Chromatofocusing of three hereditary variants of serum albumin: isolation of a new variant albumin Jaffna

Chromatofocusing of three hereditary variants of serum albumin: isolation of a new variant albumin Jaffna

Clinica Chimico Acta, 142 (1984) 131-136 Elsevier 131 CCA 02936 Short communication Chromatofocusing of three hereditary variants of serum albumin...

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Clinica Chimico Acta, 142 (1984) 131-136 Elsevier

131

CCA 02936

Short communication

Chromatofocusing of three hereditary variants of serum albumin: isolation of a new variant albumin Jaffna C.R. Tillyer ‘*, B. Dowding

b and A.L. Tarnoky

b

a Department of Chemical Pathology, The Royal Marsden Hospital, Fulham Palace Road, London W6 8RF and b Department

of Clinical Biochemistry, Royal Berkshire Hospital, Reading RGI 5AN (UK) (Received July 26th, 1983; revision May 16th. 1984)

Key words: Albumin variants; Albumin ‘Jaffna:’ Chromatofocusing

Introduction Large numbers of hereditary albumin variants have been described in human sera but very few have been studied in detail [1,2]. The exact nature of the changes in primary structure has been elucidated in only four different variants: albumin B (‘Oliphant’, ‘Ann Arbor’) [3], the proalbumins Christchurch [4-61 and Lille [7] and albumin Mexico-2 [15]. One reason for the lack of study is the difficulty in isolating the variant albumin in quantities sufficient for biochemical analysis. Differential elution from DEAE-Sephadex, preparative polyacrylamide gel electrophoresis and cellulose acetate electrophoresis have in some cases been successful [4-71. Chromatofocusing is a column chromatography technique which separates proteins according to their p1 [8,9] and avoids undue sample dilution. We have applied the technique to the separation of three variant albumins and found it provides a convenient means of separating and purifying albumin ‘Kashmir’, as reported elsewhere [lo], and the new variant albumin ‘Jaffna’ [ll], but fails to separate a third albumin ‘LH’. Methods Albumin purification Samples. Routine serum samples were obtained from three patients in whom bisalbuminaemia had been revealed on cellulose acetate electrophoresis, and from one normal person. All bisalbuminaemia samples had cathodic (slow-moving) variants in addition to the normal albumin band, and were well separated. * To whom correspondence

0009-8981/84/$03.00

should be addressed.

0 1984 Elsevier Science Publishers B.V.

