422

I t Mall?

pi

a/

Ernst Malle' Horst HeR2 Gerhard Miinscher' Gabriele hipping' Armin Steinmetz'

Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing

' Department of Internal Medicine, Division of Endocrinology and Metabolism, Philipps University, Marburgl Lahn Behringwerk AG, MarburgILahn Institute of Medical Biochemistry, Karl-Franzens University, Graz

The present work was aimed at isolating human serum amyloid A, (SAA), an acute-phase protein mainly complexed to high density lipoproteins, directly from human plasma without sequential ultracentrifugation of lipoproteins and subsequent delipidation of the apolipoprotein moiety. Hydrophobic-interaction fastprotein liquid chromatography on Octylsepharose, using stepwise gradient elution profiles under dissociating conditions, followed by fast-protein liquid-gel permeation chromatography on a Superdex TM" column revealed a higher than 95 O/o purity of isolated SAA. Further purification of SAA from coeluting apolipoproteins C and A-I1 was achieved by preparative isoelectric focusing between pH 5-7 using a Rotofor apparatus. Separation of the main SAA isoforms, SAAl (p16.5) and SAAl des-Arg ( p l 6.0, lacking the N-terminal arginine), was achieved by anion-exchange fast-protein liquid chromatography on a Fractogel E M D DEAE 650-S column. The purity of the SAAl and SAAl des-Arg isoforms, thus isolated, was checked by immunochemical techniques and amino acid analysis. With the described method various SAA isoforms can be isolated, purified and separated directly from human plasma/serum without prior ultracentrifugation.

1 Introduction Serum amyloid A (SAA) is an acute-phase protein of 104 amino acids corresponding to a calculated molecular mass of 11.5 kDa [l-31. Imniunochemical techniques [4] and the results of amino acid analyses, peptide maps and N-terminal sequence analysis [1,5-71 revealed SAA as the precursor of the amyloid A protein (molecular mass: 8.6 kDa, 76 amino acids), which forms the bulk of the fibrils in secondary, reactive aniyloidosis [8]. Moreover, human SAA is a nonglycated but polymorphic protein and different isoelectric focusing patterns have been reported [9]. In each pattern,isoforms with pl6.5 (SAA1) and p16.0 (SAA1 des-Arg, 103 amino acids, lacking the N-terminal arginine) occur. In addition, individuals may express either isoforms with p l 7.5/pl 7.0 (SAA2aISAA2a des-Arg) [lo] or isoforms with pI8.0/pl7.4 (SAA2P/SAA2b des-Arg) [lo] or may be heterozygous for all three pairs of isoforms with PI'S of 8.01 7.4-7.5l7.0-6.5/6.0. In the SAAl and SAAl des-Arg isoforms, ini'iially called SAA, and SAA, [6],an alanine/valine polymorphism at positions 52/57 [1,7] and an isoleucine/ leucine amino acid exchange in position 58 [7] has been reported. Recently, an additional allelic variation in the SAAl gene expressing the normal isoforms at p l 6 . 5 / p l 6 . 0 and isoforms at p l 6 . l / p l 5.7 named SAAhlARBllRG [ l l ] corresponding to a glycine/aspartate substitution at position 72 has been described 1121. The majorsite ofhuinan SAA(SAAl,SAA2a,and SAA2P)synthesis appears to be the liver [2,13]. After secretion to circulation part of a n acute-phase response to infarction, infection, or inflammation [8,14,15] SAA is mainly (90% or Correspondence: Ur. E. Malle, lnstitute of Medical BiochemisLry, KarlFraiizens-Univzrsity. liarrachgasse 21, A-801 0 Graz, Austria Abbreviations: apo. apolipoprotein; FPLC, fast protein liquid chromatography, H D L . high density lipoproteins; HIC, hydrophobic interaction chromatograph) : IEF, isoelectric focusing; SAA, serum amyloid A protein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis L'CH ~crlafhgesell,.chaft m h H . D-6940 Weinheim, 1992

more) complexed to high density lipoproteins (HDL) 1161 where it may account for up to 87% of the H D L apolipoprotein moiety [14]. SAA-enriched H D L subfraction-3 (HDL,) was found to be larger than normal HDL, (d=1.125-1.25 g/mL), having a radius of4.5-5.3 nm that extended into the size range ofHDL, (d=1.063-1.125 g/mL) [14,17]. As SAA behaves like an apolipoprotein (apo SAA), ultracentrifugation in the density range of total H D L (d=1.063-1.21 g/mL) was often used as the first step in SAA-isolation followed by different purification techniques. In the present study isolation of SAA was performed directly from human plasma under dissociating conditions by hydrophobic interaction chromatography (HIC), fast-protein liquid-gel permeation chromatography, and preparative isoelectric focusing (IEF). The separation of the main SAA isoforms, SAAl and SAAl des-Arg, was achieved by anion exchange chromatography.

