German Clinical Trials Register: DRKS00005993.

Developing an artificial oxygen carrier for use in humans, we polymerize native haemoglobin and myoglobin, using bifunctional, amino group specific cross-linkers, to soluble, socalled hyperpolymers. These polymers, like other polymerized globular proteins, are members of a new class of macromolecues which consist of macromolecular base units. They all have, due to the mechanisms of the chemical reaction, broad distributions of molecular weights. Fractions of hyperpolymers of human haemoglobin were obtained by employing preparative gel-permeation (size-exclusion) chromatography. The calibration curve of analytical gel-permeation chromatography (GPC) for haemoglobin hyperpolymers was determined using mean molec$ar weights of some fractions, as assessed by osmometric and light scattering measurements. In analogy to native globular proteins, the calibration curve for haemoglobin polymerswithin the range of molecular weights considered here, and within the experimental accuracyis a straight line. All fractions of haemoglobin polymers were further characterized with the aid of calibrated analytical GPC. Mean nonuniformity was about 0,6. The dependence of the logarithm of the intrinsic viscosity [4] on the logarithm of the viscosity-average molecular weight M, of the fractions (the curve in the "structure-in-solution diagram") also is a straight line, which is true for haemoglobin and for myoglobin polymers as well. Its first derivative is the exponent a of the Mark-Houwink function; for haemoglobin and myoglobin polymers the values are 0,39 and 0,46, respectively. Haemoglobin and myoglobin hyperpolymers, as members of the new class a) Diese Arbeit enthalt Teile seiner Dissertation.

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".

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