Separation of Collagen Chains and Chain
Fragments by Gel Filtration Methods of gel filtration are described for the separation of collagen u-chains from B and y components. These methods are also adapted to separating peptides of different sizes cleaved from collagen chains. The polypeptides are eluted in volatile buffers from heated columns of Sepharose CL or Sephacryl beads.
The collagen molecule is built from three colinear, hydrogen-bonded, polypeptide chains of 95,000 molecular weight. In the course of fibril formation the chains in the molecules become linked through intra-and intermolecular bonds with the formation of B (2ar-chain) and y (3cy-chain) components and larger aggregates. The hydrogen bonds maintaining the organization of the chains in the molecule may be disrupted by heating to 40ยฐC or by treatment with denaturants and the (Y, /3, and y components that are liberated can be detected by physical methods. A valuable and widely used technique for analyzing and separating these polypeptides was described by Piez (1). In the original procedure collagen was dissolved and denatured in a warmed solution of 1 M CaCl,, 0.05 M Tris, pH 7.5 (this concentration of calcium ions lowers the denaturation temperature of collagen from 37 to near 20"(Z), and the mixture of polypeptides was then applied to a column of 8% agarose gel beads in the calcium chloride-Tris buffer. A single passage at room temperature through a long column sufficed to separate the (Y chains from the /3 components. The same procedure on an agarose or Bio-Rad P150 polyacrylamide bead column was useful for separating peptides of different sizes cleaved from the collagen chains by chemical or enzymatic means. The disadvantage of these procedures is the necessity to remove the CaCl, before the collagen chains or peptides can be recovered by lyophilization or selective precipitation; CaCl, is normally removed by dialysis or gel filtration but either way this procedure is time consuming. Since the original publication the conditions of the experiment have been modified by various workers; 4 or 6% agarose gels have been substituted for the 8% at times, and guanidine hydrochloride (e.g., ref.
- and other denaturants have been substituted for CaCl,, but all of these procedures suffer from the same defect, the time-consuming step necessary to remove the denaturant.
Collagen can be readily denatured by heating to 40-45"C, but the agarose gel column is not satisfactory under these conditions. With the availability of a cross-linked agarose gel it is feasible to perform the chain separation at denaturing temperatures and in a volatile buffer so that the effluent