No description available.

Milk proteins represent an important source of protein ingredients due to their distinctive physico-chemical, nutritional, technological and functional properties. Casein content of milk represents about 80% of milk proteins. The distinguishing property of phosphorylation provides important properties to caseins. The objectives of this research were to investigate enzymatic dephosphorylation of caseins, to characterize products of dephosphorylation and to examine the effects of dephosphorylation on biological properties of caseins. / Bovine whole casein, alpha-casein and beta-casein were dephosphorylated with potato acid phosphatase; optimum dephosphorylation conditions were 37°C, pH 5.8 for 6 h. The extents of dephosphorylation accounted for 71.6, 89.2 and 73.7% for whole casein, alpha-casein and beta-casein, respectively. The apparent Vmax and apparent K m for dephosphorylation of whole casein were 0.283 mumol P/mg casein min and 9.951 mg casein/l, respectively. SDS-alphaPAGE, urea-PAGE, RP-HPLC and ESI-MS demonstrated effects of dephosphorylation on the caseins. Urea-PAGE and ESI-MS confirmed the identities of the individual fractions. ESI-MS established (a) the molecular weight for alpha-casein and beta-casein as 23, 612 and 24, 017 Da, respectively; (b) random removal of 1, 2, 4, 6, 7 and 8 phosphate groups from alpha-casein and 1, 2, 3, 4 and 5 phosphate groups from beta-casein and (c) effects of incubation conditions. The effects of dephosphorylation of alpha-casein and beta-casein on the action of pepsin and trypsin were evaluated. Peptide mapping by RP-HPLC indicated that both proteases generated a complex mixture of peptides, with dephosphorylated peptides showing an increase in retention time. LC-ESI-MS and MS/MS in conjunction with the use of advanced bioinformatics software resulted in the identification of the peptides generated. Dephosphorylated alpha-casein and beta-casein showed the presence of peptides in which phosphate groups were removed, and were not observed in peptides from the corresponding native protein. Several of the peptides identified contained sequences that have been reported to be biologically active. Residual allergenicity of dephosphorylated whole casein, alpha-casein and beta-casein as well as peptic and tryptic products of these caseins was determined by an ELISA technique. The results demonstrated that removal of phosphate groups from whole casein, alpha-casein and beta-casein reduced allergenicity by 33, 31.2 and 24.4%, respectively. Proteolysis and dephosphorylation resulted in a significantly (p < 0.05) higher reduction in the antigen-antibody binding capacity compared to non-hydrolyzed and non-dephosphorylated caseins, particularly in the highly allergenic alpha-casein.

No description available.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [88]-92).

Thesis. / Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1942. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 79-83).

The main object of this thesis was to specifically modify histidine residues in κ-casein and KA1 and KA2 with the reagent 2-phenyl-1 ,4-dibromoacetoin (PDA) for the overall purpose of testing the hypothesis that histidine plays an important role in the stabilizing ability of κ-casein. Experiments designed to test the hypothesis that PDA causes κ-casein to aggregate by cross-linking were also carried out and these included the preparation of 2-phenyl-4-bromcacetoin (PMA) and reacting this with κ-casein and KA1 and KA2, as well as reacting PDA with histidine. Preliminary objectives of this thesis were to purify large amounts of κ-casein by scaling up the method of Zittle (66), to improve the electrophoresis technique described by Perrin (4l) and Peveridge (2) so that κ-caseir; subfractions could be seen, and to purify the sub-fractions by DEAE cellulose chromatography using the method of Mercier (28). The lowest yield of κ-casein during four attempts of scaling up Zittle's method was 6.0% and the highest yield was 7.5%. Two preparations of κ-casein resembled those published by McKenzie (23) both in amino acid composition and electrophoretic heterogeneity. Modifications in the electrophoresis technique which gave promising results included steps to increase the voltage through the gel equilibrating the gels by carrying out preliminary runs without samples, lowering the ionic strength of the system, changing the bridges, and increasing the concentration of urea in the gel. Suggestions by Mercier (28) which proved to be important in the separation of the subfractions of κ-casein by DEAE cellulose chromatography included sifting the cellulose before use, recrystallizing the urea, and using concentrations of NaOH and HCl below 1.0 molar during regeneration of the cellulose. Reactions of PDA and PMA with κ-casein, KA1 and KA2 did not support the hypothesis that histidine plays an important role in the stabilizing ability of κ-casein because l) PDA κ-casein was prepared, in low ionic strength buffer, which dissolved, normally and had the same stabilizing ability as untreated κ-casein, even though 0.6 histidine residue was modified; and 2) PMA KA1 and PMA KA2 were prepared which, although having 1.05 histidine and 1.60 lysine residues less than untreated KA1 and KA2, nevertheless had the same stabilizing ability as the untreated protein. Rather, these results support the contentions that l) the low stabilizing ability of PDA-κ-casein results from aggregation caused, by cross-linking of histidine residues on separate κ-casein molecules; and 2) the cross-linking may be dependent upon the ionic strength of the reaction. Removing one bromine from PDA to make PMA seemed to result in a change of selectivity from histidine to lysine. PMA did not react on κ-casein in the same way as it did on KA1 and KA2 and this supports the statements that future work on the modification of amino acids in κ-casein and studies on the interaction of this protein with κ-casein should be carried out on the subfraction rather than on κ-casein. Reactions involving PDA and histidine did not lead to an explanation of the mechanism of action of PDA because the yields of the products were too low to allow chemical analysis. Products having Rf ‘s of 0.62, 0.58, and 0.40 were produced and purified by cellex and paper chromatography in Jones' solvent (33) for the thin layer chromatographic separation of amino acids. These products reacted positively to Pauley's reagent, indicating that they were histidine addition products. / Land and Food Systems, Faculty of / Graduate

