Application of polyacrylamide slab gel electrophoresis to the analysis and small-scale purification of amyloid proteins

Analytica Chimica Acta 372 (1998) 161±172

Review

Application of polyacrylamide slab gel electrophoresis to the analysis and small-scale puri®cation of amyloid proteins B. Kaplan1 Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer 52621, Israel

Abstract Polyacrylamide slab gel electrophoresis (PAGE) is widely used in different ®elds of amyloid research. We review the uses of PAGE for the determination of molecular mass and degree of purity of isolated amyloid proteins, for their immunochemical identi®cation by immunoblotting, and for their chemical characterization using microsequencing strategies. The review demonstrates the role of PAGE in the studies of the processing of amyloid precursor proteins, describes the application of PAGE for detection and typing of amyloid proteins in small biopsy tissues, and discusses the utility of PAGE for small-scale puri®cation of amyloid proteins. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Amyloid protein(s); Polyacrylamide slab gel electrophoresis; Immunoblotting; High performance liquid chromatography; Microsequencing; Small-scale puri®cation

1. Introduction First introduced by Raymond and Wang in 1960 [1], polyacrylamide gel electrophoresis (PAGE) is presently one of the most widespread techniques used in the analysis of mixtures of proteins, lipoproteins, glycoproteins and nucleic acids. PAGE is widely applied in the examination of amyloid, a proteinaceous ®brillar material, whose deposition in various tissues causes illness and death by physical encroachment on normal organ structures. In the early seventies, when the biochemical studies of amyloid ®brils began, PAGE was an indispensable analytical tool for determination of the molecular weight and degree of purity of the isolated amyloid proteins. Three decades later, with the marked advances in the methods of 1

Tel.: 00 972 3 5303363; fax: 00 972 3 5307002.

0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00336-5

chromatographic separation and structural analysis of proteins and peptides, PAGE continues to play an important role in detection, characterization and puri®cation of amyloid proteins. This review describes the application of slab PAGE in different ®elds of amyloid research. 2. Amyloid and amyloidosis Amyloidosis is a name given to a heterogeneous group of disorders characterized by an extracellular deposition of a proteinaceous material, amyloid, in various tissues and organs. Amyloid deposits in different forms of amyloidosis show a typical green birefringence when stained with Congo red and viewed under polarized light. Amyloid deposits consist of parallel arrays of rigid ®brils each with a diameter of 7.5±10 nm, having a b-pleated sheet

162

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

con®guration [2,3]. In spite of morphological and tinctorial similarities of amyloid ®brils in different clinical entities, differences were found in the chemical composition of the major ®bril constituents ± low molecular weight proteins ranging from about 3 to 30 kDa. It was shown that in most cases the nature of these small molecular weight proteins is related to distinct clinical forms of amyloidosis. The identi®cation of these ®bril proteins made possible the chemical classi®cation of amyloid proteins and amyloidoses. A steadily increasing number of such proteins (17 at the present time) has been characterized by their amino acid sequence [4]. The in¯ammation-related amyloid (AA), immunoglobulin-related amyloids (AL), transthyretin amyloid (ATTR), Alzheimer's disease-related amyloid (amyloid b) and b2-microglobulin amyloids are the most widely studied amyloid proteins. Amyloid ®brils derived from presumably normal proteins, such as immunoglobulin light chains in idiopathic, myeloma- or macroglobulinaemia-associated amyloidosis (AL amyloidosis), serum amyloid A (SAA) in familial Mediterranean fever and reactive amyloidosis (AA amyloidosis), transthyretin in familial polyneuropathy, cardiomyopathy, systemic senile amyloidosis (TTR amyloidosis), amyloid beta precursor protein (AbPP) in Alzheimer's disease. The mechanisms leading to ®bril formation are not fully understood, but could possibly include proteolytic degradation of the precursor proteins, their overproduction, point mutations, or post-translational modi®cations [5]. In addition to major ®bril proteins amyloid deposits contain non-®brillar constituents such as amyloid P component, amyloid enhancing factor, apolipoprotein E, proteoglycans, and glycosaminoglycans. These non-®brillar components of amyloid deposits appear to be common elements in different forms of amyloidosis. During the last few years there has been an increased interest in the potential roles of these components in the pathogenesis of amyloidosis [6,7]. 3. PAGE in analysis of amyloid proteins 3.1. Amyloid fibril proteins Although amyloidosis was recognized more than 300 years ago [8], the study of amyloid proteins on a

chemical level started only about 30 years ago when Pras et al. [9] developed the water extraction technique for the isolation of amyloid ®brils from amyloid bearing tissues, and Glenner et al. [10] showed the possibility of solubilizing amyloid ®brils using chaotropic agents and purifying the major ®bril proteins by gel permeation chromatography. At the present time the classical approach in examination of amyloid proteins is still based on these techniques: amyloid ®brils prepared by the water extraction technique are subjected to the column gel chromatography, and the isolated proteins are analyzed immunochemically and chemically. The ®rst electrophoretic examinations of amyloid proteins were performed using rod gels [10±12], but later this technique was replaced by slab gel electrophoresis. The slab gel con®guration was found to be more suitable for the molecular mass determination and allowed electrotransfer of proteins from gels onto thin blotting membranes for their further analysis. The vertical slab gel apparatus is commonly used in amyloid research. Sodium dodecyl sulfate PAGE (SDS±PAGE) is undoubtedly one of the most popular techniques used for electrophoretic separation of amyloid ®bril proteins and determination of their molecular mass. As a rule, the discontinuous buffer system originally described by Laemmli [13] is applied [14±24]. The proteins are usually run on the polyacrylamide gels of uniform concentration (15±17%) or of linear gradient concentration (from 5% to 20%). The Laemmli buffer system [13] utilized in these studies consists of: 1. 0.125 M Tris±HCl (pH 6.8) stacking gel buffer containing 0.1% SDS, 2. 0.375 M Tris±HCl (pH 8.8) running gel buffer (0.1% SDS), and 3. reservoir buffer containing 0.025 M Tris, 0.192 M glycine and 0.1% SDS. Since the resolution of amyloid proteins with a molecular weight lower than 10 kDa could be difficult when using the original Laemmli method [13], some other modified techniques have been applied. The techniques utilizing 6±8 M urea in the discontinuous buffer PAGE system of Ornstein [25] and Davis [26], in the continuous Tris±phosphate buffer SDS±PAGE system of Swank and Munkres [27], and in the discontinuous Tris±glycine [13], Tris±tricine [28] and

