α_s0-Casein: its preparation and characterization

Abstract

It has been reported previously (Annan & Manson, 1969) that chromatography of the a s -casein complex of bovine milk on columns of sulphoethyl Sephadex C-50 resulted in fractionation of the complex into a main component, asj-casein and a number of minor components one of which was designated Os 0 -casein. The present communication describes the preparation of Os 0 -casein from unfractionated casein in an amount and state of purity which render possible its further characterization. EXPERIMENTAL Acid-precipitated whole casein. Milk was obtained from individual Ayrshire cows of the Institute herd which were in the middle period of lactation and were free from mastitis. They were also selected as yielding one only of the known genetic variants of a si -casein, viz. type B as determined by electrophoresis performed on milk samples using the procedure described below. Whole casein was prepared from milk thus selected, as described previously (Manson & Annan, 1971), and dissolved in distilled water by addition of 1 N-NaOH in such a way that the pH value never exceeded 7-5. The solution was clarified by filtration through a filter disk (HP/EKS obtained from Carlson-Ford Ltd, Ashton-under-Lyne, England) under an applied pressure of 50 kPa (0-5 kg/cm 2 ) and its protein content when determined by measurement of its absorbance at 278 nm was approximately 6 %. tx S0 -Casein. a S0 -Casein was prepared from solutions of acid-precipitated whole casein by chromatography on columns (50-0 x 5-0 cm) of sulphopropyl Sephadex C-50 (Pharmacia, Great Britain, Ltd, London) which were made up in and equilibrated with Na formate/formic acid buffer (4-0 g NaOH, 11-5 g formic acid/1) containing 7-5 M-urea and having a pH of 4-0. In an earlier paper (Annan & Manson, 1969) this buffer was wrongly described as containing 4-0 g Na formate/1. Before application to the column the whole casein was first reduced by treatment with 2-mercaptoethanol (ME) at pH 8-6 following the procedure described by Anfinsen & Haber (1961). The reduction was terminated by addition of formic acid to pH 4-0 and the mixture was adjusted in composition to approximately that of the chromatographic buffer by addition to each 100 ml of 67-5 g urea, 1-7 g formic acid and 0-6 g NaOH followed by distilled water to 150 ml. The mixture was then dialysed against formate/urea buffer after which NaCl was added to a final concentration of 0-04 M. In a typical fractionation performed at 20 °C this casein solution (800 ml; 32 g protein) was applied to the top of the column followed by formate/urea buffer containing 0-04 M-NaCl. The flow through the column was maintained at 60 ml/h and the effluent which was monitored spectrophotometrically for the presence of protein

Bovine alphaso Casein; a Phosphorylated Homologue of alphas1 Casein

“…Such a difference in composition between the two proteins is fully consistent with their behaviour when subjected to electrophoresis in starch/urea gels at pH 9.2 and 2.0. It was found that at pH 9.2 the aSo preparation migrated just ahead of aS1 casein B and at pH 2.0 it had a very slightly lower mobility than this protein, thus confirming earlier findings [2]. …”

“…The amino acid composition and phosphate content of M,O casein were determined on samples of the protein which had been prepared using the procedure described in the Methods section with the addition of a second cycle of the chromatographic purification. The results, which are set out in Table 1, indicate clearly that the amino acid composition of aSo casein did not differ, within experimental error, from that of a,l casein B and fail to support the earlier suggestion [2] that the two proteins differed from each other only by substitution of one histidyl by one aspartyl residue. In addition the aso casein preparations contained the equivalent of 8.7 mol phosphate/mol protein which, although close to the required value of 8.0 for aS1 casein, was nevertheless sufficiently removed from it to suggest that as0 casein contained 9 phosphate residues/mol compared with the 8 of c(,l casein.…”

“…The occurrence of a ,~ casein as a minor constituent of the as group of caseins present in bovine milk has previously been reported [l] and its preparation in a purified state described [2]. In the present communication the relationship of the primary structure of a,o casein to that already determined for a,l casein [3] is discussed.…”

“…They were also selected as yielding one only of the known genetic variants of asl casein, namely type B [4,5]. Whole casein prepared from milk thus selected by procedures described previously [2,6] was fractionated by chromatography on columns of sulphopropyl-Sephadex C-50 (Pharmacia, Great Britain, Ltd, London) in sodium formate/formic acid buffer containing 7.5 M urea, pH 4.0 [2]. The chromatography was conducted in a linear gradient of NaCl extending from 0.04 M to 0.1 M and both .aso and a,l caseins were eluted at approximately 0.08 M NaC1, although aSo casein appeared significantly in advance of the asl component.…”

“…The presence in aSo casein of one phosphate residue more than occurs in a ,~ casein was confirmed by comparative degradative studies performed on both proteins. From these it was concluded that aSo casein may be considered as being aSl casein which has been modified by phosphorylation of the seryl residue located at position 41.The occurrence of a ,~ casein as a minor constituent of the as group of caseins present in bovine milk has previously been reported [l] and its preparation in a purified state described [2]. In the present communication the relationship of the primary structure of a,o casein to that already determined for a,l casein [3] is discussed.…”