2 Materials and methods 2.1 Reagents Sodium dodecyl sulfate (SDS), N,N,N',N'-tetramethylenediamine, N,N'-methylenebisacrylamide, Amberlite MB-6 and urea were from Serva (Heidelberg, Germany). Acrylamide (4 x recrystallized) was obtained from Roth KG (Karlsruhe, Germany). Guanidine hydrochloride (guanidine/HCl) was purchased from Fluka (Buchs, Switzerland). Octylsepharose and electrophoresis calibration kit (14.494 kDa) were from Pharmacia (Freiburg, Germany) whereas carrier ampholytes were obtained from Pharmacia (Freiburg, Germany) and Serva (Heidelberg, Germany). Immobilon polyvinylidene difluoride-membranes were from Millipore (Eschborn, Germany) and nitrocellulose was purchased form Schleicher & Schull (Dassel, Germany). Anti-sheep and anti-rabbit immunoglobulin (peroxidase conjugated) antibodies, and 4-chloro-1-naphthol were from Sigma (Taufkirchen, Germany). Agarose (Typ L) and bovine serum albumin (BSA) were from Behringwerke AG (Marburg, Germany). All other chemicals were ob0173-0835/92/0707-0422 $3.50+.25/0

E/pctrophore.sis

Isolation of serum amyloid A isoforms

1992, / 3 , 422-428

tained from Merck (Darmstadt, Germany) as analytical grade reagents. 2.2 Materials Human plasma was obtained from patients undergoing plasmapheresis for therapeutic purposes. Quantitation of SAA in plasma was performed by laser nephelometry (Behringwerke AG, Marburg, Germany) as described [18]. 2.3 HIC on Octylsepharose Separation of SAA from hydrophilic plasma proteins was achieved by chromatography on an Octylsepharose CL 4B column using a fast protein liquid chromatography (FPLC)system (Pharmacia). The FPLC-system employed two P-5000 high-pressure pumps and an LCC-500 liquid chromatography controller to form the gradient. The elution was controlled by a one-way UV monitor. A chart recorder with two channels was used to monitor the UV absorbance, the programmed gradient and the collected fractions (FRAC-100 fraction collector, Pharmacia). The column (Amicon G column, 44 X 250 mm, gel bed volume 44 X 140 mm) was equilibrated with buffer A (phosphate buffered saline, pH 7.0) at a flow rate of 2.0mL/min at 25°C. Plasma (200mL) dialyzed against buffer A was applied onto the Amicon G column and then eluted for 100 rnin at a flow rate of 2.0 mL/min and 25" C. A segmented gradient of 0% to 50% buffer B (buffer A containing 4M guanidine/ HC1/30% w/v ethylene glycol) for 200 min, resting for 300rnin at 50% buffer B. Then the column was eluted stepwise by increasing the gradient to 80% bufferB for 100 min, holding at 80% buffer B for 300 min and increasing the gradient to 100% buffer B for 100 min. Fractions of 10 mL were collected. All buffers used for FPLC were freshly prepared with deionized water,filtered through 0.2 pm membrane filters (Nalge, New York, NY, USA) and degassed before use. Urea was treated with mixed bed ion exchange resin on an Amberlite MB-6 column before use. 2.4 Chromatography of SAA by gel filtration A prepacked Superdex I M 75 prep grade column (35 X 600 mm, Pharmacia) was equilibrated at 25" C with 10 mM Tris/HCI buffer (pH 8.0) containing 5 M guanidine/HCl and 0.05 O/o NaN, w/v at a flow rate of 2.4 mL/min. A 30-40 mg protein sample (7.5-10 mL containing about 35 O/o w/v SAA) initially eluted from the Octylsepharose column was applied on the Superdex column at the same flow rate and fractions of 10 mL were collected. 2.5 Preparative IEF Preparative I E F of SAA prepurified by gel filtration was performed using the Rotofor (Bio-Rad, Munchen, Germany) at 12 W for 5 h. Buffers (pH 7.0)containing 6~ urea (10% w/v glycerol and 6.4% w/v carrier ampholytes) of different pH range (pH 3-9, pH 5-6, pH 5-7, Pharmacia, or pH 6-8, Serva) were used. Electrode solutions we-re either 0.1 M NaOH or 0.1 M H,PO,. The pH was routinely measured in all fractions (1-20) collected.

