Molar Masses and Structure in Solution of Haemoglobin Hyper-Polymers - A Common Calibration of Size Exclusion Chromatography of These Artificial Oxygen Carriers

Abstract

We are developing artificial oxygen carriers for medical use, based on synthetic polymers--so-called hyperpolymers--obtained by cross-linking mammalian haemoglobins. One requirement with respect to the polymers is that they should not increase the oncotic pressure of blood remarkably--this can be realized by high molecular weights of the polymers with a narrow distribution. They may act as a oxygen transporting blood additive, and--in combination with a plasma expander--as a blood substitute. Another important and desired property of the artificial oxygen carrier is a low viscosity, which--first--is due to a high degree of uniformity of the polymer size (or molar mass) distribution and--second--is influenced by the so-called structure in solution of the haemoglobin hyperpolymers. In this paper former determinations of molar masses--with size exclusion chromatography (SEC)--and of the structure in solution--using viscometric measurements--of hyperpolymers of human haemoglobin, synthetized with glutaraldehyde and with bis(thioisocyanato) benzenesulfonic acid as cross-linkers, were extended to hyperpolymers of bovine and pyridoxylated porcine haemoglobin, cross-linked with glycolaldehyde. These determinations were done by applying a new iterative procedure. Within a range of error all SEC calibration curves found were the same for all hyperpolymers investigated. So-called MARK-HOUWINK or structure in solution diagrams (logarithm of intrinsic viscosity versus logarithm of molar mass) yield equal straight lines for all the haemoglobin polymers. The first derivatives of these lines are the MARK-HOUWINK exponents which has a mean value of 0.38. These results indicate that there exists a common SEC calibration line for all different polymer haemoglobins produced with comparable preparative procedures. This calibration line differ significantly from that of native globular proteins--haemoglobin hyperpolymers are less compact--so a calibration of SEC with globular proteins for the determination of molar masses of haemoglobin polymers is erroneous. Furthermore, the structure in solution of the hyperpolymers is clearly different from that of flexible, randomly coiled, linear artificial polymers: hyperpolymers are more compact. A possible explanation is that the hyperpolymers--according to a great number of functional amino groups of haemoglobin-contain many intra-polymeric cross-links, and thus are at least import branched polymers or even macromolecular networks of the constituting haemoglobin "monomers".

Haemoglobin Hyperpolymers, a New Type of Artificial Oxygen Carrier – Concept and Current State of Development

“…The picture gives the impression that the hyperpolymer is very bulky and that the molecule is not as compact as a globular protein. In accordance with this, the polymer chemical Mark-Houwink coefficient for the hyperpolymer in aqueous solution is 0.38 [14]. Characteristic values for the oxygen binding of the artificial carrier together with those for other haemoglobin preparations are given in table 1.…”

“…The transmission electron microscope (TEM) bright-field micrograph shown in figure 4 is produced by M. Rosenbaum [18] who moreover verified the structure using iron spectroscopy. Molar masses (molecular weights) were determined by volume exclusion chromatography (using the gel Sephacryl S-400 HR, Amersham Biosciences Europe GmbH, Freiburg i.Br., Germany), the calibration of which was carried out indirectly with a method [14] based on the measurement of light scattering [18] and oncometry. Analysis of the oxygen equilibrium binding of the haemoglobin polymers in solution, in particular the characteristic values for p50 and n50, was carried out in an apparatus (the so-called binding curve chamber) using the method described by Barnikol et al [19], which is a modification of the method of Niesel and Thews.…”

Supramolecular assembling systems formed by heme–heme pocket interactions in hemoproteins

“…For example, the reaction of lysine residues with glutaraldehyde forms randomly linked aggregates because there are several reaction points. 109 The reaction of a maleimide dimer via a flexible linker with a protein having at least two cysteine residues on its surface, has been also investigated. 110 However, this reaction gives only small oligomers, with the largest being B7 mers.…”

Chapter 5 Size-exclusion chromatography

“…The analysis of poly(bisphenol A carbonate) by SEC/ MALDI was reported by Puglisi et al [231 ]. Potzschke et al [234] studied the molar masses and structure of hemoglobin hyperpolymers, commonly used in calibrations for SEC of these artificial oxygen carriers. Two slightly differing SEC calibration plots were obtained, due to the smaller hydrodynamic volume of the polycarbonate cyclic chains relative to the linear ones.…”