Merton L. Groves

The major component of human casein: A protein phosphorylated at different levels

Abstract A single protein, with 0–5 atoms of P per molecule, makes up the major portion of human casein. Also present is a fraction similar to bovine kappa-casein in its ability, in the presence of Ca2+, to solubilize the phosphoprotein us micelles.

Preparation of some iron-binding proteins and α-lactalbumin from bovine milk

Abstract The preparation of the iron-binding milk proteins— “red protein,” blood transferrin, and lactoperoxidase (donor: H 2 O 2 oxidoreductase, EC 1.11.1.7) — by chromatography on DEAE-cellulose and phosphocellulose is described. The “red protein” is distributed in the casein and whey fractions of milk. In some fractions it forms complexes with other proteins which drastically change its chromatographic behavior. The transferrins found in the milk and blood of an individual cow are shown to be the same by gel electrophoresis. The preparation of an electrophoretically homogeneous α-lactalbumin by chromatography on DEAE-cellulose is also described.

The separation of the components of α-casein. I. The preparation of α1-casein

Abstract A method is described for preparing α 3 -casein from α-casein involving the use of calcium chloride and ultracentrifugation. It was homogeneous by electrophoresis and sedimentation and contained 0.35% phosphorus, 1.2% sialic acid, and 0.75% hexose. The properties and composition of α 3 -casein are discussed in relation to the other components of α-casein.

Polymorphism of γ-casein in cow's milk

Abstract γ-Casein occurs in two forms, designated A and B, as measured by disc-gel electrophoresis. The results of the typing of milk samples from 165 cows for β- and γ-casein suggest a genetic basis for the polymorphism of γ-casein and a close relationship in the synthesis of β- and γ-casein.

Preparation of β- and γ-caseins by column chromatography

Abstract A new method for preparing β- and γ-casein is described consisting of fractionation of that portion of casein which is soluble at pH 4 at 2° followed by chromatography on ion-exchange cellulose. The homogeneity of the preparations is evaluated by means of starch-gel electrophoresis in the presence of urea.

Evidence from amino acid analysis for a relationship in the biosynthesis of γ- and β-caseins

Abstract γ-Casein, like β-casein, is polymorphic as shown by gel electrophoresis. Two variants of γ-casein, A 2 and B, have been isolated from samples of bovine casein genetically typed β-casein A 2 and B. The β-caseins were also isolated from the same samples. By comparison of the composition of the proteins it was shown that γ -A 2 differs from γ-B only in content of single residues of four amino acids and two substitutions, Ser/Arg and His/Pro are postulated. β -A 2 differs from β-B in the same manner, implying a close relationship in the synthesis of these milk proteins. The γ-caseins differ considerably from the β-caseins in content of proline and phosphorus. Comparison of the composition of four polymorphs of β-casein indicated that, in addition to the Ser/Arg and His/Pro substitutions, a third, Glu/Lys, may occur in the β-casein variants.

Environmental effects on disulfide bonding patterns of bovine kappa-casein.

Bovine κ-casein, the stabilizing protein of the colloidal milk protein complex, has a unique disulfide bonding pattern. The protein exhibits varying molecular sizes on SDS-PAGE ranging from monomer to octamer and above in the absence of reducing agents. Heating the samples with SDS prior to electrophoresis caused an apparent decrease in polymeric distribution: up to 60% monomer after 30min at 90°C as estimated by densitometry of SDS-PAGE. In contrast, heating the samples without detergent at 90 or 37°C caused a significant increase in high-molecular-weight polymers as judged by electrophoresis and analytical ultracentrifugation. In 6 M urea, the protein could be completely reduced, but upon dialysis, varying degrees of polymer reformation occurred depending on the dialysis conditions. Spontaneous reoxidation to polymeric forms is favored at low pH (<5.15) and low ionic strength. The results are discussed with respect to the influence of the method of preparation on the polymer size of κ-caseins and on their resultant physical chemical properties.

Plasmin cleaves human β-casein

Abstract Plasmin cleaves isolated human β-casein to form specific fragments in a manner similar to the generation of γ 1 -, γ 2 -, and γ 3 -caseins from the bovine homologue. Identification of a protein previously isolated from human milk as a specific plasmin cleaved portion of β-casein indicates that endogenous plasmin is active in whole milk. These findings suggest that protease activity should be considered in casein quantitation or isolation of components from human milk.

Separation of the components of α-casein II.: The preparation of α3-casein

Abstract A method is described for preparing α 3 -casein from α-casein involving the use of calcium chloride and ultracentrifugation. It was homogeneous by electrophoresis and sedimentation and contained 0.35% phosphorus, 1.2% sialic acid, and 0.75% hexose. The properties and composition of α 3 -casein are discussed in relation to the other components of α-casein.

The calcium-dependent electrophoretic shift of α-lactalbumin, the modifier protein of galactosyl transferase

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.