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

Tris±bicine [29] SDS±PAGE systems were successfully applied in different amyloid studies [30±37]. A protocol including tricine as a tracking ion [24] (instead of glycine in the Laemmli system [13]) was found to be effective for resolving of smaller proteins in the absence of urea and was widely used for the examination of amyloid b (Ab) proteins [38±44]. Since the introduction of electrophoretic transfer of proteins out of polyacrylamide slab gel onto a sheet of nitrocellulose [45], this technique gained a wide application in immunochemical examination (immunoblotting) of amyloid proteins. Development of sensitive immunodetection systems utilizing enzyme labeling, radioactivity or chemiluminescent tag was especially important for these studies. Most studies have been performed using the commercially available transfer chambers and nitrocellulose paper. Typical transfer buffer consisted of 20 mM Tris and 150 mM glycine, pH 8.3 (Towbin buffer [45]). Methanol was usually added to the transfer buffer (20% by volume) in order to minimize the swelling of the gel and to increase the binding capacity of nitrocellulose to protein. Addition of 0.1% SDS to transfer buffer was often used to increase the ef®ciency of protein transfer. Immunoblotting was shown to be effective for determination of type and size of amyloid proteins when using the appropriate antibodies raised against different amyloid ®bril proteins and their precursor proteins [14±16,18,22±24,38±40,42,43,46±49]. The importance of PAGE in examination of amyloid proteins has increased further with the development of microsequencing techniques and introduction of polyvinylidenedi¯uoride (PVDF) membranes as electroblotting and sequence supports [50]. It allows N-terminal sequencing of the electrophoretically separated proteins electroblotted onto PVDF membranes to be performed [51]. It also made possible to obtain the internal sequences by the enzymatic cleavage of the electrophorezed proteins in situ in the gels or on the membranes [52]. Thus, PAGE became a very promising alternative approach in the puri®cation of amyloid ®bril proteins for their chemical analysis because the open-column gel chromatography commonly used for puri®cation of amyloid ®bril proteins (see below) is a time-consuming procedure requiring large amounts of starting tissue material. In contrast, SDS±PAGE, being more fast and simple, permits analysis of partially puri®ed protein samples and it

163

is ideal for preparation of microgram quantities of protein suf®cient for their microsequencing. This SDS±PAGE-based microsequencing strategy has been applied for the chemical characterization of different amyloid proteins [15,23,40,53±60]. 3.2. Amyloid precursor proteins Detailed examination of the structure of amyloid precursor proteins and their processing in normal state and disease is extremely important for understanding amyloidogenesis. In this respect, most data are accumulated with regard to the precursor proteins implicated in the formation of AA, AL, and Ab proteins. 3.2.1. Serum amyloid A The structural identity of protein AA (8 kDa) with the N-terminal of serum amyloid A (SAA, 12 kDa) suggests that AA is a split product of SAA. SAA are highly homologous apolipoproteins, representing some of the most dramatic acute phase reactants. Two-dimensional electrophoresis, especially the combination of SDS±PAGE and isoelectrofocusing (IEF) in polyacrylamide gels, followed by immunoblotting contributed signi®cantly to the elucidation of SAA structure. Most IEF studies have been performed using carrier ampholyte-generated pH gradients, but in some studies an immobilized pH gradient was applied [61± 77]. Application of these techniques has shown that in humans there are six possible isoforms, which are the products of two genes, SAA1 and SAA2. SAA1 proteins arise from ®ve possible alleles: each of the SAA1 proteins has two isomers differing by one amino acid and focusing at pI 6.0 and 6.4, respectively. SAA2 proteins are encoded by two alleles, each of which gives rise to two isomers focusing at pI 7.0 and 7.5, and pI 7.4 and 8.0. In the acute phase plasma of mice two major isomers were found: SAA1 (pI 6.35) and SAA2 (pI 6.2) [78]. Only one of them, SAA2, is amyloidogenic, leading to the formation of AA proteins in the experimentally induced murine amyloidosis, as it was revealed using immunohistochemical, electrophoretic and immunoblotting techniques [23,36,79,80]. Contrary to that several isomeric forms of SAA are found to be involved in the pathogenesis of AA amyloidosis in humans [81]. In vitro studies of the enzymatic SAA degradation were undertaken to reveal the possible mechanisms of

164

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

protein AA formation. SDS±PAGE and immunoblotting were used to analyze proteolytic products following incubation of SAA with blood monocytes [82], Kupffer cells [83], different serine proteases [84], and acid proteases [85]. Studies were carried out to elucidate the sequence of events leading to amyloidosis: either SAA is ®rst degraded enzymatically and only then deposited in tissues, or SAA is ®rst incorporated into amyloid ®brils and afterwards processed to AA. The data of electrophoretic, Western blotting and microsequencing analyses of the amyloid deposits in mice strongly supported the second possibility [23,36,79]. 3.2.2. Immunoglobulin light chain proteins In AL amyloidosis, the amyloid deposits consist of monoclonal light chains, i.e. Bence Jones proteins, and fragments consisting primarily of the light chain variable domain. A lot of effort has been devoted to comparative structural examination of light chain amyloid deposits and that of Bence Jones proteins from patients with and without amyloidosis. The electrophoretic systems of Laemmli [13] and Shragger and von Jagow [28], and SDS±PAGE-based microsequencing techniques were the major analytical methods in those studies [54±58,86,87]. A number of amino acid substitutions in the framework region, or complementarity-determining regions, or the presence of glycosylated residues in the light chain variable region were revealed. However, the observed alterations have not yet made it possible to differentiate between the ``amyloidogenic'' and ``non-amyloidogenic'' forms of these proteins. It has been proposed that multiple rather than single amino acid alterations modify the tertiary structural features of the light chain, causing it to be amyloidogenic under appropriate conditions [88]. 3.2.3. Amyloid b precursor proteins Cerebral deposition of 4 kDa amyloid b (Ab) peptide is an early and invariant feature of Alzheimer's disease (AD). Numerous studies were aimed to elucidate the mechanism of Ab formation from the much larger amyloid b precursor protein (AbPP), a membrane-spanning glycoprotein (110±120 kDa). Most information on the structure of AbPP and its processing was obtained by using cultured cells [89±93]; some of the studies were performed by analyzing

