Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies.
The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 A, the casein micelles. Removal of Ca2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D2O was used instead of H2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, v, and correlation times, tau c, were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and v was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though tau c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.
Kumosinski
T F
TF
Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia, Pennsylvania 19118.
Pessen
H
H
Prestrelski
S J
SJ
Farrell
H M
HM
D002364:Caseins / Q000302:isolation & purification; D003903:Deuterium; D017666:Deuterium Oxide; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008823:Micelles; D008970:Molecular Weight; D011487:Protein Conformation; D014867:Water
D002364:Caseins; D046911:Macromolecular Substances; D008823:Micelles; D014867:Water; D003903:Deuterium; D017666:Deuterium Oxide