Formation and Structure of Gels and Fibrils from Glucagon

Abstract

Glucagon in acid solution aggregates to produce gels and ultimately fibrils of ,&chains. The kinetics of the aggregation have been followed by viscometry, and are found to exhibit a sigmoidal profile with a long lag phase, associated with the formation of nuclei for polymerisation. This has been demonstrated by the elimination of the lag when fresh solutions are seeded with a preformed glucagon gel. The aggregation is promoted by salt, increased pH (within the acid range of solubility) and increased temperature up to 30". The addition of 5 0 / / , of dioxan completely inhibits the aggregation. The interaction has evidently a large hydrophobic driving force, and is opposed by coulombic repulsion. The latter effect can be minimised by acetylation of the amino groups, when the polymerisation is greatly accelerated. This also occurs when only the terminal amino group is blocked by carbamylation. By contrast esterification of the carboxyl groups completely prevents aggregation. At the working pH this does not involve a change in charge, and thus appears to be a specific steric effect. Optical rotatory dispersion shows no conformational difference between native glucagon and the derivatives. The transition from a gel to a fibrillar form is promoted by salt and increased temperature. A possible explanation for the appearance of the fibrillar form in the electron microscope is that it contains tubular structures 50-70 in diameter, with about 40 extended chains around the circumference lying in antiparallel manner along the axis.Glucagon, which is a polypeptide hormone containing 29 residues, possesses the property of aggregating at acid pH, where its solubility in water is high, with the formation ultimately of gels and/or fibrils. We have previously shown [l] that both these aggregated forms are in the antiparallel /3-conformation, and thus differ from other fibrillar structures, such as for example, insulin [2]. I n alkaline solution the molecule behaves in an altogether different manner : here there appears to be a specific concentrationdependent association equilibrium, resulting in the formation of a-helical structures [3-51. Glucagon thus presents a remarkable example of a molecule delicately balanced between three different conformational states, between which it can transform in response to only modest changes in the overall charge. The object of this investigation is to determine what factors lead to the preferential stabilisation of one particular form, and also to examine the nature of the acid aggregation reaction, and the interrelation between the two types of P-containing aggregates. MATERIALS AND METHODSGlucagon was the crystalline elcctrophoretically pure product of the Novo Research Institute, generously supplied to us by Dr. J. Schlichtkrull. Reagents were analytical grades. Dioxan was purified by distillation from sodium, followed by fractional crystallisation.Acetylation of glucagon was performed by addition of a 100-fold molar excess of acetic anhydride in two portions to a suspension a t 12 mg/ml i...

An unusual bovine pancreatic protein exhibiting pH-dependent globule-fibril transformation and unique amino acid sequence.

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Interaction of the anthracycline 4'-iodo-4'-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis.

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Optimization of the Native Glucagon Sequence for Medicinal Purposes

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