cerebral cortex tissue homogenates [89]. Immunoprecipitation, SDS±PAGE, immunoblotting, and microsequencing were widely applied in these studies. It has been shown that normal secretion of AbPP involves a cleavage within Ab region by an enzyme termed asecretase, releasing a large soluble extramembraneous portion and retaining a smaller C-terminal fragment in the membrane. This secretory pathway precludes the formation of Ab. Current efforts are directed to study the alternative proteolytic pathway (by enzyme b-secretase), that can generate the intact b-peptide bearing fragments from full length AbPP. A 22 kDa AbPP fragment was selectively detected in microvessels puri®ed from cerebral cortex of healthy persons and AD patients; the size and immunoreactivity of this protein indicate that it is a stable fragment of bAPP containing intact Ab [94]. The 11.5 kDa membrane-bound AbPP fragment detected immunochemically, by using the transfected 293 cell system expressing AbPP cDNA, was also supposed to contain an intact Ab [95]. In a further study of potentially amyloidogenic C-terminal derivates of AbPP, the membrane associated proteins of human cerebral cortex were prepared, af®nity puri®ed and separated by Tris±tricine SDS±PAGE. Immunoblotting showed that AbPP is normally processed into a complex set of 8±12 kDa C-terminal derivates [96,97]. It is believed that formation of the potentially amyloidogenic fragments occurs intracellularly, by an endosomal/lysosomal pathway. Incubation of living endothelial cells with an AbPP antibody revealed reinternalization of mature AbPP from the cell surface and its targeting to endosomes/lysosomes. Immunoblotting of the puri®ed lysosomes showed the presence of intact AbPP and Ab containing proteolytic products [98]. On the other hand, recent immunochemical studies revealed the presence of a full length AbPP in culture supernatants of human neuroblastoma and kidney cell lines [95] and in the media of PC12 cell culture [99,100]. The secretion of full length AbPP by insect cells infected with recombinant baculovirus was also demonstrated by immunoblotting [101]. These data strongly suggest that some processing of AbPP may occur extracellularly. A third type of cleavage, by g-secretase, processes AbPP to Ab. First considered as a pathological event, it is now reassessed in the light of the observations that Ab is detected in soluble form in vitro and in vivo

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

during the normal proteolytic processing. The presence of 4 kDa Ab proteins in conditioned medium of variety cultured cell lines, in cerebrospinal ¯uid, plasma and urine of healthy persons and AD patients was demonstrated by immunoblotting and con®rmed by the microsequencing of the electroblotted proteins [102±108]. 3.3. Common elements 3.3.1. Amyloid enhancing factor The ability to accelerate the deposition of amyloid in experimental animals by injection of cells or tissue extracts is described in numerous reports. However, the biochemical nature of this accelerating substance called amyloid enhancing factor (AEF) is still one of the most intriguing subjects in the amyloid research. Efforts were made to purify and characterize the AEF using chromatographic, electrophoretic and immunoblotting techniques, but they still have not yielded a clear answer. In some studies the AEF activity was found in a high molecular weight substance [109,110]. However, other researchers have found that AEF could represent small fragments of amyloid ®brils [111], or low molecular weight proteins or glycoproteins of 16 [112], 15 [113], 22 [114], and 9±11 kDa [115]. The identi®cation of AEF as ubiquitin was demonstrated in amyloidic liver and spleen of mice [116±118] and in the brains of AD patients [119,120]. Thus, the question was raised whether AEF represents a single biological entity [6,121]. In any case, biochemical characterization of AEF extracted from different tissues remains to be important for understanding of mechanisms involved in amyloidogenesis. 3.3.2. Glycosaminoglycans, proteoglycans and other extracellular matrix components Glycosaminoglycans (GAGs), the linear polysaccharides composed of a characteristic disaccharide repeat (chondroitin sulfate, heparane sulfate, keratane sulfate, and others), and proteoglycans (PGs), i.e. proteins containing at least one GAG chain, are found to be associated with amyloid deposits in different types of amyloidosis (AA, AL, b-microglobulin, ATTR, amyloid b). SDS±PAGE and immunoblotting were applied to characterize PGs and their core proteins. Different molecular mass core proteins immunoreactive with antibodies against basement membrane

165

heparane sulfate PGs were identi®ed: 60 kDa protein in amyloidotic liver [122], 15 and 80 kDa ± in kidney, and 25, 30 and 65 kDa ± in lymph node [123]. On the other hand, loss of keratane sulfate PGs in AD was described recently; the de®ciency of this PG was supposed to re¯ect a speci®c functional defect of neurons in this disease [124]. Presence of other extracellular matrix components, such as laminin, collagen IV, ®bronectin, or vitronectin in amyloid deposits is described in several reports. However, these studies have mainly been performed using immunohistochemical techniques, and further biochemical data are needed to con®rm these ®ndings [7]. 3.3.3. Serum amyloid P component (SAP) SAP is a normal serum glycoprotein belonging to the family of pentraxin proteins. It was found to be associated with different forms of amyloid deposits, as was demonstrated by the immunohistochemical and electrophoretic techniques. In SDS±PAGE, amyloid P component was found to represent a protein of about 25 kDa, running as a single band under reducing conditions [125,126]. Although SAP was considered to be a homogeneous protein [126], several isoforms were tentatively identi®ed by IEF [127]. Con¯icting immunohistochemical data regarding the presence of amyloid P component within neuritic plaques have been reported previously [128,129]. Later, studies employing electrophoretic and immunoblot analyses con®rmed the presence of amyloid P component in brain parenchyma of patients with AD and other types of cerebral amyloidosis [130]. Thus, amyloid P component is thought to be a common element in all forms of amyloidosis. It is notable that amyloid P component is absent in the non-®brillar immunoglobulin light chain deposits found in light chain deposition disease [87,131]. The role of SAP in amyloid formation and deposition is not understood, but the binding of SAP to different ligands, including amyloid proteins and extracellular matrix components is supposed to be implicated in this process [132,133]. In this respect, the studies of SAP structure and its binding properties are especially important. Utilization of PAGE and IEF along with immunoelectrophoresis, ELISA, af®nity and ion-exchange chromatographies, reversed phase HPLC (RP-HPLC), and electron microscopy were found to be fruitful in these studies [126, 132±137].