Studies were conducted to observe the changes in gel filtration elution profiles of casein fractions due to temperature, pH, sodium dodecyl sulfate (SDS) concentrations, and salts. Prom these results, the optimum conditions for separation were: I) elution with 5xI0ˉ³M borate buffer at pH 10 and room temperature to separate K-casein from acid casein; 2) elution with IxIO ˉ²M phosphate buffer at pH II and 4 C to separate K-casein from skimmilk; 3) elution with IxIOˉ³M SDS solution at room temperature to separate K- casein from acid casein and skimmilk; 4) elution with 5x10ˉ⁴M SDS solution at room temperature to separate α[subscript]si-casein from acid casein. Casein fractions were characterized for purity by acrylamide gel electrophoresis, sedimentation velocity centrifugation, stability of casein fractions in the presence of calcium ions, and sialic acid content. Compared to chemical preparations of casein fractions a reasonably pure K-casein fraction was obtained by gel filtration when eluted with buffers at pH 10 and pH II. Casein fractions obtained by SDS elution yielded α[subscript]si and K-caseins almost as pure as chemically prepared and purified caseins. However, considerable decrease in stabilizing ability was observed in K-casein eluted with SDS solution. It is possible that the decrease in stability is due to a binding of SDS with casein as the sulfur content of casein fractions prepared by the SDS methods was higher than that of chemically prepared casein fractions. There was slight variation in the sialic acid content of K-casein obtained by the different methods of preparations. / Land and Food Systems, Faculty of / Graduate

A model for explaining the differences in physical chemical properties of the caseins was proposed, and the results of the experiments supporting this model are presented. κ, αsl , β and whole-casein were shown to exhibit circular dichroic measurements typical of random coiled or denatured proteins, all being similar. Modification of the proteins with Na-tetrathionate, S-mercapto-succinic anhydride, reduction and esterification or changes in the solvent system with urea or polyethylenimine effected the spectra only slightly. Significant dichroic changes were observed when pH adjustments were made. The interaction of polyethylenimine or κ-casein with αsl produced interaction products which were detectable with a nuclear magnetic resonance spectrometer. The interaction of polyethylenimine and as αsl -casein were detected by electrophoresis, ultracentrifugation, light scattering, solubility and nuclear magnetic resonance. This interaction was dependent upon pH, concentration and amino nitrogen content. αsl and κ-caseins were observed to be heat labile while their interaction product was more stable than each individual casein. The results suggest that the interaction of αsl and κ-caseins is through carboxyl and amino electrostatic bonds, and that their association is purely random due to lack of stoichiometry in the molecules. The role of urea in the dissociation of caseins is the disruption of both ionic and hydrogen bonds. / Land and Food Systems, Faculty of / Graduate

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. / Land and Food Systems, Faculty of / Graduate