Characterization of a half-molecular fragment obtained by reduction of human alpha 2-macroglobulin with dithiothreitol. A small-angle X-ray and neutron scattering investigation.
A half-molecular fragment of alpha 2-macroglobulin has been prepared by reducing and alkylating the inter-subunit disulfide bonds in the tetrameric alpha 2-macroglobulin molecule with 1 mM dithiothreitol (40 min) and 3 mM iodoacetamide (40 min). Further purification was made by gel chromatography and the homogeneous population of half-molecules has been characterized by the techniques of small-angle X-ray and neutron scattering. The radii of gyration found by the two methods are 57.0 and 58.0 A, respectively. The match point, obtained by neutron scattering from solutions with different H2O/D2O ratios, is at 43% D2O; the data are consistent with a particle having a higher scattering density at large distances from the particle centre. From the X-ray and neutron intensities scattered at zero angle, the specific volume was determined to be 0.73 cm3/g at +5 degrees C and the molecular weight to be 390,000; the latter value is associated with a relatively large error due to the uncertainty in the concentration determination. Shape analysis indicates that the best-fitting scattering-equivalent three-axial bodies are oblate shaped, with two of their axial dimensions about three to four times larger than the third one. From the volume of the best-fitting scattering-equivalent three-axial bodies, 0.72 X 10(6) A3, we obtain a water content equal to 0.38 g H2O/g protein (dry weight).
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D004229:Dithiothreitol; D006801:Humans; D009502:Neutrons; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation; D014965:X-Rays; D000511:alpha-Macroglobulins
D000511:alpha-Macroglobulins; D004229:Dithiothreitol