166

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

4. PAGE in small-scale purification of amyloid proteins Open-column gel permeation chromatography is the most common method of isolation and puri®cation of different amyloid proteins [10,15,17±19,30± 33,35,48,49,138±140]. Size exclusion (SE) and RPHPLC are currently used for puri®cation of amyloid b proteins [14,116,34,43,44,141±146]; these techniques, however, are rarely applied for the isolation of other amyloid types, such as AA, AL, ATTR [21,40,147,160]. In most of these studies, PAGE was utilized as an analytical tool to check the purity and molecular mass of the isolated proteins. In many cases, however, preparative SDS±PAGE of a crude protein mixture can achieve a level of purity that would otherwise require multiple chromatographic procedures. This is true, for example, in the case of AL proteins, which could be comprised of a number of immunoglobulin light chain fragments of a very close molecular mass. Actually, their puri®cation by conventional gel permeation chromatography is a dif®cult task requiring several rechromatography steps. Our experience showed that application of HPLC was also not effective in separation of the multicomponent AL proteins; in contrast, the use of preparative SDS± PAGE was more ef®cient (Fig. 1). Although various designs of equipment are commercially available for large-scale puri®cation of proteins by PAGE, they are rarely used in the preparative work. First, the resolution obtained is lower as compared with the analytical gel electrophoresis. Second, due to the advances in methods of protein analysis, the amounts of proteins puri®ed on analytical scale gels are often suf®cient for their further examination. A continuous-elution micropreparative polyacrylamide gel column electrophoresis system, based on a simple modi®cation of the Mini-Protean II 2D unit (Bio-Rad), was developed [148] and applied for puri®cation of small amounts of amyloid b, gelsolin-derived amyloid, and the novel amyloid of the British type [149]. In other studies polyacrylamide slab gel electrophoresis was found to be effective for isolation of proteins AA, SAA, AL and AEF, by using elution by diffusion or electroelution techniques [20,23,46,114,147,150± 152]. However, in the cases where amyloid proteins of a very similar electrophoretic mobility have to be separated or when they have to be puri®ed from other

Fig. 1. HPLC and preparative SDS±PAGE of AL proteins. Amyloid proteins were extracted from the hepatic tissue of a patient with AL amyloidosis and subjected to SE-HPLC (A), RPHPLC (B) and preparative slab gel electrophoresis (C). (A) SEHPLC: TSK-gel G3000 SWXL (TosoHaas, Stuttgart, Germany) column (3007.8 mm i.d.); 0.5 M Tris±HCl buffer, pH 6.8, containing 0.1% SDS [21]. The arrow indicates the void volume, cutoff 30 kDa. (B) RP-HPLC: Vydac 214TP54 (Alltech, Deerfield, Ê , particle IL, USA) column (2504.5 mm i.d., pore diameter 300 A size 5 mm); linear gradient from 20% to 80% acetonitrile in 0.1% TFA over 30 min [20,21]. (C) SDS±PAGE: 17% polyacrylamide slab gels, 1.5 mm thick [13]. The gel slices containing the proteins of interest were cut out and subjected to the electroelution using 0.025 M Tris/0.192 M glycine buffer, pH 8.3, containing 0.1% SDS. (1) Crude amyloid extract, and (2)±(5) the electroeluted fractions. The multiple protein bands of AL proteins (from about 10 to <30 kDa) were revealed and separated by preparative slab gel electrophoresis. In contrast, these proteins were insufficiently resolved when using SE- and RP-HPLC.

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

proteins of the same molecular mass, preparative SDS±PAGE should be followed by other separation modes. Consecutive use of preparative slab gel electrophoresis and chromatographic techniques, such as ion exchange or RP-HPLC, could be especially promising for small-scale puri®cation, as it combines the resolution power of analytical gels with the high separation speed of the HPLC columns. In this methodological approach SDS has to be removed from the proteins after their elution from the SDS gels: presence of SDS interferes with RP-HPLC by decreasing the resolution and retarding the elution of proteins [153]. Since SDS could not be completely removed by dialysis, several SDS removal techniques were developed. However, not all of them are suitable for small-scale puri®cation of amyloid proteins. The techniques based on acetone or TCA precipitation are ef®cient only at protein concentrations above 100 mg/ml and at SDS concentration not higher than 0.05% [52]. Thus, it might be necessary to reduce the SDS level by dialysis (especially in the case when the proteins are concentrated by Speed Vac) which is less appropriate if working with small amounts of protein. Use of ion-exchange resins with elution buffers containing urea should be also followed by TCA precipitation or extensive dialysis [154]. Application of ion-exchange columns with aqueous elution buffers containing no chaotropic agents is unsuitable for puri®cation of amyloid proteins due to their insolubility in these media [155]. A gel permeation technique in an aqueous 50% acetonitrile solution containing 0.1% tri¯uoroacetic acid (TFA) was applied for SDS removal from amyloid proteins by using small Fractogel TSK HW-40(F) columns (Merck) [20,156,157]. The technique is based on the dissociation of SDS±protein complexes in this solvent and allows to recover proteins in a soluble state free from SDS. The solvent is volatile and can be easily removed by lyophilization, thus avoiding the problems arising with acetone or TCA precipitation. This SDS removal technique was applied for small-scale puri®cation of AA proteins: the amyloid ®brils were run on slab SDS±polyacrylamide gel, the resolved AA proteins were electroeluted, puri®ed from SDS and separated by RP-HPLC [20]. In contrast to conventional AA isolation techniques, it yielded electrophoretically pure AA proteins with only several milligrams of starting ®brillar material. In addition, it

