Studies on the functions of disulfide linkages in k-casein
Creators: name: Toma, Sadiq Jawad
Publisher: name: University of British Columbia
Year: 2010

description: A three part investigation is described in which a method for measuring SH and SS contents of K-casein and some other food proteins, utilizing Ellman's reagent, was developed. The role of disulfide groups in the stability of heated K-casein,i was studied after modification of those groups by desulfurization or alkylation. Sepharose gel chromatography was utilized to separate a pure K-casein fraction from whole casein or skimmilk. For the determination of SH groups in food proteins urea (6.5 M), Na dodecylsulfate (0.5%), and a mixture of 6.5 M urea and 5 M guanidine-HCl were used as dissociating agents for skimmilk, egg white, flour and gluten respectively. Disulfide groups were reduced with 1 to 2% mercaptoethanol in the presence of the same dissociating agents as for the SH determination and the proteins were precipitated by adding 8-11% trichloroacetic acid. Total SH (SH + reduced SS) was analyzed after dissolving the precipitates in 8 M. urea or 0.5% Na dodecylsulfate. (SDS) at pH 8.0. Values of SH. and SS for K-casein, ⍺s5-casein, ⍺sl-casein, β-lactoglobulin, ovalbumin, egg white, skimmilk, flour and gluten were in good agreement with literature values. Recoveries of SH and SS ranged from 91 to 98% and from 89 to 102% respectively. Modification of the disulfide groups in K-casein was performed by desulfurization with. Raney nickel under an atmosphere of hydrogen or by alkylation with iodoacetamide. Approxi- mately 50% of the SS groups were removed by the desulfurization reaction at pH 7.0 for 48 hours. The sedimentation coefficient decreased from 16 S to 12 S by desulfurization and K-casein migrated as bands by polyacrylamide-gel electrophoresis at pH 9.0 with 4 M urea compared to a smear for the untreated control. These results implicated the dissociation of K-casein by desulfurization. On heating for 30 minutes in boiling water, both the desulfurized and the alkylated K-caseins considerably decreased the ⍺sl -casein stabilizing ability whereas the control revealed no significant changes. The fluorescence polarization of modified K-casein was also decreased upon heating. Aggregated peaks at the void volume were observed when the heated modified K-casein was eluted on a Sepharose 2B column with phosphate buffer at pH 7.0, whereas untreated K-casein did not show those peaks upon heating. Interaction between 3-lactoglobulin and K-casein was observed by Sepharose gel filtration and polya-crylamide gel electrophoresis, when the mixture was heated. However, this interaction was not detected with the modified K-casoins. These results indicate the importance of disulfide groups in maintaining a structural integrity which controls heat stability of the molecules. The application of Sepharose 6B in the preparation of K-casein directly from skimmilk or whole casein is reported in the last chapter. Attempts were made to utilize the mildest possible condition for the preparation of K-casein by this technique. The effect of temperature, ionic strength and pH was studied. The best resolution and dissociation of the K-B-⍺sl-casein complex were obtained when whole casein was eluted with phosphate buffer 0.005 M, pH 9.0 at 25°C. Complete elimination of ⍺sl-casein was difficult. Introducing 3 M urea into the system eliminated all the ⍺sl,-casein contaminant and produced a pure K-casein fraction. Further improvement in the resolution was obtained by increasing the urea concentration to 6.6 M and an a ⍺s₅-casein rich fraction was obtained when whole casein or directly skimmilk were applied to the column. A relatively high yield was obtained, about 200 mg K-casein from 2 gm whole casein from a single run on a column of 4X77 cm length. K-casein fraction obtained by this method was capable of stabilizing 95% of the ⍺sl-casein at a ratio of 0.13 K — to 1.0 of ⍺sl-casein. The sedimentation coefficient was 14.0 S. The molecular weight of ⍺s₅-casein and its sensitivity to calcium was investigated and it was found that only 2 mM CaCl₂ was required for precipitation of 80% of the ⍺s₅-casein compared to 8 mM for ⍺sl-casein. The turbidity of ⍺s₅-casein in 10 mM CaCl₂ was 4.5 fold greater than that of ⍺sl-casein. Calcium ⍺s₅-caseinate was stabilized by K-casein to a lesser extent than Ca-⍺sl-caseinate. The molecular weight determined by the differential boundary method in an ultra-centrifuge were 65,750 and 31,800 for ⍺s₅--casein and for the mixture of ⍺s3 -and ⍺s4-casein respectively.