This article was downloaded by: [University of Otago] On: 22 July 2015, At: 20:27 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Biomaterials, Artificial Cells and Immobilization Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ianb18
Stabilities and Properties of Multilinked Hemoglobins a
a
a
K. W. Olsen , Q. Y. Zhang , H. Huang , G. K. a
Sabaliauskas & T. Yang
a
a
Department of Chemistry Loyola, University of Chicago, 6525 North Sheridan Road, Chicago, IL, 60626, USA Published online: 30 Mar 2015.
To cite this article: K. W. Olsen, Q. Y. Zhang, H. Huang, G. K. Sabaliauskas & T. Yang (1992) Stabilities and Properties of Multilinked Hemoglobins, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 283-285 To link to this article: http://dx.doi.org/10.3109/10731199209119644
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [University of Otago] at 20:27 22 July 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
B I O M A T . , ART. CELLS & IMMOB. BIOTECH., 2 0 ( 2 - 4 ) ,
283-285
(1992)
Downloaded by [University of Otago] at 20:27 22 July 2015
STABILITIES AND PROPERTIES OF MULTILINKED HEMOGLOBINS K. W.Olsen, Q.-Y. Zhang, H. Huang, G. K. Sabaliauskas and T. Yang Department of Chemistry Loyola University of Chicago 6525 North Sheridan Road Chicago, IL 60626, USA Crosslinking of both human and dog hemoglobin has been done with a variety of reagents to produce singly, doubly and multiply crosslinked hemoglobins. Succinate and glutarate diaspirins did not crosslink deoxy human hemoglobin in good yield, in contrast to the fumarate analog (DBSF). Deoxy dog Hb did not react well with DBSF, but oxy dog Hb did react, giving crosslinked tetramers as well as dimers on SDS electrophoresis. Crosslinking with a short, rigid reagent (difluorodinitrobenzene) resulted in a similar product for both oxy and deoxy hemoglobin that had high stability and oxygen affinity. The trilinker, trischloroethylamine, produced a more stable product than the corresponding crosslinker, bischloroethylamine. Double crosslinking oxy Hb with DBSF and dimethylpimelimidate or with DBSF followed by deoxygenation and recrosslinking with DBSF gave products with higher denaturation temperatures. The diaspirin double crosslinked product had high oxygen affinity.
INTRODUCTION Crosslinked hemoglobins have different properties than normal hemoglobin that make them potential blood substitutes. One of these important functional changes is the increase in stability that usually accompanies crosslinking (1-3). Additional stability allows a rapid separation of modified hemoglobin from the uncrosslinked species by thermal denaturation and inactivation of harmful viruses in the preparation (4). This communication will summarize the effects of crosslinking on the stabilities and properties of both human and dog hemoglobin. The modifications will include crosslinking with a single reagent, double crosslinking with two different reagents or with the same reagent under two different reaction conditions and multilinking with a reagent that reacts at three different sites simultaneously.
MATERIALS AND METHODS Human hemoglobin was prepared by ion exchange chromatography from outdated blood (5). Dog hemoglobin was supplied by M. W.Rooney (Loyola University Medical Center). The diaspirins were synthesized (6). Bischloroethylamine (BCEA) and 203 Copyright 0 1992 by Marcel Dekker, Inc.
284
OLSEN ET A L .
EibM
Increased Denaturation Tem w r m e s of Crosslinked Hemoelobb Modification DBSF-OXY DBSS-OXY DBSG-OXY DFDNB-OXY DFDNB-DEOXY DBSF-DOG Hb (dimer) DBSF-DOG Hb (dimer tetramer) BCEA TCEA DMP DBSF+DMP /3,aXL DBSF
Downloaded by [University of Otago] at 20:27 22 July 2015
+
AT ("C) 15.6 12.9 13.2 19.4 19.8 18.0 18.0 0.1 7.2 15.6 20.3 19.8
trischloroethylamine (TCEA) were purchased from Aldrich, difluorodinitrobenzene (DFDNB) from Sigma and dimethylpimelimidate (DMP) from Pierce. Modifications with the diaspirins were done with the standard methods for both oxy (7) and deoxy (8) hemoglobins. Other modification reactions were done with literature methods. The modified hemoglobins were purified on Sephadex A-50 (5) except for the diaspirin double crosslinked species which required chromatography on MonoQ resin. The thermal denaturations were done as previously reported (1,2). Oxygen binding was measured on a Hemox Analyzer.
