Structure sous‐unitaire de la thyroglobuline de mouton

Abstract

Listen

Translate article

Full reduction of the disulfide bridges of pure 19 S sheep thyroglobulin has been obtained by 2‐mercaptoethanol in 0.1 M Tris‐acetate pH 8.6 containing 8 M urea followed by alkylation (iodoacetate or iodoacetamide) of the thiol groups formed. The S‐carboxymethylderivative is soluble only in the presence of concentrated urea or guanidine hydrochloride. Dissolved in the presence of 5 M or 6 M guanidine at pH 7.0 or 8.6 it sediments in the ultracentrifuge as a single homogeneous boundary. In 5 M or 6 M guanidine at 20° and 25° the sedimentation constants extrapolated to zero concentration are: s°20, guan 6 M= 1.2 S; s°25, guan 5 M= 2.0 S. The values for the diffusion constant and for the intrinsic viscosity are: D°25, guan 5 M= 3.4; 100 [η]20, guan 6 M= 85; 100 [η]25, guan 5 M= 78. Using these values, 0.705 for v and the values of s/D determined by equilibrium or approach‐to‐equilibrium sedimentation, the following molecular weight have been calculated: M1= 80 × 103 or 85 × 103 (equilibrium sedimentation), M2= 81 × 103 (approach‐to‐equilibrium in 0.017 M SDS at pH 8.6), M3= 70 × 103 (Svedberg equation). The molecular weight computed from the values of s and [η] in 5M and 6M guanidine were 160 × 103 and 114 × 103 respectively. By gel filtration on Sephadex G‐200 in 0.1 M Tris‐acetate pH 8.6 containing 0.017 M sodium dodecylsulphate, the S‐carboxymethyl derivative of thyroglobulin is more retarded than γ‐globulin 7 S and serumalbumin. After correction for protein‐bound sodium dodecylsulphate the apparent molecular weight calculated for S‐carboxymethyl thyroglobulin by gel filtration agrees fairly well with the molecular weight (80 × 103) computed from the hydrodynamic properties. A similar value has been obtained by gel filtration on Sephadex G‐200 in Tris‐acetate 0.1 M pH 8.6 containing 5 M guanidine. The discrepancy between the values of the molecular weight computed from the measurements of s and [η] on the one hand and by the measurement of s/D or s and D on the other hand is discussed in relation to data in the literature. It is concluded that the 12 S subunit of thyroglobulin is most probably constituted by two different peptide chains (A = B = 80 × 103) in a proportion which remains to be determined.