423

2.6 Anion-exchange chromatography Further separation of SAA isoforms following preparative I E F was performed on a Fractogel E M D DEAE-650 S (2040pm) column (10 X 150mm) using a universal glass cartridge system (Merck, Darmstadt, Germany). The column was equilibrated with buffer C (10 mM Tris/HCl, pH 8.2, containing 7~ urea) at a flow rate of 2 mL/min. A 4 mL protein sample containing 15mg of SAA was applied per separation. The column was then eluted at the same flow rate for 14 min followed by a segmented gradient of NaCl from 0 to 100 mM for 66 rnin and from 100 to 1000 mM for 20min (0 to lo%, 10 to 100% buffer D, containing lOmM Tris /HCl, 7~ urea and 1 M NaCl). 2.7 SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis (PAGE) was performed in 6.6-34 O/o w/v linear polyacrylamide gradient gels at lOOV, 50 mA, and 60 W for 1h, followed by 30 rnin at 400V, 100 mA, at 12" C in a Desaphor VA system (Desaga, Heidelberg, Germany) as described recently [ll]. 2.8 Analytical IEF Analytic a1 IEFwas carried out as described by Menzel etal. [19], slightly modified. Carrier ampholytes pH 3-5 (Serva) and pH 3 5 . 5 ,pH 5-7,and pH 7-9 (Pharmacia) were mixed in a ratio of 1:1:2:2 (v/v/v/v). IEF was performed at 10"C in a DesaphorVA apparatus (Desaga) and a Macrodrive (Pharmacia) power supply. After 15 h at 200V and 0.025 W/cm2 the voltage was increased to 600V and 0.075W/cm2 for 90 min. 2.9 Western blotting and other immunochemical procedures After I E F or SDS-PAGE,proteins were electrophoretically blotted onto an Immobilon membrane or nitrocellulose, with a semidry blotting technique, for 60 min (0.9 mA/cm2) at 25" C, using graphite electrodes (Pharmacia). A blotting buffer system, containing 48 mM Tris, 39 m~ glycine, 0.038% ~ / ~ S D S , 2 0 % v / v m e t h a n (pH9.5),was ol used.Immobilon membranes were then soaked for 30 rnin at 25" C in bufferE(50mMTris,90mMNaCI,pH8.0) containing2% w/v dried milk powder, followed by incubation for 3 h in buffer E, to which 8 pL/lO mL of polyclonal anti-human SAA antibodies (raised in sheep or rabbits)had been added. After washing with buffer E (50 mM Tris, 90 mM NaCl, pH 8.0) the membranes were incubated for 1h in buffer E containing 2 % w/v milk powder and 2.5 pL/mLperoxidase conjugated anti-sheep or anti-rabbit immunoglobulins. After extensive washing of membranes in buffer E, protein bands were visualized at 37"C in 100mL buffer F (50 mM Tris, 9 0 m ~NaCI, pH 6.5) containing lOOOpL of 1.8% w/v 4-chloro-1-naphthol in ethanol and 70 pL H 2 0 2 .

2.10 Preparation of oligoclonal antibodies Oligoclonal sequence-specific antibodies were raised in rabbits. The immunogens were synthetic peptides coupled to N-maleimido-butyryl-N-hydroxysuccinimideester-activated keyhole-limpet hemocyanin (Pierce, Chester, UK)

according to the manufacturer's recommendations. The peptides corresponded to amino acid residues 1-17,14-30, 27-44,40-63,59-72,68-84,79-94, and 89-104 of human SAAl protein [2] and each contained an additional N-terminal cysteine residue. All antibodies raised in rabbits failed to form irnniunoprecipitates in agarose gels.