167

was found that the electroeluted 8 kDa AA protein, homogeneous with respect to molecular mass, demonstrated multiple peaks on the reversed phase column. Further analysis of these components is needed to clarify whether they represent different AA species and re¯ect the heterogeneity of AA proteins revealed previously using IEF technique [158]. Consecutive use of SDS±PAGE and RP-HPLC was also effective for the small-scale separation of the major acute phase plasma reactants of mice, SAA1 and SAA2, using only milliliters of murine plasma [152]. The ef®cient removal of SDS used in this technology allowed to obtain the reproducible HPLC pro®les and made possible the ef®cient puri®cation of amyloid proteins, as it follows from their N- and C-terminal and mass spectral analyses [152]. 5. Use of PAGE for detection and determination of amyloid type in biopsy specimens Amyloidosis is diagnosed in tissue biopsies by the demonstration of green birefringence in sections stained with Congo red and examined by polarization microscopy. The heterogeneity of amyloid proteins related to different clinical forms of disease requires determination of the type of proteins forming the ®brillar deposits. Assessment of the amyloid type in biopsy specimens is important for consideration of therapy and evaluation of prognosis. For this purpose, the immunohistochemical examination of biopsy specimens is employed by using a panel of antibodies recognizing different types of amyloid. In cases where the immunohistochemical ®ndings are unexpected or questioned, more rigorous chemical or immunochemical con®rmation of amyloid type is needed. However, extraction of amyloid by the conventional techniques requires gram amounts of starting tissue material [9]. SDS±PAGE was applied for identi®cation of AA proteins in formalin-®xed paraf®n-embedded autopsy specimens obtained from patients with familial Mediterranian fever and reactive amyloidosis [159,160]. The samples were deparaf®nized, washed with saline, extracted with Laemmli sample buffer, analyzed by SDS±PAGE. The gels were stained with Coomassie blue in order to reveal the presence of 8 kDa protein bands corresponding to AA. This technique might be useful for the electrophoretic identi®cation of AA

168

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

proteins in small biopsy tissues [160], but it is not suitable for the extraction and detection of other amyloid proteins. In another study, the abdominal fat biopsy tissue specimens obtained from patients with AA and AL were unfatted, extracted with guanidine hydrochloride and subjected to gel permeation chromatography and RP-HPLC; the puri®ed proteins were characterized by N-terminal sequence analysis [161]. However, in this case the relatively larger biopsy specimens (1 cm3) were available. Our previous study showed that amyloid proteins could be extracted from tissues by using an aqueous 20% acetonitrile solution containing 0.1% TFA [22]. This solvent is volatile and can be easily removed by lyophilization without the need of dialysis. It made possible the effective extraction of amyloid proteins of different type (AA, AL, ATTR, Ab) from milligram amounts of tissue material [22,23,46,162,163]. The extracted material was analyzed by immunoblotting blotting [22,23,163] to determine the approximate molecular mass of amyloid proteins and their chemical type. This technique was applied for analysis of diagnostic biopsy specimens [24] and shown to be feasible in immunochemical typing of amyloid proteins in small biopsy tissues (Fig. 2). 6. Conclusions During the past decade numerous efforts were directed towards the development and improvement of chromatographic protein separation techniques, especially HPLC and capillary electrophoresis. Despite the considerable progress made in this ®eld, the polyacrylamide slab gel electrophoresis remains to be one of the most popular methods of protein separation. Use of PAGE in conjunction with highly sensitive immunodetection systems, microsequencing analysis, and mass spectrometry, has proved to be a powerful analytical tool for examination of many proteins, including the amyloids. PAGE is perhaps the surest method of protein separation being simple, inexpensive and available in most laboratories. However, most proteins, including the amyloid proteins, cannot be puri®ed in a single step. In this respect, the combined use of the ``old'' slab gel electrophoresis and modern separation methods seems to be especially promising. Further development of such combined techniques

Fig. 2. Determination of amyloid type in biopsy tissues by immunoblotting blotting. The non-fixed biopsy specimens obtained from four patients with amyloidosis (samples 1±4) were extracted with 20% acetonitrile containing 0.1% TFA [22]. The extracted material was run on 17% polyacrylamide slab SDS-gels [13] and analyzed by immunoblotting, using antibodies to transthyretin, protein AA and immunoglobulin k and l chains. The immunoreactive proteins were visualized via enhanced chemiluminescence (ECL) detection system (Amersham, Little Chalfont, UK). In each case protein bands of the molecular mass <30 kDa reacted only with one of the four antibodies tested, thus demonstrating the presence of ATTR, AA, AL-lk and AL-k deposits in samples 1, 2, 3, and 4, respectively.

and their application for the small-scale puri®cation of amyloid proteins will contribute to elucidation of amyloid structure and of the mechanisms involved in the formation of these proteins. References [1] [2] [3] [4]

S. Raymond, V.J. Wang, Anal. Biochem. 1 (1960) 39. A.S. Cohen, E. Calkins, Nature 183 (1959) 1202. G.G. Glenner, N. Engl. J. Med. 302 (1980) 1283. J. Ghiso, R. Vidal, G. Gallo, B. Frangione, Rev. Bras. Rheumatol. 35 (1995) 93. [5] G.G. Glenner, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 2. [6] R. Kisilevski, E. Gruys, T. Shirahama, Amyloid: Int. J. Exp. Clin. Invest. 2 (1995) 128.

