Reconstitution of Amyloid Fibrils from Alkaline Extracts

Solubilization and reconstitution is one of the key steps in the purification of fibrous proteins and in the clarification of certain of their physical properties. Since the description of amyloid as a fibrous protein (1-3), several preliminary studies in this direction have been reported for the amyloid fibril. Newcombe and Cohen (4) studied the effect of pH changes on solubility of amyloid and described a precipitate obtained from the alkaline extracts (pH 9.5 in glycine buffer) by reducing the pH to the apparent isoelectric point (pH 4.5). Glenner and Bladen (5) utilized the method of Newcombe and Cohen on a different amyloid extract and succeeded in reconstituting "periodic fibrils" which they believed to be a specific component of amyloid (6) and which have recently been identified in our laboratory as the P (plasma)component of amyloid (7, 8) by electrophoretic, immunologic, and morphologic means.' Benditt and Eriksen (9) also attempted to reconstitute amyloid fibrils from a fraction of a 6-8 M urea extract of amyloidotic tissue, but the nature of the products appears to be difficult to identify. A crucial problem in the above studies and in the identification of any solubilized and reconstituted macromolecule is an understanding of the subunit structure of the molecule. Recently the present authors have attempted to define precisely the subunit structure of the amyloid fibril and concluded that (10, 11): the amyloid fibril consists of a number of 75-80-A wide filaments assembled side-by-side; the amyloid filament is made up of several subunits, amyloid protofibrils, which are arranged almost longitudinally to the long axis of the filament and surround a central core 15-20 A in diameter; the amyloid protofibril is 25-35 A wide and appears to consist of two to three subunit strands, subprotofibrillar strands, helically arranged with a 35-50 A repeat (or less likely is composed of globular subunits aggregated end-to-end). It is