2.11 Immunoelectrophoresis, double immunodiffusion and determination of protein content Purity of isolated SAA was assessed by double immunodiffusion and immunoelectrophoresis in 1 O/o agarose gels using polyclonal antibodies to human serum raised in sheep. Protein determination was performed by a commercially available test kit (Bio-Rad) with crystalline BSA as standard. 2.12 Amino acid analysis Amino acid analysis was performed according to Spackmann et ai.[20] in a Biotronic LC 7000 apparatus (column 14.5 X 0.32 cm, resin BTC 2710) on hydrolysates prepared in sealed tubes under vacuum in 6 M HC1 at 105" C for 24 h. For comparative purposes, the results are expressed as the percentage ofthe total number of pmoles of amino acids recovered, excluding ammonia and tryptophan. Correction factors (1.05 for Thr and Tyr; 1.11 for Ser) were applied for amino acids subjected to partial destruction during acid hydrolysis

3 Results To isolate the entire SAA occurring in plasma, dialyzed plasma was directly applied to Octylsepharose. Under our experimenl al conditions, Octylsepharose was capable of binding up to 75 m g of SAA/300 mL of acute-phase plasma as confirmed by laser nephelometry and a double immunodiffusion technique, using polyclonal anti-human SAA antibodies raised in sheep. Proteins were eluted by a stepwise gradient of guanidine/HCl/ethylene glycol using the FPLC system. Figure 1 shows the elution profile and 3 main peaks that were recovered: peak 2 ( 2 guanidine/ ~ HC1/15% ethylene glycol); peak 3 (3.2 M guanidine/HCl/ 24% ethylene glycol); peak 4 (4 M guanidine/HC1/30% ethylene glycol). After SDS-PAGE, staining of gels with

1

2

05

lr---/

1

00-l

i

/

20

LO

60

80

100

120

f roctions

F/gurr 1. H I C on Octylsepharose (solid line) o f 2 0 0 m L plasma containing SA.4 (25,5mg/dL), equilibrated with buffer A . After elution a segmented gradient (dashed line) was applied at a flow rate of2 mL/min. For details see Section 2.3.

Commassie Brilliant Blue revealed a 12 kDa protein in peak 2(Fig. 2, lane B).Neither peak 3 nor peak 4 contained this 12 kDa protein (Fig. 2 , lane C, D). Densitometric evaluation of gels after SDS-PAGE further showed that this 12 kDa protein was present up to 40% of the total protein content in the corresponding Octylsepharose fraction (Fig. 1, peak 2). Analytical I E F (pH 3-9) revealed two main protein bands with pl's of 6.5 and 6.0 at the same intensities only in peak 2 (Fig. 3, lane A). Western blot analysis and subsequent immunochemical detection with polyclonal anti-human SAA immunoglobulins confirmed both protein bands at p l 6 . 5 and pl6.0 to be the main SAA isoforms, SAAl and SAAl des-Arg SAA (data not shown). The SAA-containing fraction was rechromatographed by gel permeation-FPLC on a Superdex'" 75 prep grade column. Under dissociating conditions (5 M guanidine/HCl) two peaks (peak A and B) were eluted (Fig. 4). Peak A contained mainly apolipoproteins of the H D L fraction (apo A-I, apo A-IV) as confirmed by double immunodiffusion (data not shown) in addition to other plasma proteins (Fig. 2, lane E). Peak B contained the 12 kDa SAA protein (Fig. 2, lane F).Analytical IEF (pH 3-9) further confirmed the two protein bands at p l 6.5 and 6.0, representing SAAl and SAAl des-Arg present only in peak B but not in peak A (Fig. 3, lane D, E). In some preparations, impurities (less than

F/gure 2. SDS-PAGE of fractions obtained from HIC on Octylsepharose or gel filtration o n Superdex. Gels were stained with Coomassie Brilliant Blue as described [ 1 9 ] . Lane (A). molecular mass makers; (B) Octylsepharose, peak 2: (C) Octylsepharose. peak 3; (D) Octplsepharose, peak 4; ( E ) Supcrdcx Th17S. peak A; and (F) Superdex T'7S, peak B. For chromatography patterns see Pigs. 1 and 4.

