I n previous reports [l-51, we have described some of the features of the primary structure of bovine oL,l-casein. I n the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed.I n the polypeptide chain, the region containing the phosphopeptides Tml (Ti -T2), TmlT2 and TmlTl [1,4,5], had only been shghtly studied as yet. Because of the difficulties encountered in the breakdown of these phosphopeptides, hydrolysis with endopeptidases and exopeptidases were performed on both native and dephosphorylated peptides.It was confirmed that peptide TmlT2 contains three hydroxy-amino acids ( 2 Ser, 1 Thr), but, instead of three [l], two phosphorus atoms were found with purified preparations. Partial acid hydrolysis and dephosphorylation using an orthophosphoric-monoester phosphohydrolase indicate that the two seryl residues in this peptide are 0-phosphorylated. The remaining gap in the sequence of the central part of peptide TmlT2 was bridged by studying two fragments obtained by papain digestion of the dephosphorylated preparation.Instead of four serines, postulated earlier from the results of the amino-acid composition of peptide TmlTl [I], five were shown to be present after detailed analysis of the fragments. Since the five phosphate groups could be readily removed by an orthophosphoric-monoester phosphohydrolase, the five seryl residues could be 0-phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide TmlTl were further degraded using classical methods for the determination of the amino acid sequence.I n the light of all the results obtained on this phosphoprotein (a,l-casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [l-51, and the molecular weight has been calculated to be 23616. The following amino-acid composition; Asp,, Asn,, Thr,, Ser,, SerP,, Glu,,, Gln,,, Pro,,, Gly9, Ma,, Val,,, Met,, Ile,,, Leu,,, Tyr,,, Phe,, LysI4, His,, Trp,, Arg,, indicates that there is a higher number of acidic than basic residues in this protein. On the basis of the intrinsic dissociation constants of titratable groups in proteins [6], the negative net charge of the molecule was estimated to be 22 at pH 6.5 and 28 at pH 8.6. Since Bigelow's parameter for the average hydrophobicity [7] of this protein is 1170, it could be considered to be a hydrophobic protein. The high amount (8.50/,) and uniform distribution of prolyl residues indicate that this protein has limited structuralcoiling possibilities.The polypeptide chain contains three hydrophobic regions, viz. 1-44,90-113 and 132-199. The fist two are characterized by the fact that basic residues predominate over acidic residues. The third region, where most of the aromatic residues are located, contains very few basic residues and is therefore of more acidic character.Two regions, 45-89 and 114-131, are hydrophilic. The former contains more than half of the total acidic residue...
Solubility of the main proteins in 10 x acid and rennet whey retentates was studied in the pH range 2.0 to 4.0 in the presence of NaCl from 2 to 15% (w/v) final concentration, at 20°C to find fractionation conditions suitable for preparing pure P-lactoglobulin and P-lactoglobulin-free whey proteins and scaling up. At pH 2.0, 7% NaCl, 20 min holding time, nearly all P-lactoglobulin remained soluble while a precipitate (Pl) containing all other proteins was formed. Pure P-lactoglobulin was quantitatively recovered by salting-out the centrifugation supernatant at 30% NaCl (w/v) final concentration. Pl, insoluble at pHs lower than 4.0, was made soluble at any pH by dissolving at pH 9.0, dialyzing against 50 mM formic acid (pH 3.0) and freeze-drying.
In a previous report [1], we have given the complete primary structure of ϰB1‐caseinomacro‐peptide which is the soluble COOH‐terminal fragment split from bovine ϰB1‐casein by rennin. We also reported on the COOH‐terminal sequence of the NH2‐terminal fragment, the so‐called para‐ϰ‐casein. The present paper deals with the complete amino acid sequence of bovine ϰB‐casein, which has now been achieved by establishing the primary structure of para‐ϰB‐casein of which we discuss the salient features. This work has been reported briefly in a short communication [2]. SCM‐para‐ϰB‐casein and maleyl ϰBCN1, the NH2‐terminal cyanogen bromide fragment of ϰB‐casein [1], were used as starting material. The tryptic and peptic peptides of SCM‐para‐ϰB‐casein and the chymotryptic peptides of ϰBCN1 were isolated on Dowex 50 and Sephadex G‐50 or G‐25, and their sequences were determined either partially or completely by using classical methods and in some cases mass spectrometry. All these peptides and a NBS fragment of SCM‐para‐ϰB‐casein have provided all the overlaps needed for the completion of the amino acid sequence of para‐ϰB‐casein. Para‐ϰB‐casein is a single polypeptide chain containing 105 amino acid residues: Asp3, Asn4, Thr3, Ser7, PyroGlu1, Glu4, Gln12, Pro12, Gly1, Ala9, Val5, 1/2 Cys2, Met1, Ile6, Leu7, Tyr9, Phe4, Lys6, His3, Trp1, Arg5, and its molecular weight has been calculated to be 12269. The average hydrophobicity, calculated according to Bigelow [3], is 5.48 kJ (1.310 kcal) per residue, and para‐ϰB‐casein can be therefore considered to be a very hydrophobic molecule. Its net positive