Structure primaire de la caséine α sl ‐bovine

In previous reports [1–5], we have described some of the features of the primary structure of bovine α s1 ‐casein. In the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed. In the polypeptide chain, the region containing the phosphopeptides Tm1 (T1‐T2), Tm1T2 and Tm1T1 [1,4,5], had only been slightly studied as yet. Because of the difficulties encountered in the breakdonwn of these phosphopeptides, hydrolysis with endopeptidases and exopeptidases were performed on both native and dephosphorylated peptides. It was confirmed that peptide Tm1T2 contains three hydroxy‐amino acids (2Ser, 1Thr), but, instead of three [1], 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 O ‐phosphorylated. The remaining gap in the sequence of the central part of peptide Tm1T2 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 Tm1T1 [1], 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 O ‐phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide Tm1T1 were further degraded using classical methods for the determination of the amino acid sequence. In the light of all the results obtained on this phosphoprotein (α sl ‐casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [1–5], and the molecular weight has been calculated to be 23616. The following amino‐acid composition; Asp 7 , Asn 8 , Thr 5 , SerP 8 , Glu 25 , Gln 25 , Gln 14 , Pro 17 , Gly 9 , Ala 9 , Val 11 Met 5 , Ile 11 , Leu 17 , Tyr 10 , Phe 8 , His 5 , Trp 2 , Arg 6 , 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 gruops 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 (8.5%) 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 first 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 residues, in particular seven of the total eight phosphoseryl residues. Another remarkable feature is the concentration of amino acids with carboxylic side chains, particularly glutamic acid, in the vicinity of two clusters of phosphoseryl residues. It may be noticed that the phosphoseryl residues in α sl ‐casein are arranged in a manner similar to those in β‐casein [8]. In particular, in α s1 ‐casein, the 62–70 region containing four phosphoseryl residues, is similar to the 13–21 region in β‐casein [8]. It is interesting to point out that all but one of the phosphoseryl residues always occur in position n with relation to a glutamyl or a phosphoseryl residue, which is in position n + 2. From this, it would appear that there is an enzymatic phosphorylation of the polypeptide chain (carried out by a phosphoryl kinase which may require a negative charge in the phosphorylation site) rather than direct incorporation of phosphoserine into the polypeptide chain during protein synthesis. The location of the amino‐acid substitutions that characterize the α s1 ‐casein D and the β‐casein C variants has provided evidence for an enzymatic phosphorylation of casein [9]. The problem of the phosphorylated sites in α s1 ‐ and β‐caseins will be discussed in a forthcoming paper. The repeating amino‐acid sequences located in the two regions of orsl‐casein, viz. 70–84 and 110–125, are also of interest. The primary structure of α s1 ‐casein given here is that of the genetic variant B. The three other variants A, C and D have also been studied. The C and D variants differ from the B variant by the substitutions of Gly/Glu in position 192 [10] and Thr P /Ala in position 53 [9], respectively. The A variant is characterized by a deletion of thirteen amino‐acid residues from position 14 to position 26 in the hydrophobic NH 2 ‐terminal part of the polypeptide chain [ll]. Because of this deletion, there is an important change in the physico‐chemical properties of α s1 ‐casein [12,13]. Some important features of the amino‐acid‐sequence determination should be noted. Firstly, the great value of maleic anhydride as a reversible‐blocking reagent for amino groups of proteins [14], and the usefulness of thermolysin as a specific enzyme for degrading polypeptides [15]. Secondly, the difficulties encountered during the study of the phosphopeptides: these pep‐ tides were difficult to purify since they have similar anionic characters and only react slightly with ninhydrin during paper revelation; other difficulties have been the high destruction of phosphoserine during hydrolysis with HCl5.7 N, the poor yield in the substractive Edman degradation when the NH 2 ‐terminal residue is a phosphoserine, and the failure of endopeptidases and exopeptidases to split off peptide bonds that are close to phosphate groups. Fortunately, alkaline phosphatase has proved to be a useful tool for removing phosphate groups, thus avoiding most of these difficulties. Since the amino‐acid sequence of α s1 ‐casein is now known, the degradation of this protein by coagulating and other proteolytic enzymes during cheese ripening may be studied. In particular, bitter peptides originating from α s1 ‐casein may be located. As we have already briefly noted [4], it is obvious that one of the three bitter peptides isolated by Matoba et al. [16] from a tryptic hydrolysate of whole casein, was the segment 23–34 of the polypeptide chain of α s1 ‐casein. In addition, the knowledge of the primary structure will be of help in understanding many aspects of the relations between structure and physical properties in this phosphoprotein.