Jay J. Basch

Chromatographic and electrophoretic methods used for analysis of milk proteins

Current knowledge of milk proteins and their behavior in dairy foods is based on early applications of chromatography and electrophoresis. Electrophoretic identification of the number and genetic variety of milk proteins inaugurated a research effort in which chromatographic techniques were successfully applied to the isolation of each milk protein, thus facilitating the characterization and further study of milk and dairy products. This review focuses on recent applications of chromatography for separations and analysis and on analytical applications of electrophoresis.

Hydrogen ion equilibria of the genetic variants of bovine β-lactoglobulin

Abstract The hydrogen ion equilibria of β-lactoglobulins A, B, and C have been examined. The resulting titration curves can be accounted for in terms of the normal ionization of all groups, with the exception of two histidines in the C variant and two carboxyls in all three of the variants. Conformational changes at pH 4.5–6 and 6.5–9 release these abnormal groups to ionization.

Identification of the milk fat globule membrane proteins. I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cows milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.

Amino acid composition of α1-, α2-, and α3-caseins

Abstract Amino acid analyses have been made of a purified α-casein, of its principal component, α 1 -casein, and of two other purified components, α 2 - and α 3 -caseins. α 1 -Casein resembles α-casein in content of most amino acids, but both differ from α 2 - and α 3 -caseins which have their own characteristic amino acid compositions.

The isolation and amino acid composition of two peptides from chymotryptic digests of β-lactoglobulins A and B

Abstract Short-term chymotryptic digests have been carried out with β-lactoglobulins A and B, performic acid-oxidized and S-sulfonated derivatives. Differences in the peptide patterns have been found after high-voltage electrophoresis only when disulfide bonds were previously cleaved. Two peptides have been isolated from chymotryptic digests of S-sulfonated β-lactoglobulins, and their amino acid compositions have been determined to be as follows: For the peptide from the β-A derivative: Asp 2 , Thr 1 , Glu 4 , Pro 1 , Ala 3 , Val 1 , Ileu 2 , Phe 1 , 1 2 Cys 1 , Lys 4 ; and for the peptide from β-B: Asp 1 , Thr 1 , Glu 4 , Pro 1 , Ala 3 , Val 1 , Ileu 2 , Phe 1 , 1 2 Cys 1 , Lys 4 , Gly 1 . The sole difference lies in the aspartic acid-glycine content. The valine-alanine difference in β-lactoglobulins A and B remains unaccounted for in the present study.

Physicochemical comparison of goat β-lactoglobulin with the bovine varieties

Abstract β-Lactoglobulin was prepared from mixed goat milk and multiple amino acid analyses were carried out. One difference in basic residues was found from the analysis previously reported. Ultracentrifugal, light-scattering, and optical rotatory dispersion experiments showed that the goat protein is quite similar to the bovine β-lactoglobulins in most of its physical properties, but indications were obtained that it is conformationally less stable at low pH values than its bovine analog. Titration experiments indicate that six of the free carboxylic acid (Asp and Glu) residues in monomeric bovine β-lactoglobulin B do not occur in titratable form in the caprid β-lactoglobulin. These experiments also indicate a low pH-induced conformational change to a molecule of slightly greater radius.

Charge separation of proteins complexed with sodium dodecyl sulfate by acid gel electrophoresis in the presence of cetyltrimethylammonium bromide

Globular proteins, casein, and membrane proteins which were reacted with sodium dodecyl sulfate were studied by acid urea gel electrophoresis. The sodium dodecyl sulfate bound tightly to the proteins, producing a more acidic charge which prevented migration into the gel. When cetyltrimethylammonium bromide was added to the sodium dodecyl sulfate-protein complexes, the sodium dodecyl sulfate apparently reacted with cetyltrimethylammonium bromide and dissociated so that the proteins migrated in acid gel in a normal manner as compared to the proteins without any added detergent. The sodium dodecyl sulfate-cetyltrimethylammonium bromide complex could be removed from the proteins by centrifugation. Thus, cetyltrimethylammonium bromide used in conjunction with acid gel electrophoresis allows direct comparison by charge of proteins fractionated in the presence of sodium dodecyl sulfate with the starting mixture of proteins not exposed to detergent. The reaction of cetyltrimethylammonium bromide with sodium dodecyl sulfate in acidic urea also provides a simple convenient method of removal of sodium dodecyl sulfate from proteins.

Chemical and physico-chemical characterization of genetic variant D of bovine β-lactoglobulin

Abstract The recently discovered genetic variant of β-lactoglobulin, β-D, has been compared to those previously known. It has been shown that β-D varies from β-B by the substitution per subunit chain of a glutamine residue in β-D for glutamic acid in β-B. This conclusion has been verified by titration experiments. Conformational studies have shown that the structure of β-D is indistinguishable from that of the other variants. Furthermore, β-D does not form the low temperature octamer, characteristic of the A variant, while it dissociates into subunits both at acid and alkaline pH, just as the other variants.

Amino acid analysis of the β-lactoglobulins from individual cows of two phenotypes

Abstract Amino acid analyses were performed on β-lactoglobulin samples prepared from individual dairy cows, 24 of which were homozygous for β-lactoglobulin A and 9 for β-lactoglobulin B. No differences were found in individual contents of any amino acids other than the previously known β-A-β-B difference amino acids. The total analyses agreed well with previously published analyses on β-A and β-B obtained from pooled milk of many animals.

Influence of preparative method on the lipid profiles of bovine fat globule membrane

Abstract The lipid profiles of bovine fat globule membrane preparations were obtained using a procedure that separates neutral and polar lipids on a silica cartridge, analyzes components of these two lipid groups by high performance liquid chromatography, and identifies fatty acids of total lipid as well as neutral and polar lipids separately by gas chromatography. Fat globule membrane preparation involved either churning the cream, followed by ammonium sulfate precipitation of membrane, or a combined isolation-extraction with dimethyl sulfoxide. In preparations that did not include a delipidation step, the carryover of neutral lipids was significant. In contrast, preparations that were delipidated after ammonium sulfate precipitation or as part of the isolation-extraction had much lower levels of neutral lipids. For the latter samples, values for neutral and polar lipids were comparable to those reported previously. The relative ease with which fat globule membrane is obtained using the isolation-extraction procedure makes it the preferred method for subsequent isolation and purification of fat globule membrane proteins.