RESULTS AND DISCUSSION (3,s-Dibromosalicyl)succinate (DBSS) and (3,5-dibromosalicyl)glutarate (DBSG) gave very poor yields with deoxy Hb when compared with that for DBSF. This greatly decreased reactivity is probably due to the greater flexibility of these reagents. Getting the reagent into the reaction site between the or99 lysines is like threading he eye of a needle. These reagents reacted with oxy hemoglobin, where the /382 Lys site is open to the solution. The stabilities were slightly less than that of DBSF-Hb (Table I), probably also due to the greater flexibility of the saturated hydrocarbon chains. Reaction of both oxy and deoxy human HbA with DFDNB at a 10:1 reagent ratio produced dimers on SDS gels, similar mobilities on alkaline agarose gels, similar T,'s (Table I) and high oxygen affinity. In addition to intrasubunit crosslinks, this reagent appears to have reacted between subunits in the same manner regardless of the degree of oxygenation. The short, stiff crosslink produced a very stable product. DBSF did not produce intersubunit crosslinks in deoxy dog Hb but gave three species with different crosslinks in oxy Hb. The first and last peaks off the A-50
MULTILINKED HEMOGLOBIN STABILITIES AND PROPERTIES
285
column gave monomers and dimers on SDS gels, whereas the second also showed tetramers. The lack of reactivity of deoxy dog Hb may be due to Leu-Phe at (~129, which would decrease the access to the a99 lysines. The additional crosslinking in oxy dog Hb could be due to the introduction of a Lys at 0104. The additional crosslinking in this species did not lead to a significant increase in the denaturation temperature (Table I).
Downloaded by [University of Otago] at 20:27 22 July 2015
The effect of a multilinker can be seen by comparing the results for BCEA and TCEA (Table I). The trilinker is more effective at stabilizing the protein. SDS gels of these modified proteins show only monomers and dimers for the BCEA-Hb but give monomers, dimers, trimers and some tetramers for the TCEA-Hb. Double crosslinking has been done two ways. First, Hb was crosslinked with DBSF to give either the a99 or 082 crosslinked protein which was then recrosslinked with DMP. The product was slightly more stable than either of the singly crosslinked hemoglobins (Table I) but it was also very heterogeneous due to the randomness of the DMP reaction. Second, oxy Hb was crosslinked with DBSF, then deoxygenated and reacted with DBSF a second time. After purification on MonoQ, the major product gave only dimers on SDS gels and was slightly more stable than the singly crosslinked products. This 0,aXLHb had high affinity, decreased cooperativity and was unaffected by inositol hexaphosphate.
ACKNOWLEDGEMENTS The authors would like to thank Drs. M. Rooney and L. Hirsch at Loyola Univ. Medical Center for supplying the dog hemoglobin. This work was sponsored by the Research Corporation and by the American Heart Association of Metropolitan Chicago.
REFERENCES 1. White, F.L., and Olsen, K.W. (1987) Arch. Biochem. Biophys. 258, 51-57. 2. Yang T., and Olsen, K.W. (1988) Arch. Biochem. Biophys. 261, 283-290. 3. Yang, T., and Olsen, K.W. (1991) Biochem. Biophys. Res. Comm. 174, 518-523. 4. Estep, T.N., Bechtel, M.K., Miller, T.J., and Bagdasarian, A. (1989) in Substi(T.M.S. Chang and R.P. Geyer, ed.), Marcel Dekker, 129-134. 5 . Dozy, A.M., Kleihauer, E.F., and Huisman, T.H.J. (1968) J. Chrom. 32, 723-727. 6. Zaugg, R.H., King, C.L., and Klotz, I.M. (1975) Biochem. Biophys. Res. Comm. 64, 1192-1198. 7. Walder J.A., Zaugg, R.H., Walder, R.Y., Steele, J.M., and Klotz, I.M. (1979) Biochemistry 18, 4265-4270. 8. Chatterjee, R.Y., Welty, E.V., Walder, R.Y.,Pruit, S.L., Rogers, P.H., Amone, A., and Walder, J.A. (1986) J. Biol. Chem. 261, 9929-9937.
