α1-Fetoprotein: Separation of two molecular variants by affinity chromatography with concanavalin A-agarose

Biochimica et Biophysica Aeta, 317 (1973) 231-235 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA

36474

al-FETOPROTEIN: SEPARATION OF TWO MOLECULAR VARIANTS BY AFFINITY CHROMATOGRAPHY WITH CONCANAVALIN A-AGAROSE

C A R O L J. S M I T H AND P H I L I P C. K E L L E H E R

Department of Medicine, University of Vermont College of Medicine, Burlington, Vt. o54o1 (U.S.A .) (Received April 4th, I973)

SUMMARY

al-Fetoprotein present in fetal/newborn rat serum and in hepatoma-bearing human serum has been resolved into two molecular variants by concanavalin Aagarose affinity chromatography. The concanavalin A-reactive variant has been purified by gel filtration column chromatography, preparative block electrophoresis and immunoadsorption affinity chromatography.

INTRODUCTION

Most of the effective methods reported for the purification of al-fetoprotein have employed a combination of physicochemical and immunochemical approaches. Human al-fetoprotein has been purified by various combinations of the techniques of (NH4)2SO4 fractionation; immunofiltration2-*, immune precipitation 5-8, ionexchange chromatography1,83, 8 and isoelectric focusing5. Alpert et al2 described an isolation procedure for human al-fetoprotein which did not involve an immunochemical step. Rat al-fetoprotein has been isolated reportedly by geon-pevicon electrophoresis at pH 8. 9 (ref. IO) and by a complicated procedure involving salt fractionation, ion-exchange chromatography, preparative electrophoresis, molecular filtration and repeated isoelectric focusingn. Major problems in isolating al-fetoprotein have been the separation of the human or rat al-fetoprotein from albumin, the complexity of some of the methods reported and the exposure of the proteins to non-physiologic conditions, such as low pH buffers or chaotropic ions. Molecular heterogeneity of human a~-fetoprotein has been demonstrated by Alpert et al2. Two variants were resolved by isoelectric focusing and were converted to a single electrophoretically slower form by treatment with neuraminidase. No molecular variants of al-fetoprotein have been separated on a preparative scale. This paper reports the results of our studies on the interaction of concanavalin A with al-fetoprotein and describes a procedure for separating two molecular variants of rat and human al-fetoprotein. Concanavalin A-reactive al-fetoprotein has been purified by a method that avoids exposure of the protein to extremes of pH or other potentially denaturing conditions.

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MATERIALS AND METHODS

Sprague Dawley albino rats were used in these experiments. 2o-day-gestation fetal rats or 1-day-old newborn rats were bled by neck vein incision. The blood was allowed to clot, and the serum was separated by centrifugation. Hepatoma-bearing human serum generously was supplied by Dr Elliot Alpert, Massachusetts General Hospital, Boston, Mass. Antisera were prepared by injecting an emulsion (i :I, V/V) of fetal/newborn rat serum proteins or of hepatoma-bearing human serum albumin al-fetoprotein prepared by agarose gel filtration chromatography and complete Freund's adjuvant into adult rabbits at four subcutaneous sites. A booster injection was given in 3 weeks, and the rabbits were bled by cardiac puncture I week later. The antiserum of fetal/newborn rat serum proteins was rendered specific for ~qfetoprotein by absorption with normal adult rat serum proteins; the antiserum to hepatoma-bearing human serum albumin-ayfetoprotein was rendered specific for human ~h-fetoprotein by absorption with normal adult human serum proteins. These antisera were used to locate the rat and human ~q-fetoprotein-containing fractions, respectively, in the isolation steps. Fetal/newborn rat serum proteins or hepatoma-bearing human serum proteins were fractionated on a column of lO% agarose gel (Bio-Gel A o.5 m, 2oo-4oo mesh) and were eluted with o.oI M sodium phosphate buffer, pH 7.2, o.14 M NaC1, o.oi M sodium azide. Column fractions were monitored for the presence of protein by absorbance at 28o nm and for the presence of a~-fetoprotein by Ouchterhmy r' gel double immunodiffusion. Preparative electrophoresis was carried out on the rat or human al-fetoprotein-containing agarose column fractions. Starch block electrophoresis was performed using potato starch in o.o 5 M sodium barbital buffer, pH 8.6, at 8 V/cm for I8 h, and preparative agar gel electrophoresis was performed using o.75 % agarose in 0.05 M Tris-HC1 buffer, p H 8.o, at 6 V/cm for IO h. Proteins were eluted from the starch or agarose with o.15 M NaC1 and were quantified by the method of Lowry trt a/. la. RESULTS AND DISCUSSION