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172 [7] J.H. Magnus, T. Stenstad, Amyloid: Int. J. Exp. Clin. Invest. 4 (1997) 121. [8] R.A. Kyle, J. Intern. Med. 232 (1992) 507. [9] M. Pras, M. Schubert, D. Zucker-Franklin, A. Rimon, E.C. Franklin, J. Clin. Invest. 47 (1968) 924. [10] G.C. Glenner, W. Terry, M. Harada, C. Isersky, D. Page, Science 172 (1971) 1150. [11] G.C. Glenner, D. Page, C. Isersky, M. Harada, P. Cuatrecasas, E.D. Elanes, R.A. de Lellis, H.A. Bladen, H.R. Keiser, J. Histochem. Cytochem. 19 (1971) 16. [12] M. Pras, T. Reshef, Biochim. Biophys. Acta 271 (1972) 193. [13] U.K. Laemmli, Nature 227 (1970) 680. [14] D.J. Selkoe, C.R. Abraham, M.B. Podlisny, L.K. Duffy, J. Neurochem. 46 (1986) 1820. [15] F. Prelli, E. Castano, G.G. Glenner, B. Frangione, J. Neurochem. 51 (1988) 648. [16] C.L. Joachim, L.K. Duffy, J.H. Morrisand, D.J. Selkoe, Brain Res. 474 (1988) 100. [17] F. Prelli, M. Pras, S. Shtrasburg, B. Frangione, Scand. J. Immunol. 33 (1991) 783. [18] C.P.J. Maury, K. Alli, M. Bauman, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark, Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 405. [19] K. Sletten, J.B. Natvig, P. Westermark, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark, Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 477. [20] B. Kaplan, M. Pras, M. Ravid, J. Chromatogr. 573 (1992) 17. [21] B. Kaplan, M. Pras, J. Liq. Chromatogr. 15 (1992) 2467. [22] B. Kaplan, G. German, M. Pras, J. Liq. Chromatogr. 16 (1993) 2249. [23] S. Yakar, B. Kaplan, A. Livneh, B. Martin, K. Miura, Z. AliKhan, S. Shtrasburg, M. Pras, Scand. J. Immunol. 40 (1994) 653. [24] B. Kaplan, S. Yakar, A. Kumar, M. Pras, G. Gallo, Amyloid: Int. J. Exp. Clin. Invest. 4 (1997) 80. [25] L. Ornstein, Ann. NY Acad. Sci. USA 121 (1964) 321. [26] B.J. Davis, Ann. NY Acad. Sci. USA 121 (1964) 40. [27] R.T. Swank, K.D. Munkres, Anal. Biochem. 39 (1971) 462. [28] H. Schagger, G. von Jagow, Anal. Biochem. 166 (1987) 166. [29] J. Wiltfang, N. Arold, V. Neuhoff, Electrophoresis 12 (1991) 352. [30] G.G. Glenner, C. Wang, Biochem. Biophys. Res. Commun. 120 (1984) 885. [31] K. Higuchi, A. Matsumara, S. Takeshita, T. Yonezu, A. Honma, K. Higuchi, M. Hosokawa, T. Takeda, in: G.G. Glenner, E.F. Osserman, E.P. Benditt, E. Calkins, A.S. Cohen, D. Zucker-Franklin (Eds.), Amyloidosis, Plenum Press, New York, 1986, p. 669. [32] B. Skogen, K. Sletten, T. Lea, J.B. Natvig, in: G.G. Glenner, E.F. Osserman, E.P. Benditt, E. Calkins, A.S. Cohen, D. Zucker-Franklin (Eds.), Amyloidosis, Plenum Press, New York, 1986, p. 11. [33] A. Husebekk, B. Skogen, G. Husby, G. Marhaug, in: G.G. Glenner, E.F. Osserman, E.P. Benditt, E. Calkins, A.S.

[34] [35]

[36] [37] [38] [39]

[40] [41] [42]

[43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53]

169

Cohen, D. Zucker-Franklin (Eds.), Amyloidosis, Plenum Press, New York, 1986, p. 245. W.M. Pardridge, H.V. Vinters, J. Yang, J. Eisenberg, T.B. Choi, W.W. Tourtellotte, V. Huebner, J.E. Shively, J. Neurochem. 49 (1987) 1394. R.P. Linke, J. Floege, F. Lottspeich, R. Deutzman, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 369. R. Kisilevski, S. Narindrasorasak, C. Tape, R. Tan, L. Boudreau, Amyloid: Int. J. Exp. Clin. Invest. 1 (1994) 174. H.W. Klafki, J. Wiltfang, M. Staufenbiel, Anal. Biochem. 237 (1996) 24. S.J. Frucht, E.H. Koo, J. Neuropath. Exp. Neurol. 52 (1993) 640. Y. Harigaya, M. Shoji, T. Kawarabayashi, M. Kanai, T. Nakamura, T. Iizuka, Y. Igeta, T.C. Saido, N. Sahara, H. Mori, S. Hirai, Biochem. Biophys. Res. Commun. 211 (1995) 1015. E.M. Castano, F. Prelli, M. Pras, B. Frangione, J. Biol. Chem. 270 (1995) 17610. K. Yanagisawa, A. Odaka, N. Suzuki, Y. Ihara, Nature Med. 1 (1995) 1062. J.K. Teller, C. Russo, L.M. DeBusk, G. Angelini, D. Zaccheo, F. Dagna-Bricarelli, P. Scartezzini, S. Bertolini, D.M.A. Mann, M. Tabaton, P. Gambetti, Nature Med. 2 (1996) 93. M. Lalowski, A. Golabek, C.A. Lemere, D.J. Sekoe, H.M. Wisniewski, R.C. Beavis, B. Frangione, T. Wisniewski, J. Biol. Chem. 271 (1996) 33623. T. Wisniewski, M. Lalowski, M. Bobik, M. Russel, J. Strosznajder, B. Frangione, Biochem. J. 313 (1996) 575. J. Towbin, T. Stachelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 4350. S. Yakar, B. Kaplan, G. German, A. Livneh, K. Miura, S. Shtrasburg, M. Pras, Amyloid: Int. J. Exp. Clin. Invest. 2 (1995) 167. D. Brancaccio, G.M. Ghiggeri, P. Braidotti, A. Garberi, M. Gallieni, V. Belloti, U. Zoni, R. Gusmano, G. Coggi, J. Am. Soc. Nephrol. 6 (1995) 1262. J.M. Campistol, D. Bernard, G. Papastoitsis, M. Sole, J. Kasirsky, M. Skinner, Kidney Int. 50 (1996) 1262. W. Li, S.L. Chan, S. Chronopoulos, A. Bell, Z. Ali-Khan, Exp. Parasitol. 83 (1996) 1. P. Matsudaira, J. Biol. Chem. 261 (1987) 10035. Q. Charbonneau, in: P. Matsudaira (Ed.), A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, San Diego, 1993, p. 17. K.L. Stone, K.R. Williams, in: P. Matsudaira (Ed.), A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, San Diego, 1993, p. 45. O.P. Veiby, K. Sletten, G. Husby, K. Nordstoga, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 551.