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Serum samples (1-2 ml) were equilibrated with ‘starting Sample preparation. buffer’ either histidine-HCl (pH 6.2; 0.025 mol/l) or piperazine-HCl (pH 5.5; 0.025 mol/l) on a 15-ml Sephadex G-25 column. Fractions (1.6-1.8 ml) from the column were pooled and concentrated in Minicon TM (Amicon, Stonehouse, Gloucs., UK) clinical sample concentrators to a volume of 1-2 ml. Chromatofocusing. Polybuffer exchange columns (PBETM94-Pharmacia, Milton Keynes, UK) of about 15 ml packed volume were equilibrated overnight with 12-15 column volumes of starting buffer. The concentrated samples from the G-25 column were applied to the PBE 94 column after the initial application of 5-8 ml of ‘eluting buffer’ (an aqueous 1 : 12 dilution of Polybuffer 74TM-HC1, pH 4). Fractions were then eluted with 3 column volumes of ‘eluting buffer’, at a flow rate of approximately 30 ml/h. Fractions containing albumin were pooled, concentrated as above, and equilibrated with phosphate-buffered saline (NaCl 0.137 mol/l, KC1 0.025 mol/l, Na,HPO, 0.008 mol/l, KH,PO, 0.0015 mol/l) on a G-25 column and concentrated again in a MiniconTM concentrator. An anti-human transferrin column was prepared with Affinity chromatography. rabbit anti-human transferrin antiserum (Seward London, UK) and cyanogen bromide activated Sepharose T”4B (Pharmacia) according to the manufacturers’ recommendations. 2 g of gel were swollen and washed in 10V3 mol/l HCl; 2 ml of antiserum, in 10 ml of buffer (consisting of 0.1 mol/l NaHCO, and 0.5 mol/l NaCl) were mixed with the gel by gentle rotation at room temperature for 2 h. The gel was washed with the same buffer and then left in 1 mol/l ethanolamine, pH 8, for 2 h. The gel was then washed alternately with 0.1 mol/l acetate-l mol/l NaCl buffer, pH 4, and 0.1 mol/l borate-l mol/l NaCl buffer, pH 8, three times and finally resuspended in phosphate-buffered saline, pH 7.4. After chromatofocusing albumin samples were directly applied to the affinity column (8 ml packed gel volume), eluted with phosphate-buffered saline, fractions collected, pooled and concentrated to a final volume of l-2 ml. Fractions were assessed by cellulose acetate electrophoreAssessment of purity. sis using a routine method [12], the ‘Helena’ gel system [lo], polyacrylamide gel electrophoresis [13] and immunoelectrophoresis [12]. Albumin and transferrin assays. Albumin and transferrin were measured by rate nephelometry using the Beckman ‘ICS’ system. TFypsin digest. The Jaffna serum (0.05 ml) was incubated at 37’C with 0.1 ml of a 0.1% solution of Trypsin (TPCK-Trypsin, Worthing Biochemicals Twyford, Berks., UK) in 0.1 mol/l Tris-HCI buffer, pH 8.0 for l-3 h. The samples were then diluted with an equal volume of Tris-HCl buffer and examined by cellulose acetate electrophoresis. Results

Chromatofocusing of normal human serum produces the pattern shown in Fig. 1. The first peak to elute contains y-globulins and consists of proteins which do not

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bind to the column at the pH of the ‘starting buffer’. The second main peak contains albumin and is preceded by a smaller peak, which usually appears as a shoulder on the albumin peak, containing transferrin (Fig. 1). The bisalbuminaemic sera showed markedly different patterns on chromatofocusing. The albumin ‘Jaffna’ serum showed almost complete separation of normal and variant albumins (Fig. 2), the albumin ‘Kashmir’ serum only partially separated peaks (Fig. 3) and the ‘LH’ serum a single peak which contained both albumins (Fig. 4). Histidine-HCl or piperazine-HCl as the ‘starting buffer’ gave virtually the same pattern for Kashmir, Jaffna and normal sera with only a marginally better peak separation when using the shallower pH gradient of piperazine-HCl. Normal albumin eluted at pH 4.2-3.9. The variant albumins Kashmir and Jaffna eluted at slightly higher pH, which may be expected if the variant has a slightly higher p1 than the normal albumin. Transferrin eluted at pH 4.8-4.4. The pH at which these proteins elute is well below their reported PI’S which are 4.7 for albumin and 5.9 for transferrin [14]. The only distinct protein contaminating the variant albumin peak was transferrin. For isolation of albumin Jaffna an anti-transferrin affinity column removed almost all of this contaminant giving a 95% pure variant albumin as determined by densitometric scanning of cellulose acetate electrophoresis (Fig. 2, insert b). The

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transferrin Fig. 1. Chromatofocusing profile of normal human serum showing albumin (m-m), and pH (- - -). Fractions from peaks labelled 1, 2 and 3 0), absorbance (280 nm) ( -) (Owere pooled, concentrated and ‘Helena’ cellulose acetate electrophoresis of these concentrated peaks are shown with a normal serum (N) for comparison.