Because of the absence of carbohydrate moieties, albumin does not react with concanavalin A (ref. I4). When the rat al-fetoprotein-albumin preparation from agarose column chromatography was incubated with soluble concanavalin A, both al-fetoprotein and albumin were present in the soluble phase of the mixture. The precipitate did not contain any al-fetoprotein. Examination of the immunoelectrophoretic precipitin arcs of albumin and al-fetoprotein, however, revealed that tile al-fetoprotein's electrophoretic mobility and immunoprecipitate shape had been altered by the presence of concanavalin A (Fig. I). The albumin immunoprecipitin arc was not affected. Addition of glucose to the cormanavalin A-protein mixture caused the al-fetoprotein's electrophoretic mobility and immunoprecipitate shape to revert to normal. These results demonstrated that concanavalin A and al-fetoprotein formed a soluble complex in the presence of excess concanavalin A, which is in contrast to the insoluble complexes which form between m a n y carbohydrate-containing macromolecules and concanavalin A (refs 15, 16) and is consistent with a single concanavalin A reactive site. The instability of the concanavalin A al-fetoprotein

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Fig. I. Microimmunoelectrophoretic analysis of a fetal rat s e r u m albumin-ax-fetoprotein p r e p a r a tion. The sample in the u p p e r well on each slide was incubated with soluble concanavalin A. Antis e r u m was to whole fetal rat s e r u m proteins; a n t i s e r u m in the lower slide was a b s o r b e d with whole n o r m a l a d u l t r a t s e r u m proteins.

complex made separation of the complexed al-fetoprotein from non-complexed serum proteins by gel filtration impossible. The interaction of concanavalin A chemically coupled to agarose with polysaccharides and glycoproteins has been described by Lloyd 17. al-Fetoprotein-containing fractions from the starch block or agar gel electrophoresis were dialyzed against o.o5 M Tris-HC1 buffer, pH 7.4, i.o M NaCI, o.o2% I,i,I-trichloro-2-methyl2-propanol (w/v) (chloretone), in order to remove all traces of soluble carbohydrate. The rat or human protein preparations were chromatographed on a o. 9 cm × 3 ° cm column of Glycosylex-A (Miles-Yeda Laboratories, Kankakee, Ill., (U.S.A.) and Rehovot, Israel) or Con A-Sepharose (Pharmacia Fine Chemicals, Piscataway, New Jersey) which is concanavalin A chemically linked to a 4% agarose gel (Fig. 2). Initial experiments were performed with o.oi M sodium azide rather than chloretone as the bacteriocidal agent in these buffers, but it was learned subsequently that the use of sodium azide results in the partial loss of the carbohydrate-binding capacity of the concanavalin A (Rashi, M., Miles-Yeda Ltd, Rehovot, Israel, personal communication). The fraction that was elated with the initial buffer contained all the albumin and the variant of at-fetoprotein which does not react with concanavalin A, while the fraction eluted by the glucose-containing buffer included the concanavalin A-reactive al-fetoprotein present in the sample. Upon rechromatography the retained

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Fig. 2. Concanavalin A-agarose c o l u m n c h r o m a t o g r a p h y of a fetal r a t s e r u m albumin-a~-fetoprotein p r e p a r a t i o n . S t a r t i n g buffer was o.o 5 M Tris-HC1, p H 7.4, I.O M NaC1, o.o20/o chloretone, o.ooi M Ca 2+, o.ooi M Mn2L A r r o w indicates the addition of o.I M glucose in o.o 5 M Tris-HC1, p H 7.4, I.O M NaC1, o.o2% chloretone.