170

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

[54] E. Rodilla Sala, H.D. Kratzin, A.I. Pick, N. Hilcshman, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 161. [55] H.D. Kratzin, A.I. Pick, N. Hilcshman, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 165. [56] G.S. Foss, R. Nilsen, G.G. Cornwell III, G. Husby, K. Sletten, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 235. [57] J. Nyquist, H.M. Ramstad, K. Sletten, G. Husby, P. Westermark, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 247. [58] A.M. Berg, R.F. Troxler, G. Grillone, J. Kaszniica, K. Kane, A.S. Cohen, M. Skinner, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 256. [59] A.F. Ronning, K. Sletten, R. Lopez, T. Skarra, K. Nordstoga, G. Husby, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 238. [60] L.F. Hermansen, T. Bergman, H. Jornvall, G. Husby, I. Ranlov, L. Sletten, Eur. J. Biochem. 22 (1995) 772. [61] G. Marhaug, K. Sletten, G. Husby, Clin. Exp. Immunol. 50 (1980) 382. [62] G. Margauh, G. Husby, Clin. Exp. Immunol. 45 (1981) 97. [63] J.S. Hoffman, E.P. Benditt, J. Biol. Chem. 257 (1982) 10510. [64] J.S. Hoffman, L.H. Ericsson, N. Ericsen, K.A. Walsh, E.P. Bendit, J. Exp. Med. 159 (1984) 641. [65] G.A. Coetzee, A.F. Strachan, D.R. van der Westhuyzen, H.C. Hoppe, M.S. Jeenah, F.C. de Beer, J. Biol. Chem. 261 (1986) 9644. [66] G. Hoche, H. Kaffarnik, J. Chromatogr. 526 (1990) 203. [67] L. Bini, B. Magi, C. Cellesi, A. Rossolini, V. Pallini, Electrophoresis 13 (1992) 743. [68] E. Malle, H. Heb, G. Munscher, G. Knipping, A. Steinmetz, Electrophoresis 13 (1992) 422. [69] C.F. Bruun, K. Sletten, G. Husby, G. Marhaug, Electrophoresis 14 (1993) 1372. [70] J.G. Rynes, K.P.W.J. MacAdam, N.F. Totty, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 84. [71] B.P.C. Hazenbery, P.C. Limburg, J. Bijzet, M.H. van Rijswijk, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 90.

[72] G. Marhaug, V. Syversen, M. Rygy, C.F. Bruun, G. Husby, S.B. Dowton, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 99. [73] A. Steinmetz, C. Pfeiffer, S. Schwarz, S. Brand, C. Schmitz, T. Muller, G. Minscher, E. Malle, R. Hackler, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 131. [74] S.P. Alsemgeest, A. Horadagoda, C.K. Hulskamp-Koch, P.C. Tooten, D.H. Kim, T.A. Niewold, E. Gruys, Scand. J. Immunol. 41 (1995) 407. [75] A. Ducret, C.F. Bruun, E.J. Bures, G. Marhaug, G. Husby, R. Aebersold, Electrophoresis 17 (1996) 866. [76] C.F. Bruun, K. Sletten, A. Mehlum, G. Marhaug, J. Chromatogr. 685 (1996) 360. [77] T. Yamada, T. Miida, Y. Itoh, T. Kawai, M.D. Benson, Clin. Chim. Acta 251 (1996) 105. [78] S. Yakar, A. Livneh, B. Kaplan, M. Pras, Seminars in Arthritis and Rheumatism 24 (1995) 255. [79] M. Shiroo, E. Kawahara, I. Nakanishi, S. Migita, Scand. J. Immunol. 26 (1987) 709. [80] K. Miura, S.T. Yu, A.S. Cohen, T. Shirahama, J. Immunol. 144 (1990) 610. [81] D.J. Faulkes, J.C. Betts, P. Woo, Amyloid: Int. J. Exp. Clin. Invest. 1 (1994) 255. [82] G. Lavie, D. Zucker-Franklin, E.C. Franklin, J. Exp. Med. 148 (1978) 1020. [83] D. Zucker-Franklin, A. Fuks, in: G.G. Glenner, E.F. Osserman, E.P. Benditt, E. Calkins, A.S. Cohen, D. Zucker-Franklin (Eds.), Amyloidosis, Plenum Press, New York (1986) p. 225. [84] A. Husebekk, B. Skoggen, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 107. [85] T. Yamada, B. Kluve-Beckerman, J.J. Liepnieks, M.D. Benson, Scand. J. Immunol. 41 (1995) 570. [86] E.A. Myath, F.A. Westholm, D.T. Weiss, A. Solomon, M. Schiffer, F.J. Stevens, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 3034. [87] G. Gallo, F. Goni, F. Boctor, R. Vidal, A. Kumar, F.J. Stevens, B. Frangione, J. Ghiso, Am. J. Pathol. 148 (1996) 1397. [88] A. Solomon, D.T. Weiss, Amyloid: Int. J. Exp. Clin. Invest. 2 (1995) 269. [89] D.J. Selkoe, M.B. Podlisny, C.L. Joachim, E.A. Vickers, G. Lee, L.C. Fritz, T. Oltersdorf, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 7341. [90] W.E. Van Nostrand, A.J.M. Rozemuller, R. Chung, C.W. Cotman, S.M. Saporito-Irwin, Amyloid: Int. J. Exp. Clin. Invest. 1 (1994) 1. [91] B.A. Rowe, R.S. Siegel, M.F. Murphy, T.S. Vedvick, S.L. Wagner, Amyloid: Int. J. Exp. Clin. Invest. 3 (1996) 100. [92] F.S. Esch, P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Culwell, T. Oltersdorf, D. McClure, P.J. Ward, Science 248 (1990) 1122.