charge at pH of native milk (about 6.8) is very close to 4.5. The high content (11.5%) and rather uniform distribution of prolyl residues are incompatible with much α‐helical organization of the molecule, as previously shown for ϰ‐casein [4]. Both hydrophobic and charged amino acid residues are distributed non‐uniformly along the chain. Two regions, 1–24 and 80–105, are hydrophilic: the very hydrophilic former, with NH2‐terminal pyroglutamic acid, contains a cysteinyl residue located inside a cluster of eleven ionizable residues including six out of the seven total dicarboxylic amino acids; the 80–105 region, which contains the second cysteinyl residue in position 88, is rather hydrophilic, except at the COOH‐terminal end which is hydrophobic in spite of the presence of a cluster of four basic residues. These two hydrophilic regions are very likely to be on the outisde of the molecule and this may favor the formation of intermolecular S‐S bonds. The very hydro‐phobic central part of the chain, viz., 25–79, where most of the aromatic residues are located, has a para‐ϰ‐casein‐like behaviour in aqueous solution, and it may be responsible for the aggregation ability of para‐ϰ‐casein. According to the sequence data of both ϰB1‐caseinomacropeptide [1] and para‐ϰ‐casein, it is concluded that bovine ϰB‐casein is made up of a single polypeptide chain containing 169 amino acid residues: Asp4, Asn7, Thr14, Ser12, SerP1, PyroGlu1, Glu12, Gln14, P...
Summary -After a short description of bovine milk proteins, the various methods of current or potential use for detecting and determining them in dairy products are reviewed. This includes, first, the determination of total protein from nitrogen analysis, dye-binding capacity, infra-red spectrometry and amino acid analysis. The methods that allow determination of sorne milk protein fractions of interest (whole casein, whey proteins,~-Iactoglobulin) are then given. They include the Aschaffenburg-Rowland procedure, dye-binding and infra-red methodologies. A description of the various methods (electrophoresis, column chromatography, immunochemical or enzymatic tests), that can be used for detecting and individually quantitating the various caseins and whey proteins is presented. Finally, sorne applications of various analytical procedures to the analysis of different classes of dairy products are given.
This paper describes the elucidation of the primary structure of the three genetic variants of goat asl-casein, asl-Cn D, E and F, which have been found to be associated with reduced amounts of asl-casein in milk. Variant E has the same electrophoretic mobility as variant B, but differs from the latter by the substitutions of Arg for Lys and of Thr for Ala at positions 100 and 195. A genetically controlled event which does not affect the amino acid sequence of this variant might be responsible for its lower rate of synthesis compared to that of asl-casein B. The deletion of 11 amino acids at positions 59 -69 and of 37 amino acids at positions 59 -95 in variant B leads to variants D and F. In both cases the deletions, which start at the same position of the polypeptide chain, include the major phosphorylation site of the protein. On the basis of sequence data for casein genes and cDNAs, it was concluded that the deletions occurring in the D and F variants are due to the exclusion of one and several exons, respectively. The observed deletions in the proteins could thus be the consequence of splice site mutations which would induce altered RNA processing and hence reduce the rate of synthesis of the casein.Early work on the casein family, which includes three phosphoproteins, asl-, as2-, and fl-casein, and one glycophosphoprotein, k--casein, was performed on proteins isolated from cow's milk. The primary structures of these four polypeptides, which contain 169 -209 amino acid residues, have already been established [l]. Whereas genetic polymorphism is widespread for asl-, fl-, and k--casein, it is more restricted and is breed-dependent for as2-casein [2, 31. Segregation studies led to the conclusion that the four proteins are controlled by a cluster of closely linked genes. In this cluster no recombination was observed in a total of 448 informative families [4]. The unit of inheritance is thus the haplotype which is a combination of individual alleles of each of the four clustered loci.Although the same classes of caseins have been identified in goat's milk [5], so far, in this species, only asl and as2-casein are known to exhibit genetic polymorphism [6]. Segregation studies have confirmed that asl-and as2-casein genes are closely linked suggesting that, as in the cow, casein expression in the goat is controlled by a cluster of genes.Electrophoretic patterns of goat asl-casein are unusually complex due to differences in the mobilities of different variants and to varying intensities of electrophoretic bands which reflect different amounts of protein [6]. It is now established [6, 71 that polymorphism of asl-casein is under the control of at least seven alleles denoted as: asl-CnA, asl-CnB, asl-Cnc, asl-CnD, asl-Cn", asl-CnF and asl-Cno. Alleles asl-CnA, asl-
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