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Studies on the structure and interaction of bovine k-casein, using fluorometric techniques

University of British Columbia

The structure and interactions of κ -casein were studied using spectrofluorometric techniques including, fluorescent yield, emission wavelength, energy transfer, and fluorescence depolarisation. The effect of denaturing factors, temperature, sodium dodecyl sulfate, ionic strength, urea and pH, on the fluorescent characteristics of a conjugate of κ -casein and 8-anilinonaphthalene-l-sulfonate are indicative of structural changes in the protein. The importance of hydrogen bonding and hydrophobic interactions for the maintenance of the structural integrity of the molecule have been indicated. The structural changes are effected without the use of disulfide reducing reagents. The dissociating agents, urea and sodium dodecyl sulfate, had very significant effects upon the conformation of the protein. Temperature induced changes were related to the bonding which maintained the conformation. Completely dissociated or random structures did not exist except at high pH, in 8M urea, or in 0.005M sodium dodecyl sulfate. The interaction between α[sub s 1⁻] and κ -casein was studied by the fluorescence polarisation technique. The thermodynamic parameters Δ S and Δ H increased with temperature. Δ S and Δ H were positive. Δ F was negative and decreased further with increasing temperature. These data suggest that the ease or spontaneity of interaction increases with temperature, and that the interaction is hydrophobic in nature. The results of the interaction studies at 40 and 50°C demonstrated a 1:1 mole ratio for the interacting proteins. Blocking of the sites for electrostatic interaction, the negative charges on the α[sub s 1⁻] -casein, by polyethylimine did not inhibit the reaction or affect the interaction ratio. It was therefore concluded that α[sub s 1⁻] and κ -caseins interact through hydrophobic interactions in a 1:1 mole ratio. Kappa-casein was modified in a stepwise manner by (1) carbamylation of the ε -amino groups of lysine with potassium isocyanate (2) esterification of the free carboxylic groups using the carbodiimide method and glycine methyl ester, structural changes in the κ -casein, thus modified, were followed by measuring changes in fluorescent properties of a conjugate of the proteins with 8-ahilino-naphthalene-1-sulfonate. These structural changes were indicative of decreasing hydrophobicity of the caseins and loss of stabilising ability of α[sub s 1⁻] -casein against calcium ion precipitation. It is concluded that the charged groups are responsible for the maintenance of the protein structure, but may not be directly related to stabilising ability or interaction with α[sub s 1⁻] -casein.

Text

2011-03-31

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http://hdl.handle.net/2429/33137

Food Science

University of British Columbia

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