integrating the chromatograms of the reaction products. The results were obtained from the average of three repeated measurements. Fig. 7. The plot of pH as the function of the content of mannite in borate buffer. The results were obtained from the average of three repeated measurements. conjugation, was reasonable to minimize the ineffective self conjugation while promoting the effective conjugation between HSA and Hb. 3.3. Acid-adjusted two-step conjugation 3.2.2. The effect of pH on the effective conjugation of HSA and Hb The effect of pH on the effective conjugation of HSA and Hb was also investigated at different pH conditions. Fig. 5 indicates that, the pH between two pIs of the reactant proteins (the pIs of HSA and Hb are 4.7 and 6.8, respectively) promoted the formation of the effective conjugation. Therefore, it was assumed that, pH value influenced the conjugation process not only via the protonated/de-protonated condition of the functional groups, but via the net charge of reactant proteins. While the reactant proteins were oppositely charged, the conjugation between them was promoted by the electrostatic interaction. Thus, a pH-switch strategy that first activating HSA with glutaraldehyde at pH 9 and then changing pH to 5.7 (average of the pI of HSA and Hb) by mannite and adding Hb for further Fig. 6. Characterization of the resultant mixture prepared by acid-adjusted conjugation reaction. The sample was loaded on the Shodex gel filtration column, followed by elution of 50 mM PBS, plus 0.1 M Na2SO4. Firstly, acid-adjustment was tried to change the pH of the reaction system. However, after the addition of hydrochloric acid reduced the pH of the reaction system to pH 5.7, as shown in Fig. 6, compared with the conventional single-step method (Fig. 2), the crosslinking degree was controled, but yield of the conjugate of HSA and Hb was low. 3.4. Conjugation reaction in Borax-borate buffer–mannite system Borate has the properties of coordinating with polyhydroxyls resembling mannite, and the pH of borax-borate buffer decreases as the increase of mannite added, as shown in Fig. 7. The pH value of the borate buffer containing 3% Fig. 8. Characterization of the resultant mixture prepared in the Borax-borate buffer–mannite system. The sample was loaded on the Shodex gel filtration column, followed by elution at a flow rate of 0.5 ml min 1 of 50 mM PBS, plus 0.1 M Na2SO4. C. Zheng et al. / Process Biochemistry 42 (2007) 303–309 307 Fig. 9. The conjugate yield as the function of duration of reaction. The conjugation reaction was carried out at 4 8C, and terminated by adding excessive lysine. (w/v) approximated 5.7, the working pH chosen to increase the electrostatic interaction of the reactant proteins and then improve the conjugation of them. Compared with the acidadjusted conjugation reaction (Fig. 6), the conjugate yield of the conjugation reaction in the borate–mannite system was markedly increased (Fig. 8). The effect of time duration of the reaction over 30 h periods was also investigated to achieve the best performance of the conjugation reaction. Fig. 9 shows the kinetics of the conjugate yield as a function of reaction time. After 6 h, the formation of 1:1 conjugate of HSA and Hb began to plateau, while the accumulation of the conjugates of other ratio relations of HSA and Hb continued to increase in another 12 h. Thus, the duration of 6 h was used to ensure the maximal 1:1 conjugate yield and limit the crosslinking degree of conjugates in other stoichiometry. Fig. 11. Characterization of the purified conjugates by the DEAE Sepharose Fast Flow ion-exchange chromatography (a), native HSA (b), and native Hb (c). The samples were loaded on the Shodex gel filtration column, followed by elution at a flow rate of 0.5 ml min 1 50 mM PBS, plus 0.1 M Na2SO4. 3.5. Purification of the conjugates The reaction mixture was loaded on a DEAE Sepharose Fast Flow column. Peak (b) in Fig. 10 appeared by elution with 0– 0.1 M NaCl gradient in buffer A. The sample corresponding to peak (b) was pooled and subjected to the gel filtration HPLC determination. As presented in Fig. 11a, the result indicated that the uncoupled Hb was removed. And most of activated HSA was conjugated with Hb. Fig. 12 shows the SDS-PAGE profile of the reaction mixture (lane 6), peak (a) (lane 1) and peak (b) (lane 5) in Fig. 10. After the purification, the uncoupled Hb with potential nephrotoxicity was removed in peak (a), and for peak (b) (lane 5), the lack of the band with about 130 kDa appeared in trace amount in lane 4 and band with 32 kDa appeared in lane 6 demonstrated that the both the self conjugate of HSA and that of Hb were inhibited. The concentration of the preparation was determined by Bradford method, and the total yield of the conjugates was about 50%. 