Biomaterials, Artificial Cells and Immobilization Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ianb18
Stabilities and Properties of Multilinked Hemoglobins a
a
a
K. W. Olsen , Q. Y. Zhang , H. Huang , G. K. a
Sabaliauskas & T. Yang
a
a
Department of Chemistry Loyola, University of Chicago, 6525 North Sheridan Road, Chicago, IL, 60626, USA Published online: 30 Mar 2015.
To cite this article: K. W. Olsen, Q. Y. Zhang, H. Huang, G. K. Sabaliauskas & T. Yang (1992) Stabilities and Properties of Multilinked Hemoglobins, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 283-285 To link to this article: http://dx.doi.org/10.3109/10731199209119644
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [University of Otago] at 20:27 22 July 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
B I O M A T . , ART. CELLS & IMMOB. BIOTECH., 2 0 ( 2 - 4 ) ,
283-285
(1992)
Downloaded by [University of Otago] at 20:27 22 July 2015
STABILITIES AND PROPERTIES OF MULTILINKED HEMOGLOBINS K. W.Olsen, Q.-Y. Zhang, H. Huang, G. K. Sabaliauskas and T. Yang Department of Chemistry Loyola University of Chicago 6525 North Sheridan Road Chicago, IL 60626, USA Crosslinking of both human and dog hemoglobin has been done with a variety of reagents to produce singly, doubly and multiply crosslinked hemoglobins. Succinate and glutarate diaspirins did not crosslink deoxy human hemoglobin in good yield, in contrast to the fumarate analog (DBSF). Deoxy dog Hb did not react well with DBSF, but oxy dog Hb did react, giving crosslinked tetramers as well as dimers on SDS electrophoresis. Crosslinking with a short, rigid reagent (difluorodinitrobenzene) resulted in a similar product for both oxy and deoxy hemoglobin that had high stability and oxygen affinity. The trilinker, trischloroethylamine, produced a more stable product than the corresponding crosslinker, bischloroethylamine. Double crosslinking oxy Hb with DBSF and dimethylpimelimidate or with DBSF followed by deoxygenation and recrosslinking with DBSF gave products with higher denaturation temperatures. The diaspirin double crosslinked product had high oxygen affinity.
INTRODUCTION Crosslinked hemoglobins have different properties than normal hemoglobin that make them potential blood substitutes. One of these important functional changes is the increase in stability that usually accompanies crosslinking (1-3). Additional stability allows a rapid separation of modified hemoglobin from the uncrosslinked species by thermal denaturation and inactivation of harmful viruses in the preparation (4). This communication will summarize the effects of crosslinking on the stabilities and properties of both human and dog hemoglobin. The modifications will include crosslinking with a single reagent, double crosslinking with two different reagents or with the same reagent under two different reaction conditions and multilinking with a reagent that reacts at three different sites simultaneously.
MATERIALS AND METHODS Human hemoglobin was prepared by ion exchange chromatography from outdated blood (5). Dog hemoglobin was supplied by M. W.Rooney (Loyola University Medical Center). The diaspirins were synthesized (6). Bischloroethylamine (BCEA) and 203 Copyright 0 1992 by Marcel Dekker, Inc.
284
OLSEN ET A L .
EibM
Increased Denaturation Tem w r m e s of Crosslinked Hemoelobb Modification DBSF-OXY DBSS-OXY DBSG-OXY DFDNB-OXY DFDNB-DEOXY DBSF-DOG Hb (dimer) DBSF-DOG Hb (dimer tetramer) BCEA TCEA DMP DBSF+DMP /3,aXL DBSF
Downloaded by [University of Otago] at 20:27 22 July 2015
+
AT ("C) 15.6 12.9 13.2 19.4 19.8 18.0 18.0 0.1 7.2 15.6 20.3 19.8
trischloroethylamine (TCEA) were purchased from Aldrich, difluorodinitrobenzene (DFDNB) from Sigma and dimethylpimelimidate (DMP) from Pierce. Modifications with the diaspirins were done with the standard methods for both oxy (7) and deoxy (8) hemoglobins. Other modification reactions were done with literature methods. The modified hemoglobins were purified on Sephadex A-50 (5) except for the diaspirin double crosslinked species which required chromatography on MonoQ resin. The thermal denaturations were done as previously reported (1,2). Oxygen binding was measured on a Hemox Analyzer.
