Molecular weight distribution curves determined by electron microscopy exhibit a deviation when approaching a molecular weight of 10'. Picturing of single molecules therefore was improved by the introduction of shadowing by electron beam. Techniques of preparation (by freeze-drying) and shadowing are described in detail. The scanning size of the electron beam shadowing is in between 15 and 20 A and is given by the structure of the supporting film. By double shadowing from different sides it is demonstrated, that there is no "decoration effect" in electron beam shadowing. The lower limit of an exact determination of the dimensions is a molecular weight of ca. lo5. It is shown with latex particles that there is a change of shape with time of the particles after sedimentation on the support which is dependent on the kind of polymer and of the conditions. Their volume though remains constant within the limits of error.

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.

ZUSAMMENFASSUNG:Bei der elektronenmikroskopischen Abbildung von Einzelmolekulen treten eine Reihe von praparativen, Schwierigkeiten auf, welche dazu gefuhrt haben, daS man trotz zahlreicher Arbeiten diese Methode bisher noch nicht zu einem Standardverfahren entwickeln konnte. Ein Haupthindernis stellt das Auftreten von Artefakten dar, die den nichtkristallinen Polymermolekiilen tauschend ahnlich sehen.Zur Erkennung und Vermeidung dieser ,,ScheinmolekCle" ist es notwendig, quantitative Praparations-und Kontrollmethoden zu entwickeln. Es eignen sich zwei Verfahren : einmal die sogenannte Tropfenmethode und zum anderen die Gefriertrocknuugsmethode. Mit Hilfe dieser Verfahren gelingt es, das Artefaktproblem zn losen und die Homogenitat der Praparate zu testen. SUMMARY:The imaging of single polymer molecules by electron microscopy faces a lot of difficulties with regard to preparation, thus preventing this method from developing to a standard technique, in spite of numerous attempts. A main obstacle is the appearance of artefacts which look strikingly similar to polymer molecules.It is necessary to develop preparation methods enabling quantitative analysis in order

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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 (2,5-diisothiocyanatobenzenesulfonate (DIBS); 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate (DIDS); 1,3-butadiene diepoxide (BUDE); glutaraldehyde (GDA)) in concentrated (case 1) and diluted (case 2) hemoglobin solutions. With high concentration hyperpolymer and with low concentration only monomer products were obtained. P50 and n50 of the products were determined at pH = 7.4, PCO2 = 40 mmHg, temp. = 37 degrees C. The difference of properties in both cases are regarded as the influence of polymerization per se. Considering this difference we found with almost all combinations of hemoglobin and crosslinker an increase of O2 affinity, with DIBS and DIDS cooperativity was not changed and with BUDE and GDA it was decreased. As compared with native hemoglobin loss of cooperativity is considerable in any combination and condition, but comparing human and bovine hemoglobin the first seems to maintain better cooperativity. In contrast bovine hemoglobin as compared with human hemoglobin maintains better or even decreases its O2 affinity upon reaction with the crosslinkers forming both, monomer and hyperpolymer products, especially in the deoxy state. DIBS and DIDS react very similarly.(ABSTRACT TRUNCATED AT 250 WORDS)