Eiectt-ophorr.si.5 1992, 13, 422-428

Isolation of serum amyloid A isoforms

5 O h ) due to apo C-II/C-I11 (p/ 4.8-5.1) and to a lesser degree apo A-I1 (p14.9-5.0) were observed (Fig. 5, lane 1). Therefore, preparative I E F was applied as a further step in the purification of SAA. Carrier ampholytes of a suitable pH range (pH 5-7) allowed the purification of SAA from apo C proteins (Fig. 5, lane 2-6, pH range: 5.1 to 5.6). However, no clear separation of total SAAl and SAAl des-Arg could be a chieved (Fig. 5, lane 10-20, pH range: 6.0-6.8), even when narrow-range pH 5-6 carrier ampholytes were used in the Rotofor (data not included). Separation of SAAl and SAAl des-Arg was achieved by anion exchange chromatography using a Fractogel E M D DEAE 650-S gel bed (Fig. 6). Two peaks, peak C eluting at 0.25% w/vNaCl, 42.7 mM, peak D eluting at 0.4% w/v NaC1,

A

B C

D E

6 8 . 4 m ~correspond , to SAAl (pl6.5) and SAAl des-Arg (PI 6.0), respectively. This was confirmed by analytical I E F and Western blot analysis using both polyclonal antihuman SAA immunoglobulins (raised in sheep) or different oligoclonal antibodies (raised in rabbits), directed to different epitopes of the SAA protein. Both the polyclonal antibodies (Fig. 7, lane 18) as well as the oligoclonal antibodies (Fig. 7 , lane 19-26) further confirmed that SAA is not degraded during the entire isolation process. Immunoelectrophoresis of purified SAA revealed a single band close to the IgM migrating position ( y I -position), using polyclonal anti-human SAA antibodies (Fig. 8). Polyclonal anti-human serum antibodies neither reacted with SAAl nor with SAAl des-Arg-containing protein samples (data not included). Amino acid analysis of SAA subspecies purified by the described isolation procedure revealed n o remarkable differences between the expected and the observed amino acid composition (Table 1) in comparison to the purification technique of apo SAA reported previously from our laboratoy 1211 or published by other groups [1,221.

0

l.7

6.5 -

425

6.0 -

10 20 30 LO

fractions

F/g:ire 3. Analytical I E F (pH 3-9, 200V, 3 W, 15 h at 8°C) of fractions eluted after IIIC on Octylsepharose (peak 2-4) and subsequent gel filtration of peak 2 on Superdex T'75 prep grade gel bed. Gels were stained with Coomassie Brilliant Blue as described [19], Lane (A), Octylsepharose. peak 2; (B) Octylsepharose, peak 3; (C) Octylsepharose, peak 4; (D) Superdex T'75,peak A; and (E) Superdex r'75. peak B. For chromatography see Figs. 1 and 4.

1

5

Figure 4. Rechromatography on a Superdex T"75 prep grade column of peak2 eluted by H I C on Octylsepharose. For details see Section 2.4.Two peaks,Aand B; were eluted.PeakBcorresponds to an apparent molecular mass of 7-15 kDa.

20

- 6. 5 - 6*

Figure 5. Preparative I E F of SAA (lane l), purified by Octylsepharose (Fig. 1, peak 2) and subsequent gel permeation chromatography on Superdex (Fig. 4, peak B). Lane (2)-(6) show the separation ofapolipoproteins ofC-series still present as impurities. Lane (6)-(20) contain SAA corresponding to SAA1, p16.5, and SAAl des-Arg,pI 6.0, focused in the pH 5.6-6.8 range.

Electrophorrsis 1992. 13, 422-428

Table 1. Amino acid composition of S A A l isolated by H I C on Octylsepharose, gel filtration on Superdex TM75,preparalive IEF, and subsequent anion exchange chromatography on Fractogel E M D D E A E 650-S Amino acid 05 -

Asp

0 0

025-

lOOmM NoCl

0 00-

10 20

30 LO

fractions

of pooled fractions 10-20 obtained by preparative I E F (Fig. -5) on a Fractogel E M D D E A E 650-S column. For details see Section 2.6. Two peaks, corresponding to S A A l (peak C) and S A A l des-Arg (peak D) were eluted. Figure 6. Rechromatography

f

Asn

Th; Ser Glu i- Gln Pro GIy Ala Val CYs Met Ile Leu Tyr Phe LY s His Trp Arg

(a) 14 0 7 9 4 12 16 1

0 2 3 3 5 8 4 3 3 10

Composition given in "In (mole/100 moles) (b) (c) (d )