RESULTS AND DISCUSSION (3,s-Dibromosalicyl)succinate (DBSS) and (3,5-dibromosalicyl)glutarate (DBSG) gave very poor yields with deoxy Hb when compared with that for DBSF. This greatly decreased reactivity is probably due to the greater flexibility of these reagents. Getting the reagent into the reaction site between the or99 lysines is like threading he eye of a needle. These reagents reacted with oxy hemoglobin, where the /382 Lys site is open to the solution. The stabilities were slightly less than that of DBSF-Hb (Table I), probably also due to the greater flexibility of the saturated hydrocarbon chains. Reaction of both oxy and deoxy human HbA with DFDNB at a 10:1 reagent ratio produced dimers on SDS gels, similar mobilities on alkaline agarose gels, similar T,'s (Table I) and high oxygen affinity. In addition to intrasubunit crosslinks, this reagent appears to have reacted between subunits in the same manner regardless of the degree of oxygenation. The short, stiff crosslink produced a very stable product. DBSF did not produce intersubunit crosslinks in deoxy dog Hb but gave three species with different crosslinks in oxy Hb. The first and last peaks off the A-50
MULTILINKED HEMOGLOBIN STABILITIES AND PROPERTIES
285
column gave monomers and dimers on SDS gels, whereas the second also showed tetramers. The lack of reactivity of deoxy dog Hb may be due to Leu-Phe at (~129, which would decrease the access to the a99 lysines. The additional crosslinking in oxy dog Hb could be due to the introduction of a Lys at 0104. The additional crosslinking in this species did not lead to a significant increase in the denaturation temperature (Table I).
Downloaded by [University of Otago] at 20:27 22 July 2015
The effect of a multilinker can be seen by comparing the results for BCEA and TCEA (Table I). The trilinker is more effective at stabilizing the protein. SDS gels of these modified proteins show only monomers and dimers for the BCEA-Hb but give monomers, dimers, trimers and some tetramers for the TCEA-Hb. Double crosslinking has been done two ways. First, Hb was crosslinked with DBSF to give either the a99 or 082 crosslinked protein which was then recrosslinked with DMP. The product was slightly more stable than either of the singly crosslinked hemoglobins (Table I) but it was also very heterogeneous due to the randomness of the DMP reaction. Second, oxy Hb was crosslinked with DBSF, then deoxygenated and reacted with DBSF a second time. After purification on MonoQ, the major product gave only dimers on SDS gels and was slightly more stable than the singly crosslinked products. This 0,aXLHb had high affinity, decreased cooperativity and was unaffected by inositol hexaphosphate.
ACKNOWLEDGEMENTS The authors would like to thank Drs. M. Rooney and L. Hirsch at Loyola Univ. Medical Center for supplying the dog hemoglobin. This work was sponsored by the Research Corporation and by the American Heart Association of Metropolitan Chicago.
REFERENCES 1. White, F.L., and Olsen, K.W. (1987) Arch. Biochem. Biophys. 258, 51-57. 2. Yang T., and Olsen, K.W. (1988) Arch. Biochem. Biophys. 261, 283-290. 3. Yang, T., and Olsen, K.W. (1991) Biochem. Biophys. Res. Comm. 174, 518-523. 4. Estep, T.N., Bechtel, M.K., Miller, T.J., and Bagdasarian, A. (1989) in Substi(T.M.S. Chang and R.P. Geyer, ed.), Marcel Dekker, 129-134. 5 . Dozy, A.M., Kleihauer, E.F., and Huisman, T.H.J. (1968) J. Chrom. 32, 723-727. 6. Zaugg, R.H., King, C.L., and Klotz, I.M. (1975) Biochem. Biophys. Res. Comm. 64, 1192-1198. 7. Walder J.A., Zaugg, R.H., Walder, R.Y., Steele, J.M., and Klotz, I.M. (1979) Biochemistry 18, 4265-4270. 8. Chatterjee, R.Y., Welty, E.V., Walder, R.Y.,Pruit, S.L., Rogers, P.H., Amone, A., and Walder, J.A. (1986) J. Biol. Chem. 261, 9929-9937.
