Kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin as measured by small-angle neutron scattering.
The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into two half-molecular fragments was investigated at 21.0 degrees C by using small-angle neutron scattering. The relative change in molecular mass that occurs upon dissociation was monitored by recording the forward scattering of neutrons as a function of time. All these kinetic data can be explained by a reaction that is first-order with respect to the concentration of undissociated alpha 2-macroglobulin. The velocity constant is a function of urea concentration and it varies within wide limits. For instance, the half-life of the reaction at the lowest concentration of [2H]urea studied (2.70 M) is 328 h, whereas the same value at the highest concentration of [2H]urea (6.24 M) is only 8 min. Measurements were made both with [1H]urea in 1H2O and with [2H]urea in 99% 2H2O, and it was found that there is a pronounced kinetic isotope effect, i.e. the dissociation is 4 times faster in the 1H-containing medium as compared with the 2H-containing medium at the same molar concentration of urea. From the angular dependence of the neutron scattering it can be concluded that the dissociation is associated with a drastic change in structure. This is directly shown by the radius of gyration, which increases from about 7.4 nm immediately after the addition of urea up to about 9.4 nm when the protein is fully dissociated. A structural analysis shows that the scattering curve of urea-dissociated alpha 2-macroglobulin can best be explained by that of a Gaussian coil with a radius of gyration equal to 9.44 nm. These data indicate that the so-called non-covalent interaction of alpha 2-macroglobulin probably is more complicated than just a pure hydrophobic interaction. Finally, it is also shown that the dissociation is accompanied by a loss in trypsin-binding activity, which is directly related to the fraction of dissociated protein.
Sjöberg
B
B
Department of Medical Biochemistry, University of Göteborg, Sweden.
Pap
S
S
Järnberg
S E
SE
Mortensen
K
K
D000818:Animals; D006801:Humans; D007554:Isotopes; D007700:Kinetics; D009502:Neutrons; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000494:pharmacology*; D000511:alpha-Macroglobulins / Q000187:drug effects / Q000378:metabolism*
D007554:Isotopes; D000511:alpha-Macroglobulins; D014508:Urea; D014357:Trypsin