14.0 0.35 6.93 10.56 4.22 11.69 15.62 I .67 0 2.19 3.14 3.12 5.50 7.86 4.9 2.8 N.D. 9.58

13.47 1.47 7.76 12.30 7.17 13.40 16.15 1.58 0 2.02 3.08 5.04 5.29 8.00 5.89 3.03 N.D 9.89

13.9 0.4 5.8 9.1 3.9 12.4 15.4 0.9 0 1.9 2.7 3.6 4.9 7.3 4.1 2.9 1.9 10.2

(c) 13.8 Trace 9.3 11.5 3.7 12.3 14.3 1.5 0 1.6 2.5 3.7 4.6 6.9 4.4 3.6 0.8 7.8

(a) Expected composition (b) Present study (average of 3 determinations); (c) apo SAA isolated by FPLC [21] (d) SAA proteins purified by DEAE-chromatography [22]; mean of 4 preparations from a normal subject and a patient with Waldenstriim's macroglobulinemia. ( e ) SAA proteins purified by DEAE-chromatography [22]; single determination from a normal subject. N.D.. not determined

6.56.0-

4 Discussion

+

Up to now, the isolation of apo SAA from human plasma was mainly based on sequential-flotation centrifugation of H D L followed by delipidation and separation of apolipoproteins under dissociating conditions by gel filtration at different cross-linked gels beds, e. g . Sephadex (3-100, [22261, Sephacryl S-200 [22,27] or Sepharose A [22]. In a previous paper we reported the purification of apo SAA from apo A-I containing plasma particles after isolation of HDL, at density intervals of 1.12-1.25 g/mL, recentrifugation of 6 M guanidine-treated HDL, and subsequent delipidation of the HDL, apolipoprotein moiety [21]. Other isolation procedures were also based on sequential-flotation centrifugation, followed by preparative SDS-PAGE of apolipoproteins and subsequent electroelution of apo SAA- containing bands [3,17,28]. Other authors reported the direct isolation of SAA from plasma acidified with formic acid (10% v/v) and subsequent gel filtration of apoproteins on Bio-Gel P-60 and Sephadex (3-75 [ 14,29,30], Sephadex G-50 [29] or Sephacryl S-200 [31] Superfine gels.

Figure 8. Ininiunoelectrophoresis (1O/o agarose,4 h,30 mA, 10OC) of SAA purified by NIC, gel filtration, preparative IEF, and anion exchangeFPLC. Polyclonal anti-human SAA antibodies raised in sheep were used as antiserum.

Raynes and McAdams [32] first described the isolation of SAA by a combination of gel permeation chromatography and HIC using different hydrophobic gel beds. The recovery of SAA isolated o n Sepharose gels was higher [32] than purification after ultracentrifugation [ 171. Affinity chromatography of SAA-enriched plasma on cholesteryl hemisuccinate and elution with 6 M guanidineIHC1 (0.55 M Tris/ HCI, pH 7.0), however, yielded even a lower recovery of SAA isolated directly from serum than obtained from HDL, after centrifugation of serum [33]. As SAA is associated with various lipoprotein species [23,27] and/or with

Figure 7. Vertical IEF and Western blotting followed by immunochemical detection of S.4A of fraction 10 of preparative IEF. Blots were incubated with polyclonal anti-human SAA antibodies (lane 18) or oligoclonal antibodies directed to h u m a n S A A l epitopes of the following amino acid sequence: 1- 17 (lane 19), 14-30 (lane 20), 27-44 (lane 21), 40-63 (lane 22). 59--72 (lane 23), 68-84 (lane 24), 79-94 (lane 25), and 89-104 (lane 26).

