Vernetzte globuläre Proteine — eine neue Klasse halbsynthetischer Makromoleküle: Charakterisierung ihrer Struktur in Lösung am Beispiel hyperpolymeren Hämoglobins und Myoglobins mittels Volumenausschluß‐Chromatographie, Viskosimetrie, Osmometrie und Lichtstreuung

Abstract

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.

Oxygen Carriers (“Blood Substitutes”)Raison d'Etre, Chemistry, and Some PhysiologyBlut ist ein ganz besondrer Saft

“…Detailed O 2 binding studies suggested that α,α-cross-linking reduced O 2 affinity by affecting the intrinsic ligand binding properties of the heme rather than by changing allosteric contributions . Modification of Cys-93β usually resulted in increased O 2 affinity. ,,,, Polymerization and conjugation with neutral polymers quasi-generally increased O 2 affinity. ,,,,,,,, Oxygen affinity increased consistently with degree of polymerization and more so when the reaction was performed on oxyHb rather than deoxyHb. Interestingly, O 2 affinity was also increased by simple, non-cross-linking “decoration” of Hb with glutaraldehyde, indicating that the chemical modification itself, rather than direct conformational freezing by polymerization, may be responsible for the effect .…”

“…Further negatively charged cross-linkers used with Hb include DIBS 4.38 , , DIDS 4.39a , , its dihydrostilbene analogue 4.39b , and several series of carboxyl-group-bearing dialdehydes . Reaction of human HbA with DIBS 4.38 yielded a predominant product cross-linked within the tetramer between the NH 2 termini of the α chains .…”

“…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 .…”

“…Such polymerization was achieved using the carbodiimide EDCI 4.43 on human Hb internally cross-linked (XL-Hb, 4.83) with sebacoyl residues and bovine Hb internally cross-linked with adipoyl residues. 811 688,732 or porcine Hb. 469 Divinyl sulfone 4.58, for example, when allowed to react directly with deoxygenated human RBCs (i.e., on a highly concentrated Hb solution), produced, after hemolysis, neutralization of excess reagent, fractionation, and concentration, highly polydisperse "hyper"polymerized Hb with high P 50 values.…”

“…Comparison with intratetrameric cross-linked Hb products produced from diluted Hb solutions under otherwise similar conditions indicated that O 2 affinity had increased 2-to 5-fold upon polymerization and that there was considerable loss in cooperativity. Detailed studies have been published on the viscosimetry and structure in solution 732 and on MW determinations 813 for polymers obtained by reaction of glutaraldehyde or DIBS with RBCs.…”