3.6. Bioactivity examination of the preparation Fig. 10. Purification of the conjugate using ion-exchange chromatography on DEAE Sepharose Fast Flow and monitored by the UV absorbance at 280 and 405 nm (the special absorbance wavelength of Hb). The column was equilibrated with buffer A. After the sample was loaded on the column, the column was eluted with buffer A, followed by elution with 60 min, 0–0.1 M NaCl gradient in buffer A. The flow rate was 0.5 ml min 1. The O2 equilibrium curves of the conjugate and native bovine Hb were shown in Fig. 13. It was evident that the curves of the conjugate changed in position and shape. By comparison with that of native bovine Hb (P50 = 27.1 mmHg, n = 2.76), the P50 value and the Hill coefficient were 16.1 mmHg and 1.82, 308 C. Zheng et al. / Process Biochemistry 42 (2007) 303–309 Fig. 12. Characterization of the conjugate using SDS-PAGE. Lane (1), peak (a) in Fig. 10; lane (2), native Hb; lane (3), standard marker; lane (4), native HSA (loaded in large amount for the trace HSA dimmer); lane (5), peak (b) in Fig. 10; lane (6), the reaction mixture before separation. The stacking and running gels were 5% and 15% in acrylamide, respectively. control the conjugation process. It assumedly exerted two functions in the conjugation process of HSA and Hb: (1) pH switcher: in the original pH 9 borax-borate buffer, the HSA was first activated by crosslinker glutaraldehyde, and the conversion of functional groups on HSA to aldehyde blocked further self-polymerization of the HSA molecules in the following reaction. After mannite was added, the pH of the system was switched to the pH range that could inhibit the self-polymerization of Hb. In the Borax-borate buffer– mannite system with pH 5.7, the hemoglobin was added to the system and reacted with the activated HSA. Therefore, both the ineffective by-products of HSA self-polymer and the Hb self-polymer were inhibited in the Borax-borate buffer– mannite system with mannite functioning as a pH switcher. Moreover, (2) as a reaction promoter, when added to the borax-borate buffer, mannite coordinated with the compounds and formed a easily dissociating complex, which changed the micro-environment of the reactant proteins and facilitated the conjugation between HSA and Hb. On the other hand, the addition of mannite also reduced the ion strength of the system. Since the decreasing the ionic strength could increase the attraction between the oppositely charged proteins and/or molecules [14]. It was speculated that the reduction of ion strength and the following increase of attraction between HSA and Hb, oppositely charged under the given pH condition, also contributed to the increase of conjugation yield. This might be also a practical and facile method to conjugate other proteins or peptides with novel engineered merits. Acknowledgement The authors are grateful for the support from National Natural Science Foundation of China (grant no. 20136020). References Fig. 13. O2 equilibrium curves of native bovine hemoglobin (a) and the conjugates (b). respectively. It showed that the coupling of HSA somewhat impair the cooperativity of the subunits of hemoglobin. Besides, the conjugate was saturated at the normal physiological range of O2 pressure (90–100 mmHg), indicating a good performance of the conjugate for O2 delivery. 4. Discussion The Borax-borate buffer–mannite system with flexible pH range and special dissociation properties was first applied to [1] Rameshraja Palaparthy BS, Huashan Wang MD, Anil Gulati MD. Current aspects in pharmacology of modified hemoglobins. Adv Drug Deliv Rev 2000;40:185–98. [2] Levy JH, Goodnough LT, Greilich PE, Parr GVS. Polymerized bovine hemoglobin solution as a replacement for allogeneic red blood cell transfusion after cardiac surgery: results of a randomized, double-blind trial. J Thorac Cardiovasc Surg 2002;124:35–42. [3] Nishi K, Yamakawa T, Yano K, Ohta T. Transfusion of polyethylene glycol conjugate of pyridoxylated human hemoglobin in dogs with hemorrhagic shock. Biomater Art Cells Art Org 1988;16(1–3):653–5. [4] Lu XL, Zheng CY, Shi XD, Wang YQ, Suo XY, Yu PZ, et al. Conjugate of bovine hemoglobin and human serum albumin as a candidate for blood substitute: characteristics and effects on rats. Art Cells Blood Subs Biotech 2005;33(2):83–99. [5] Theodore P. All about albumin: biochemistry, genetics, and medical applications Academic Press; 1995. [6] Hai TT, Pereira DE, Nelson DJ, Catarello J, Srnak A. Surface modification of diaspirin cross-linked hemoglobin (DCLHb) with chondroitin-4-sulfate derivatives. Part 1. Bioconjug Chem 2000;11:705–13. [7] Bonhard KH, Boysen UF. Preparation of coupled hemoglobin molecules. US Pat 4336348 (1981). [8] Hermanson GT. Bioconjugate techniques Rockford, IL, USA: Pierce Chemical Company; 1996. C. Zheng et al. / Process Biochemistry 42 (2007) 303–309 [9] Azhitskii GIu, Troitskii GB, Malyi KD. Human serum albumin: microheterogeneity and hemin-binding ability. Biokhimiia 1976;41(4):597–603. [10] Rodnikova NV, Rybinskaya MN, Ostrovskaya GV. Chemical analytical methods for boron- and barium-zircon ceramic glazes. Glass Ceram 1963;20(11):597–600. [11] Kolotilina NK, Dolgonosov AM. Ion-chromatographic determination of borates and sulfides with the use of a developing column. J Anal Chem 2005;60(8):738–42. 309 [12] Cheung LC, Storm CB, Gabriel BW, Anderson WA. The preparation of stroma-free hemoglobin by selective DEAE/cellulose absorption. Anal Biochem 1984;137:481–4. [13] Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [14] He LZ, Gan YR, Sun Y. Adsorption–desorption of BSA to highly substituted dye–ligand adsorbent: quantitative study of the effect of ionic strength. Bioproc Eng 1997;17:301–5.