Molecular structure and physical properties of type IV collagen in solution David E. Birk* and Frederick H. Silver Department of Pathology, University of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854, USA (Received 1 April 1986) The physical properties of intact type IV collagen from the mouse EHS sarcoma were studied in acid solution using laser light scattering and viscometry. The experimentally observed values of molecular weight, translational diffusion coefficient, particle scattering factor at 175.5 ° and a wavelength of 633 nm and intrinsic viscosity at 22°C were 532000, 0.66 x 10-7 cm 2 s-l, 0.492 and 74.7 ml/g respectively. Plots of Kc/Ro versus collagen concentration were linear with a slope of approximately O, indicating that under the conditions studied, type IV collagen molecules do not form supra-molecular aggregates. Experimentally determined translational diffusion coefficients closely approximated the calculated value for a rod-like molecule 424 nm long and 1.5 nm in diameter. Based on this observation, it is concluded that the type I V collagen molecule translates like a bent rigid rod similar to the interstitial collagens. However, the low intrinsic viscosity and larger value of the particle scattering factor for type IV collagen molecules in comparison with the interstitial collagens indicate that type I V collagen is considerably more flexible. Physical measurements on molecules in solution are consistent with a model of the type I V molecule containing numerous flexible bends with bend angles less than 125%It is concluded that the type IV collagen molecule behaves like a worm-like rod in solution. Keywords: TypeIV collagen;basementmembrane;lightscattering;EHS sarcoma Introduction Type IV collagen is the major structural protein in basement membranes 1-3. This ~;ollagen type is distinct from the interstitial collagens (types, I, II and III) in a number of ways. First, it does not form 67nm striated fibrils characteristic of the interstitial collagens. Secondly, the type IV molecule has distinct non-collagenous terminal regions and the helical region contains non-collagenous interruptions 3'4. Also, it is longer and more flexible than the fibril forming collagens 5-8. Finally, there is evidence that type IV collagen is incorporated into the extracellular matrix without prior processing9'1° A network model of type IV collagen within basement membranes has been proposed, primarily as a result of electron microscopic studies of rotary shadowed type IV molecules and aggregates4'1~. This model requires the interaction of type IV collagen with itself in two distinct ways. An interaction between two carboxy terminal globular ends and an interaction involving the amino terminal, disulphide rich 7S region of four molecules. More recently, a third type of interaction has been demonstrated where type IV collagen forms laterally associated complexess. We have studied the physical characteristics of type IV collagen in solution using laser light scattering techniques and viscometry. These data will contribute to the understanding of the interactive properties of the type IV collagen molecule and the differences between fibril * To whomcorrespondence shouldbe addressed. forming, interstitial collagens and non-fibril forming, basement membrane collagens. Materials and methods The EHS sarcoma was grown in C57/6J mice as previously described 12. The mice were made lathyritic by the inclusion of 0.1% fl-aminopropionitrile fumarate in the drinking water. Extraction and purification Type IV collagen was extracted as described by Kleinman et al. 1°. Briefly, the tumours were dissected, extracted and purified at 4°C in the presence of 10mu Na 2 EDTA, lOmM N-ethylmaleimide, 1 #g/ml leupeptin, 1 mM benzamidine-HC1. In addition, the initial washing and homogenization steps contained 0.1 M e- aminocaproic acid. The tumours were homogenized in 3.4M NaC1, 50mM Tris-HC1 at pH 7.4 followed by sequential extractions with: 0.5 M NaCI, 50 mu Tris-HCl, pH 7.4; 2.0M guanidine-HCl, 50mM Tris-HCl pH 7.4 and finally 2.0M guanidine-HCl, 50mM Tris-HC1 pH 7.4 with 2mM dithiothreitol (DTT). The final extract containing the type IV collagen was brought to 3.5 M NaCI to precipitate the type IV collagen. The pellets were resuspended in 0.5 M acetic acid, dialysed against the same solvent and the collagen precipitated by the addition of solid NaCI to 1.2M. The pellets were redissolved in 2.0M guanidine-HCl, 50mM Tris-HCl pH 7.4 with 2 mM DTT containing inhibitors and stored this way until used. This purified collagen was 0141-8130/87/010007-04503.00 © 1987 Butterworth & Co. (Publishers) Ltd Int. J'. Biol. Macromol., 1987, Vol 9, February 7