and non-retained ~q-fetoproteins were eluted in their respective original elution positions. Tile concanavalin A-reactive rat and human al-fetoproteins were free from albumin by the criteria of cellulose acetate electrophoresis and immunoelectrophoresis using wide spectrum antisera to whole adult rat and whole fetal rat serum proteins and to hepatoma-bearing serum albumin al-fetoprotein, respectively. Based on the conditions of the immunoelectrophoretic analyses, albumin would have been detected if it had constituted more than 2.5% of the total protein of the concanavalin Areactive fraction. Small amounts of adult rat serum proteins contaminating the fetal rat serum concanavalin A-reactive a~-fetoprotein were removed by passage of the sample through an immunoadsorbent column of rabbit antibodies to normal adult rat serum proteins coupled to cyanogen bromide-activated Sepharose 4 B (Pharmacia Fine Chemicals). The ax-fetoprotein fraction was eluted from the column with o.o5 M sodium phosphate buffer, pH 7.o, o.5 M NaC1; tile bound proteins were eluted with 3 M NaCNS in o.o 5 M sodium phosphate buffer, pH 6.o. Ouchterlony gel double immunodiffusion using specific antiserum to fetal rat serum al-fetoprotein demonstrated complete immunologic identity among all the concanavalin A-agarose retained a~-fetoproteins, among all the non-retained atfetoproteins and among the retained and non-retained am-fetoproteins from six fetal rat litters. Coneanavalin A-agarose chromatography of the maternal rat sera corresponding to the fetal litters also revealed the two forms of a~-fetoprotein. Electroimmunodiffusion analysis using antiserum specific for fetal rat serum al-fetoprotein demonstrated differences in the relative concentrations of the fetal and maternal non-retained and retained ai-fetoproteins. The ratio of the concentrations of the non-retained to retained a~-fetoproteins from the fetal sera ranges from o.75 to 1.75

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235

(mean 1.32); from m a t e r n a l sera o. 4 to i . i (mean o.73 ). The fetal r a t serum nonr e t a i n e d a : f e t o p r o t e i n c o n c e n t r a t i o n is approx. 35 times higher t h a n t h a t of t h e m a t e r n a l n o n - r e t a i n e d a l - f e t o p r o t e i n , the fetal r e t a i n e d a l - f e t o p r o t e i n concentration, a p p r o x . 20 times higher t h a n t h a t of the m a t e r n a l r e t a i n e d a : f e t o p r o t e i n . The results of the c o n c a n a v a l i n A-agarose column c h r o m a t o g r a p h y h a v e p r o v e n r e p r o d u c i b l e a n d d e m o n s t r a t e t h a t this m e t h o d is suitable for p r e p a r a t i v e scale s e p a r a t i o n of two molecular v a r i a n t s o f a : f e t o p r o t e i n for use in physicochemical, f u n c t i o n a l a n d genetic comparisons. I n addition, the c o n c a n a v a l i n A-reactive a : f e t o p r o t e i n v a r i a n t is s e p a r a t e d c o m p l e t e l y from albumin. The concanavalin A-nonr e a c t i v e a n d reactive v a r i a n t s m a y be purified b y passage t h r o u g h i m m u n o a d s o r b e n t columns using a n t i b o d i e s to concanavalin A - n o n - r e a c t i v e a n d reactive a d u l t r a t serum proteins, respectively. The a d v a n t a g e s of our m e t h o d over those r e p o r t e d to d a t e are its r e l a t i v e simplicity, high recovery, s u i t a b i l i t y for p r e p a r a t i v e scale work, m i l d conditions of p H values a n d salt concentration a n d lack of exposure of the a : f e t o p r o t e i n to ethanol, h e a v y metals, chaotropic ions a n d non-physiologic p H values. ACKNOWLEDGEMENTS This p r o j e c t was a i d e d b y G r a n t D R G 1184 from the D a m o n R u n y o n Memorial F u n d for Cancer Research.

REFERENCES 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17

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