I I

Isolation of’ serum amyloid A isoforms

Elpctruphorests 1992, 13, 422-428

albumin or plasma proteins of high molecular weight [23,29] HIC in combination with FPLC seems a suitable technique for rapid isolation of entire on SAA from plasma. Raynes and McAdams [32] observed that almost the total amount of serum cholesterol (> 98.7%) and triglycerides (>93.5Yo) was bound to hydrophobic gel beds even when elution of SAA from serum was performed by means of a linear gradient of guanidine/HCl/ethylene glycol/NaOH. Under our experimental conditions using either plasma or pure lipoproteins, such as very low density lipoproteins (d=0.94-1.006 g/mL) or low density lipoproteins (d=1.006-1.063 g/mL) isolated by sequential ultracentrifugation, we also observed that apo B-100 was completely adsorbed to 0ctylsepharose.These findings underline the prefereritial use of H I C in apolipoprotein isolation. The use of Octylsepharose makes it possible to recover up to 85% of the apo A-I and apo A-IV and up to 100% of the SAA. This high binding capacity of SAA to hydrophobic gel beds is obviously due to a weak amphipathic a-helix of the N-terminal 11 amino acid residues which form the functionally important lipid-binding site of SAA [27,34,35]. A stepwise gradient of guanidine/HCl/ethylene glycol (Fig. 1) followed by a baseline separation of SAA from other plasma proteins on a Superdex TM75column (5 M guanidine/HCl, pH 8.0, Fig. 4) allowed a 90-95 O/o recovery of SAA. Guanidine/HCI, however, altered the apparent diameter of gel beds and thus the apparent molecular mass of each of the apoproteins. By means of this denaturating agent a baseline separation of SAA from apo A-I (28.1 kDa) but not from apo A-I1 (17 kDa) and from apo C-proteins (about 8.8 kDa) was observed. However, as a result of preparative I E F (Fig. 5 ) no further impurities in SAA preparations were present. The total recovery of SAA thus isolated ranges between 50-70%, depending on the separation of SAA from apolipoproteins (mentioned above) by the preparative I E F Rotofor system, represents the crucial step in SAA purification. Although preparative IEF is an excellent technique for the isolation of various plasma proteins, the separation of SAAl and SAAl des-Arg can only be achieved by subsequent anion exchange chromatography even when carrier ampholyte mixtures of different pH ranges were applied in the Rotofor system. Separation of both isoforms has been described on DEAE 52 and DEAE-Sephacel [29,36], or Mono Q H R [21].We applied Fractogel E M D DEAE 650-S under denaturing conditions ( 7 urea) ~ for the isolation of SAAl and SAAl des-Arg by means of a combination of ion exchange chromatograpy-FPLC. After analytical IEF and subsequent silver staining of gels in some SAA preparations, a faint band at pZ6.05 and pZ5.65, coeluted with the corresponding main SAA isoforms at pZ6.5 and pZ6.0, respectively (Fig. 5). These protein bands at PI’S 6.0515.65 could be observed even in a previous report [21] when SAA was isolated by completely different techniques, and in the present study when sera from patients having SAA levels higher than 40 mg/dL were subjected to I E F and SAA isoforms could be detected with polyclonal anti-human SAA antibodies after Western blotting (data not shown). Both bands at pZs 6.05/5.65, reacted with all the different oligoclonal anti-SAA peptide antibodies. It is more likely that these findings suggest carbamylation byproducts or deamination processes similar to those observed for apo A-I [37] than proteolytic processing of SAAI/SAAI des-Arg. First, the loss of the Ser residue (position 2 of SAA) from the

427

N-terminus does not alter the pZ of SAA. Secondly, no human SAA isoforms in which NH,-terminal trimming has proceeded beyond position 2 [3] or degradation from the C-terminal side of the protein have been reported so far [22]. Thirdly, slices of gels with the SAA-like “degradation product at pZ5.6” revealed a similar apparent molecular mass as observed for SAA when applied to SDS-PAGE [ l l ] . All these findings taken together d o not suggesta drastic heterogeneity of acute-phase SAA in molecular mass as previously reported [38]. In summary, we have presented a n isolation protocol for SAA and its main isoforms, SAAl and SAAl des-Arg, by a combined methodology of chromatographic techniques and preparative IEF. This method may prove useful not only for the isolation of different apolipoproteins but, more likely, also for the isolation and separation of SAA isoforms such as SAAlISAAl des-Arg (pZ6.5/6.0), SAA2uISAA2a des-Arg (p18.0/7.4), or SAA2B/SAA2P des-Arg (p17.5/7.0) using carrier ampholytes of suitable pH range prior to ion exchange chromatography-FPLC. This work was supported by grants f r o m the Austrian FWF to G.K. (P 8249 MED), the DFG and the Kempkes-Stlftung to A.S. and the E Lanyar Foundation to E.M. The excellent technical assistance of Petra Vesper and Christa Weiherhaeuser is gratefully acknowledged. Received February 26, 1991

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