Non‐Aggregated Tropocollagen at Physiological pH and Ionic Strength

Abstract

Collagen was isolated from the skins of normal and lathyritic rats. Amino acid analysis, sugar analysis and analysis of the subunit composition indicated that the two types of collagen differed only with regard to the degree of cross-linking. Investigations by physical chemical methods showed that the lathyritic collagen preparations behaved as free monomers below 15 "C a t physiological ionic conditions with the following characteristics: s :~,~ = 2.92 S, D:,,w = 0.78x lo-' cm2/sec, [rj ] = 11.1 dl/g. Mr from light scattering, sedimentation equilibrium and sedimentation-diffusion was 310000-320000. Light scattering data agreed with a rod-like structure and the molecular length from light scattering, frictional ratio and intrinsic viscosity was 300 nm. Preparations of normal collagen were aggregated under physiological ionic conditions and different reasons for the aggregation are discussed. It is suggested that lathyritic collagen should be a good model substance in studies a t the molecular level of fibrillogenesis and interactions between collagen and other macromolecules. Tropocollagen, which can be isolated by various extraction procedures [6] from different types of tissues, has the inherent ability to form fibers of native type, when the temperature is raised to 37 "C and the pH is near 7 [7]. The fiber formation seems to be influenced by the extent of cross-linking of the tropocollagen molecule [8,9] For studies in vitro, under physiological conditions, of fiber formation from tropocollagen as well as of interactions between tropocollagen and glycosaminoglycans it is desirable to use tropocollagen in monomeric form. The isolation of native tropocollagen in monomeric form is, however, cumbersome and has so far only been successful a t acid pH [2,6,17]. When studied in neutral salt solutions, the tropocollagen molecules have invariably been aggregated [17,18] and light scattering measurements have given molecular weights corresponding to polymeric forms of tropocollagen, in spite of previous strenuous centrifugation [17,18]. The lower molecular weights obtained by sedimentation equilibrium techniques [ 171 have probably been due to a removal of aggregated material from the solution. I n the present investigation, lathyritic collagen has for the first time been characterized in solution by physical chemical methods. The results show, that this collagen occurs in monomeric form a t physiological pH and ionic strength a t temperatures ranging between 4 and 15 "C. For comparison, neutral saltsoluble and citrate-soluble collagens from the skin of normal rats have been partly characterized. EXPERIMENTAL PROCEDURE Materials3-Amino-propionitrile was supplied by AB Pharmacia, Uppsala and 3-amino-propionitrile fumarate was supplied by Schuchardt (Munich).All chemicals were reagent grade or the highest quality available. Isolation and Purification of CollagenCollagen was isolated from the skins of normal and lathyritic male Sprague-Dawley rats, ranging in weight from 50-150 g. Animals were made lathyritic by feeding ...

Collagen fibrillogenesis: intermediate aggregates and suprafibrillar order.

“…1). Similar observations were made on solutions prepared from the lyophilized lathyritic chick collagens, or from any of the nonlathyritic preparations, but in addition these showed a number of early aggregates, similar to those to be described below, despite vigorous centrifugation (10). If nonlyophilized lathyritic stocks were stored in neutral condition at 40 for more than 1-2 days, aggregates were apparent.…”

The Influence of Glycosaminoglycans on the Formation of Fibers from Monomeric Tropocollagen in vitro

“…The collagen preparation (preparation 111) used in the present investigation was isolated from skins of lathyritic rats as described previously [15]. The protein occurred as free monomer under physiological ionic conditions a t temperatures between 4 " and 15 "C. Solutions of the collagen were freshly prepared every week as described [15].…”

“…The protein occurred as free monomer under physiological ionic conditions a t temperatures between 4 " and 15 "C. Solutions of the collagen were freshly prepared every week as described [15]. Prior to all experiments the light scattering of these solutions was measured [15].…”

“…The discrepancies may in part reflect differences between various collagen preparations with regard to source, method of isolation, purity and state of aggregation [15]. Furthermore, the experimental conditions have varied.…”

“…The collagen employed was a well-characterized lathyritic preparation, which a t pH 7.4, I = 0. 15 and temperatures below 15 "C consisted exclusively of monomers [is]. The results obtained have been related to the polysaccharide-collagen interaction that occurs a t 4"C, when there is no spontaneous fiber formation [13].…”