John W. Holland

Maillard Reaction and Protein Cross-Linking in Relation to the Solubility of Milk Powders

Protein changes in relation to solubility, Maillard reaction (MR), and protein cross-linking in whole milk powder (WMP), skim milk powder (SMP), and whey protein concentrate (WPC) stored at different relative humidities (RHs) were investigated by chemical and electrophoretic methods. WMP and SMP reached minimum solubility rapidly, while WPC showed no change in solubility. The loss of solubility corresponded with development of high-molecular-weight protein complexes observed by two-dimensional electrophoresis. The maximal MR rate occurred at 66% RH for WMP and SMP (high lactose/protein ratios) and 84% RH for WPC (low lactose/protein ratios) based on the furosine and hydroxymethylfurfural contents. However, browning was greatest at 84% RH in all powders. The minimum solubility corresponded with the casein and fat contents. The retention of solubility and minimal protein cross-linking of WPC compared to casein-containing powders suggest that the casein content and cross-linking strongly influence the decrease in the solubility of milk powder.

A proteomic approach to detect lactosylation and other chemical changes in stored milk protein concentrate.

Milk proteins undergo chemical changes such as lactosylation, deamidation and protein cross-linking during processing and storage of milk products. A proteomic technique combining two-dimensional gel electrophoresis and mass spectrometry was used to investigate chemical modifications to proteins, in milk protein concentrate (MPC80), during storage. Lactosylation, deamidation and protein cross-linking were observed on 2-DE gels. They were storage temperature-, humidity- and time-dependent. Lactosylated whey proteins were well separated on 2-DE in vertical stacks of spots. The masses of the spots varied by multiples of 324, indicating the attachment of lactose to lysine residues in the proteins. The trypsin-digested spots of α-lactalbumin were analysed by MALDI-TOF mass spectrometry, which indicated multiple lactosylation sites. The lactose adducts on gels were quantified by image analysis, allowing development of adducts over time to be monitored. The results show that proteomics can be used for the detection and quantification of chemical modifications to proteins in stored MPC80.

Post-translational Modifications of Caseins

Publisher Summary Caseins are phosphoproteins and constitute about 80% of the protein in milk. Caseins exhibit a high degree of heterogeneity as a result of post-translational modifications (PTMs). Phosphorylation of the α- and β-caseins and glycosylation of κ-casein are the best-known modifications and are critical for the formation and stability of casein micelles. κ-Casein has long been known to exhibit a high degree of variability in glycosylation. This chapter summarizes current understanding on PTMs of the αs1-, αs2-, β-, and κ-caseins. Caseins are assembled in a colloidal complex with calcium phosphate and small amounts of other minerals. The casein micelle structure is critical in determining the physical properties of milk and for the provision of amino acids, calcium, and phosphorus for infant nutrition. A critical factor in micelle formation and stability is the presence PTMs such as phosphorylation, glycosylation, and perhaps disulfide bond formation that occur in the endoplasmic reticulum and/or Golgi complex after synthesis of the polypeptide chain. In recent years, the development of proteomic technologies has greatly enhanced our ability to analyze milk proteins, particularly with respect to PTMs. Two-dimensional electrophoresis (2-DE) provides a high-resolution methodology for displaying the heterogeneity of the major milk proteins. Advances in mass spectrometry (MS) have enhanced our ability to analyze the proteins arrayed on 2-D gels. Therefore, not only is it possible to resolve many proteins and their isoforms but it is also possible to characterize them, particularly with respect to the many PTMs that affect their electrophoretic mobility. Furthermore, the chapter includes an extended discussion on the functional significance of κ-casein heterogeneity.

Analysis of disulphide linkages in bovine κ-casein oligomers using two-dimensional electrophoresis

Disulphide bonds play an important role in protein structure and function. Bovine κ‐casein (κ‐csn), an important glycoprotein in milk, contains two cysteines that can form disulphide bonds. On 2‐D gels run under nonreducing conditions the κ‐csn in milk presented a complex pattern of monomers and disulphide‐linked oligomers. Trains of spots corresponding to monomers to hexamers were observed as a result of the participation of different glycoforms and phosphoforms in oligomer formation. The dimers and trimers ran as doublets on the gel and analysis of the disulphide‐linked peptides released from them after in‐gel tryptic digestion showed they were the result of different disulphide linkages. The linkages were confirmed by MSMS. When milks with electrophoretically distinct genetic variants of κ‐csn were mixed and run on 2‐D gels, they retained their distinct patterns indicating that disulphide exchange reactions or disulphide ‘scrambling’ was not occurring during 2‐D analysis. The patterns observed represent the native distribution of κ‐csn in milk at harvest. The role and significance of the disulphide bonding of κ‐csn are discussed.

Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring

Lactosylation in stored milk powder was quantified by multiple reaction monitoring (MRM), a mass spectrometry-based quantification method. The MRM method was developed from a knowledge of peptide fragmentation. The neutral losses of 162Da (cleavage of galactose) and 216Da (the formation of furylium ion) which were representative of lactosylated peptides were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins.

Identification of the glycosaminoglycan keratan sulfate in the prostatic secretory cell

Prostate secretory granules (PSG) represent the basic secretory unit of the prostate gland, containing many of its exocrine proteases. Recent analysis of intraluminal corpora amylacea, a proposed by‐product of PSG secretion, detected sulfated glycosaminoglycans (GAG) possibly keratan sulfate (KS), indicating a secretory mechanism for GAG in the human prostate surface epithelial cell.

Direct experimental evidence for competitive inhibition of dihydrofolate reductase by methotrexate

Steady-state enzyme kinetic techniques at very low enzyme concentration (0.4 nM) were used successfully to measure the inhibition constant (53 pM) for the dissociation of methotrexate from the ternary complex of methotrexate, NADPH and dihydrofolate reductase from Lactobacillus casei; and to demonstrate unequivocally that the inhibition was, indeed, competitive with respect to dihydrofolate.

Thermodynamic characterization of the interactions of methotrexate with dihydrofolate reductase by quantitative affinity chromatography

Affinity chromatography on methotrexate-Sepharose has been used to evaluate dissociation constants for interactions of methotrexate with dihydrofolate reductase from Lactobacillus casei. Equilibrium constants of 0.25 microM and 0.6 nM were obtained for dissociation of the inhibitor from the enzyme-methotrexate and enzyme-NADPH-methotrexate complexes, respectively, these estimates being in good agreement with the corresponding published values for dihydrofolate reductase from Streptococcus faecium. By employing a different method for evaluating the thermodynamic dissociation constant for the enzyme-NADPH-methotrexate interaction, this investigation provides independent support for the inference drawn from published fluorescence quenching studies that the interaction of methotrexate with dihydrofolate reductase-NADPH complex is governed by a dissociation constant in the vicinity of 600 pM, a value slightly higher than, but approaching, the inhibition constant of 50-60 pM obtained by enzyme kinetic techniques.

Composition of the inner mitochondrial membrane of porcine corpus luteum.

An inner mitochondrial membrane fraction was prepared from porcine corpus luteum. The concentrations of the respiratory cytochromes, cytochrome P-450scc, cholesterol, ubiquinone, cardiolipin and the total phospholipids were measured. The fatty acid compositions of cardiolipin and the total phospholipid fraction were determined. Comparative data from porcine heart and liver were obtained using the same methods. Differences in both the concentration and the fatty acid composition of the phospholipids were observed between the tissues. It appeared that the phospholipid bilayer was expanded relative to haem a in luteal mitochondria. It is proposed that in the ovary this expansion may be necessary to accommodate cytochrome P-450scc and its substrate, cholesterol.

Chapter 5 – Post-translational Modifications of Caseins

Caseins are phosphoproteins and constitute about 80% of the protein in milk. Caseins exhibit a high degree of heterogeneity as a result of post-translational modifications (PTMs). Phosphorylation of the α- and β-caseins and glycosylation of κ-casein are the best-known modifications and are critical for the formation and stability of casein micelles. κ-Casein has long been known to exhibit a high degree of variability in glycosylation. This chapter summarizes current understanding on PTMs of the αs1-, αs2-, β-, and κ-caseins. Caseins are assembled in a colloidal complex with calcium phosphate and small amounts of other minerals. The casein micelle structure is critical in determining the physical properties of milk and for the provision of amino acids, calcium, and phosphorus for infant nutrition. A critical factor in micelle formation and stability is the presence PTMs such as phosphorylation, glycosylation, and perhaps disulfide bond formation that occur in the endoplasmic reticulum and/or Golgi complex after synthesis of the polypeptide chain. In recent years, the development of proteomic technologies has greatly enhanced our ability to analyze milk proteins, particularly with respect to PTMs. Two-dimensional electrophoresis (2-DE) provides a high-resolution methodology for displaying the heterogeneity of the major milk proteins. Advances in mass spectrometry (MS) have enhanced our ability to analyze the proteins arrayed on 2-D gels. Therefore, not only is it possible to resolve many proteins and their isoforms but it is also possible to characterize them, particularly with respect to the many PTMs that affect their electrophoretic mobility. Furthermore, the chapter includes an extended discussion on the functional significance of κ-casein heterogeneity.