Influence of the Polymerization Step Alone on Oxygen Affinity and Cooperative During Production of Hyperpolymers from Native Hemoglobins with Crosslinkers
Abstract:The aim of this study was to find out how the polymerization per se changes oxygen affinity (P50) and cooperativity (n50) of various soluble huge hyperpolymers prepared from native hemoglobins by crosslinking. Increase of cooperativity would be expected considering natural hemoglobin networks. Those hyperpolymers with molecular weights of some 10(6) g/mol are candidates for artificial oxygen-carrying blood additives rather than volume substitutes. Human and bovine hemoglobin reacted with several crosslinkers (… Show more
“…1,2:3,4-Diepoxybutane 4.59 undergoes ring-opening reactions with nucleophiles, primarily thiols, but also amines and hydroxyl groups. It was used for Hb polymerization …”
Section: S-alkylation Of Hemoglobinmentioning
confidence: 99%
“…a. Glutaraldehyde. Glutaraldehyde is the reagent most frequently utilized for intermolecular cross-linking of Hb, pyridoxalated Hb, and other Hb derivatives (see, for example, refs , , , , , , , −777, 792 and 799). This highly reactive cross-linker has long been employed as a nonspecific tissue fixative .…”
Pharmaceutical Corporation in San Diego; however, the opinion expressed here are solely his and not necessarily those of the Corporation. Jean G. Riess received his chemical engineer and doctorat-e `s-sciences degrees (the latter with Professor Guy Ourisson in 1963) from the University of Strasbourg. He then spent two years with John Van Wazer at Monsanto in Saint-Louis, MO, learning some phosphorus and transitionmetal chemistry. In 1968 he became Professor at the University of Nice, France, where he founded, directed, and eventually became honorary director of the Unite ´de Chimie Mole ´culaire (associated with the Centre National de la Recherche Scientifique). His research successively involved phosphorus chemistry, transition-metal chemistry, organometallics, and eventually perfluorochemicals. His present interests are in fluorocarbons, fluorinated amphiphiles, and their colloid chemistry, including fluorocarbon emulsions for in vivo O 2 delivery, fluorinated self-assemblies, and drug delivery systems. Professor Riess has published about 360 papers and holds ca. 25 patents. He has served on numerous councils and committees and has chaired or co-chaired international conferences on phosphorus chemistry and blood substitutes. He has won awards from the French Academy of Sciences, French Chemical Society, Alexander von Humboldt Stifftung, City of Nice and Controlled Release Society, as well as Alliance's first Distinguished Contribution Award. He holds an honorary Research Associate position at the University of California at San Diego and sits on the Board of Directors of Alliance Pharmaceutical Corp.
“…1,2:3,4-Diepoxybutane 4.59 undergoes ring-opening reactions with nucleophiles, primarily thiols, but also amines and hydroxyl groups. It was used for Hb polymerization …”
Section: S-alkylation Of Hemoglobinmentioning
confidence: 99%
“…a. Glutaraldehyde. Glutaraldehyde is the reagent most frequently utilized for intermolecular cross-linking of Hb, pyridoxalated Hb, and other Hb derivatives (see, for example, refs , , , , , , , −777, 792 and 799). This highly reactive cross-linker has long been employed as a nonspecific tissue fixative .…”
Pharmaceutical Corporation in San Diego; however, the opinion expressed here are solely his and not necessarily those of the Corporation. Jean G. Riess received his chemical engineer and doctorat-e `s-sciences degrees (the latter with Professor Guy Ourisson in 1963) from the University of Strasbourg. He then spent two years with John Van Wazer at Monsanto in Saint-Louis, MO, learning some phosphorus and transitionmetal chemistry. In 1968 he became Professor at the University of Nice, France, where he founded, directed, and eventually became honorary director of the Unite ´de Chimie Mole ´culaire (associated with the Centre National de la Recherche Scientifique). His research successively involved phosphorus chemistry, transition-metal chemistry, organometallics, and eventually perfluorochemicals. His present interests are in fluorocarbons, fluorinated amphiphiles, and their colloid chemistry, including fluorocarbon emulsions for in vivo O 2 delivery, fluorinated self-assemblies, and drug delivery systems. Professor Riess has published about 360 papers and holds ca. 25 patents. He has served on numerous councils and committees and has chaired or co-chaired international conferences on phosphorus chemistry and blood substitutes. He has won awards from the French Academy of Sciences, French Chemical Society, Alexander von Humboldt Stifftung, City of Nice and Controlled Release Society, as well as Alliance's first Distinguished Contribution Award. He holds an honorary Research Associate position at the University of California at San Diego and sits on the Board of Directors of Alliance Pharmaceutical Corp.
“…1B). This procedure circumvents the problems associated with in vivo expression of genes coding for long tandem repeats of globular proteins and produces a higher degree of polymerization than other methods, such as using crosslinking agents (11) or genetic protein engineering (5).…”
Section: Experimental Procedures Synthesis Of Polymersmentioning
Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ؎ 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal͞mol.
“…However, few studies have reported on cross‐linking proteins of biological activity with EGDE. Barnikol14 tested an epoxide, 1,3‐butadiene diepoxide (BUDE), as a cross‐linker of hemoglobin (Hb) to investigate the influence of cross‐linking on protein activity. There was no further investigation.…”
Being widely applied in the chemical industry, ethylene glycol diglycidyl ether (EGDE) was tried as a protein cross-linker for cross-linking of bovine hemoglobin (Hb). The characteristics of the reaction and the products were studied using electrophoresis and multi-angle laser light scattering coupled with HPLC. At pH 7.5, EGDE could react with the sulfhydryl group and the ε-amino group, but reacted preferentially with the sulfhydryl group. As the pH increased from 7.5 to 9.5, the reaction took place mainly between the amino groups and EGDE. By controlling the pH and other conditions, intra-and inter-molecularly cross-linked products with narrow size distribution were obtained.
INTRODUCTIONChemical cross-linking of two peptide chains has many applications such as preparation of enzyme-antibody conjugates, stabilization of a protein structure or modification of protein properties. Various crosslinkers are available with different reaction chemistries. For large-scale applications, one has to consider the cost of the cross-linker and the ease of operation in addition to the suitability of the conjugated product. To date, the most popular cross-linker for proteins is glutaraldehyde which is highly active towards amine groups of a peptide chain at room temperature in aqueous solution. However, the cross-linking reaction with glutaraldehyde is complicated.1 The presence of α,β-unsaturated glutaraldehyde polymers interferes with the desired reaction. The Schiff base formed is unstable and requires subsequent reduction. As a consequence, glutaraldehyde cross-linking of hemoglobin often results in formation of a complex mixture. This leads to difficulties in process validation and quality control.Ethylene glycol diglycidyl ether (EGDE), whose structural formula is shown in Scheme 1, has been widely used in epoxy resin preparation.
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