טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBen Shoshan Einav
SubjectAssociation Behavior of Beta-casein
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Danino Dganit


Abstract

The self-association behavior of bovine's milk β-casein was studied as a function of protein concentration and solution temperature, above the isoelectric point (pI), and for the first time also below the pI. The β-casein organization, morphology, size, and shape were studied by cryogenic transmission electron microscopy (cryo-TEM) and small angle X-ray scattering (SAXS). Fluorescence with ANS as probe was used to determine the critical micellar concentration (CMC), and analytical centrifugation was applied to determine the micelles molecular weight and to evaluate the aggregation number.

The results obtained at pH 2.1 in lactic acid solution were different from those obtained at pH 7 in buffer phosphate and ionic strength of 0.1.

At pH 7 globular micelles of about 15 nm in diameter were found by cryo-TEM. An ellipsoid shape was fitted by SAXS, with a circular radius of 7.4 nm and width of 12.3 nm. The CMC values at 25 0C and 40 0C were found to be 0.7 mg/ml and 0.35 mg/ml, respectively. These values are in good agreement with data obtained from ITC. Analytical centrifugation indicated a polydispersed system at this pH. At pH 2.1, up to at least 2 mg/ml the protein was found in its monomeric state. At high concentration of 20 mg/ml elongated, flat structures were observed by cryo-TEM. A disk model was fitted by SAXS, with a radius of about 10 nm, and width of ~ 2 nm.

The results indicate a relatively low organization state of the protein at low pH, relative to that found at natural pH. This may result from the high positive charge that is distributed along the protein molecule at low pH, which increases the electrostatic repulsions between monomers. In addition, under the conditions studied, the ionic strength is very low. In contrast, at pH 7 electrostatic repulsions are decreased due to neutralization of charge both inside the molecule, and from counter ions present in the buffer.