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Fig. 2. Chromatofocusing profile of albumin ‘Jaffna’ serum. Albumin (D -m), absorbance (280 nm) pH (- - -). Fractions from peaks 1 and 2 were pooled and concentrated. Insert (a) shows ( -). ‘Helena’ cellulose acetate electrophoresis of these concentrated peaks with a normal serum (N) and the original Jaffna serum (J) for comparison. Insert (b) shows the isolated variant Jaffna (A) after further purification by affinity chromatography with the original serum (B) for comparison.

average recovery of albumin from the chromtofocusing column was 60% and 74% from the affinity column. The isolated albumin still contained traces of an qglobulin and y-globulins on polyacrylamide gel electrophoresis and immunoelectrophoresis. Incubation with trypsin had no effect on the Jaffna serum. Discussion The variant albumin Jaffna has been partially characterised (111 but the detailed molecular structure has yet to be elucidated. The failure of the variant to convert to normal after trypsin incubation suggests that the Jaffna albumin is not a proalbumin. The very different separations of variant albumins ‘Jaffna’ and ‘Kashmir’ on chromatofocusing are not readily explicable. All three variants were widely separated on cellulose acetate electrophoresis. Changing buffers to produce shallower pH gradients or changing column flow rates (from 40 to 12 ml per h) did not significantly affect separation. One cannot predict from the electrophoretic be-

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haviour of a variant on cellulose acetate whether it will separate from normal albumin on chromatofocusing. It seems unlikely that the same method of separation will be successful with different variant albumins. References 1 &hell LM, Blumberg B.S. The genetics of human serum albumin. In: Rosenoer VM, Oratz M, Rothschild MA, eds. ‘Albumin, structure, function and uses.’ London: Pergamon Press, 1977: 113-141. 2 Tarnoky AL. Genetic and drug-induced variation in serum albumin. In: Latner AL, Schwartz MK, eds. Advances in clinical chemistry, Vol. 21. New York: Academic Press, 1980: 101-146. 3 Winter WP, Weitkamp LR, Rucknagel DL. Amino acid substitution in two identical inherited human serum albumin variants: albumin Oliphant and albumin Ann Arbor. Biochemistry 1972; 11: 889-896. 4 Brennan SO, Carrel1 RW. A circulating variant of human proalbumin. Nature 1978; 274: 908-909. 5 Brennan SO, Carrel1 RW. Functional abnormality of proalbumin Christchurch. Biochim Biophys Acta 1980; 621: 83-88. 6 Rousseaux J, Abdo G, Coanon C, Vittu C, Dautrevaux M. Studies of an abnormal albumin unstable upon storage. Clin Chim Acta 1982; 124: 293-302. 7 Abdo Y, Rousseaux J, Dautrevaux M. Proalbumin Lille, a new variant of human serum albumin. FEBS Lett 1981; 131: 286-288. 8 Sluyterman LAAE, Elgersma 0. Chromatofocusing: isoelectric focusing on ion exchange columns. I. General principles. J Chromatogr 1978; 150: 17-30. 9 Sluyterman LAAE, Elgersma 0. Chromatofocusing: isoelectric focusing on ion exchange columns. II. Experimental verification. J Chromatogr 1978; 150: 31-44. 10 Tillyer CR, Dowding B, Tarnoky AL. Separation of albumin ‘Kashmir’ from normal human serum albumin - A by chromatofocusing. IRCS Med Sci 1982; 10: 636. 11 Bayliss VM, Curnow JV, Tillyer CR, Tarnoky AL. Albumin ‘ Jaffna’, a new variant, present in blood and urine. IRCS Med Sci 1983; 11: 250-251. 12 Hudson L, Hay FC. In: Practical immunology. Oxford: Blackwell Scientific Publications, 1976. 13 Davies BJ. Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 1964; 121: 404-427. 14 Schultze HE, Heremans JF. Molecular biology of human proteins with special reference to plasma proteins, Vol. 1. Amsterdam: Elsevier, 1966: 173-235. 15 Franklin SG, Wolf SI, Zweidler A, Blumberg B.S. Localisation of the amino acid substitution site in a new variant of human serum albumin, albumin Mexico-2. Proc Nat1 Acad Sci USA 1980; 77: 2.505-2509.