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172 [93] S.S. Sisodia, E.H. Koo, K. Beyreuther, A. Unterbeck, D.L. Price, Science 248 (1990) 492. [94] A. Tamaoka, R.N. Kalaria, I. Lieberburg, D.S. Selkoe, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 1345. [95] T. Oltersdorf, P.J. Ward, T. Heriksson, E.C. Beattie, R. Neve, I. Lieberburg, L.C. Fritz, J. Biol. Chem. 265 (1992) 4492. [96] S. Estus, T.E. Golde, T. Kunishita, D. Blades, D. Lowery, M. Eisen, M. Usiak, X. Qu, T. Tabira, B.D. Greenberg, S.G. Younkin, Science 255 (1992) 726. [97] T.T. Cheung, J. Ghiso, M. Shoji, X.-D. Cai, T. Golde, S. Gandy, B. Frangione, S. Younkin, Amyloid: Int. J. Exp. Clin. Invest. 1 (1994) 30. [98] C. Haass, E.H. Koo, A. Mellon, A.Y. Huaang, V.J. Selkoe, Nature 357 (1992) 500. [99] K.J. Conn, G. Papastoitsis, B. Meckelein, C.R. Abraham, Amyloid: Int. J. Exp. Clin. Invest. 1 (1994) 232. [100] J.A. Ripellino, D. Vassilacopoulou, N.K. Robakis, J. Neurosci. Res. 39 (1994) 211. [101] R. Bhasin, L. Gregori, I. Morozov, D. Goldgaber, Amyloid: Int. J. Exp. Clin. Invest. 1 (1994) 221. [102] C. Haass, M.D. Schlossmacher, A.Y. Hung, C. Vogo-Pelfrey, A. Mellon, B.L. Ostaszewski, D. Lieberburg, E.H. Koo, D. Schenk, D.B. Teplow, D.J. Selkoe, Nature 359 (1992) 322. [103] P. Seubert, C. Vigo-Pelfrey, F. Esch, M. Lee, H. Dovey, D. Davis, S. Sinha, M. Schlossmaher, J. Whaley, C. Swindlehurst, R. McCormack, R. Wofert, D. Selkal, I. Lieberburg, D. Schenk, Nature 359 (1992) 325. [104] M. Schoji, T.E. Golde, J. Ghiso, T.T. Cheung, S. Estus, L.M. Shaffer, X.-D. Cai, D.M. McKay, R. Tintner, B. Frangione, S.D. Younkin, Science 258 (1992) 126. [105] A.R. Koudinov, N.V. Koudinova, A. Kumar, R.C. Beavis, J. Ghiso, Biochem. Biophys. Res. Commun. 223 (1996) 592. [106] A.R. Koudinov, N.V. Koudinova, Cell Biol. Int. 21 (1997) 265. [107] A.L. Biere, B. Ostaszewski, E.R. Stimson, B.T. Hyman, J.E. Maggio, D. Selkoe, J. Biol. Chem. 271 (1996) 32916. [108] J. Ghiso, M. Calero, E. Matsubara, S. Governale, J. Chuba, R. Beavis, T. Wisniewski, B. Frangione, FEBS Lett. 408 (1997) 105. [109] M.A. Axelrad, R. Kisilevski, J. Willmer, S.J. Chen, M. Skinner, Lab. Invest. 47 (1982) 139. [110] M.L. Baltz, D. Caspi, C.R.K. Hind, A. Feinstein, M.P. Pepys, in: G.G. Glenner, E.F. Osserman, E.P. Benditt, E. Calkins, A.S. Cohen, D. Zucker-Franklin (Eds.), Amyloidosis, Plenum Press, New York, 1986, p. 115. [111] T.A. Nieweld, P.R. Hol, A.C.J. van Andel, E.T.G. Lutz, E. Gruys, Lab. Invest. 56 (1987) 544. [112] T. Shirahama, C.R. Abraham, S.T. Ju, K. Miura, A.S. Cohen, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 288. [113] T. Yokota, T. Ishihara, M. Takahashi, Y. Yamashita, T. Gondo, S. Kawamura, Y. Hoshii, M. Koga, T. Iwata, F. Uchino, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and

[114]

[115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] [135] [136]

[137]

171

Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 307. F. Gervais, C. Morissete, B. Frangione, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 168. S. Shtrasburg, A. Livneh, R. Gal, M. Pras, Clin. Exp. Rheumatol. 14 (1996) 37. S. Chronopoulos, K. Alizadeh-Khiavi, J. Normand, Z. AliKhan, J. Pathol. 163 (1991) 199. S. Chronopoulos, P. Lembo, K. Alizadeh-Khiavi, Z. AliKhan, J. Pathol. 167 (1992) 249. K. Alizadeh-Khiavi, J. Normand, S. Chronopoulos, A. Ali, Z. Ali-Khan, Virchows Arch. A. Pathol. Anat. Histopathol. 420 (1992) 139. K. Alizadeh-Khiavi, J. Normand, S. Chronopoulos, Z. AliKhan, Acta Neuropathol. (Berlin) 81 (1991) 280. Z. Ali-Khan, J. Normand, K. Alizadeh-Khiavi, Y. Robitaille, S. Chronopoulos, Neurosci. Lett. 139 (1992) 24. K. Ganoviak, P. Gultman, U. Engstrom, A. Gustavson, P. Westermark, Biochem. Biophys. Res. Commun. 199 (1994) 306. J.H. Magnus, T. Stenstad, G. Husby, S.O. Kolset, Biochem. J. 288 (1992) 225. T. Stenstad, J.H. Magnus, G. Husby, S.O. Kolset, Scand. J. Immunol. 37 (1993) 227. A.D. Snow, D. Nochlin, R. Sekiguichi, S.S. Carlson, Exp. Neurol. 138 (1996) 305. F.C. de Beer, M.B. Pepys, J. Immunol. 50 (1982) 17. P.N. Hawkins, G.A. Tennent, P. Woo, M.P. Pepys, Clin. Exp. Immunol. 84 (1991) 308. I.J. Sorensen, O. Andersen, E.H. Nielsenand, S.E. Svehag, Int. Arch. Allergy Immunol. 106 (1995) 25. P. Westermark, T. Shirama, M. Skinner, A.E. Cameron, A.S. Cohen, Lab. Invest. 46 (1982) 482. I.F. Rowe, O. Jensson, P.D. Lewis, J. Candy, G.A. Tennent, M.B. Pepys, Neuropathol. Appl. Neurobiol. 10 (1984) 53. F. Coria, E. Castano, F. Prelli, M. Larrondo-Lillo, S. van Duinen, M.L. Shelanski, B. Frangione, Lab. Invest. 58 (1988) 454. M.M. Picken, B. Frangione, B. Barlogie, M. Luna, G. Gallo, Am. J. Pathol. 134 (1989) 749. T. Stenstad, J.H. Magnus, K. Syse, G. Husby, Clin. Exp. Immunol. 94 (1993) 189. B. Danielsen, I.J. Sorensen, M. Nybo, E.H. Nielsen, B. Kaplan, S.E. Svehag, Biochim. Biophys. Acta 1339 (1997) 73. E.H. Nielsen, I.J. Sorensen, K. Vilsgaard, O. Andersen, S.E. Svehag, APMIS 102 (1994) 420. I.H. Sorensen, O. Andersen, E.H. Nielsen, S.E. Svehag, Scand. J. Immunol. 41 (1995) 263. J.G. Raines, G.B. O'Sullivan, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 156. N.H. Heergaard, P.M. Heergaard, P. Roepstorff, F.A. Robey, Eur. J. Biochem. 238 (1996) 850.

172

B. Kaplan / Analytica Chimica Acta 372 (1998) 161±172

[138] M. Levin, E.C. Franklin, B. Frangione, M. Pras, J. Clin. Invest. 51 (1972) 2773. [139] M. Pras, F. Prelli, E.C. Franklin, B. Frangione, Proc. Natl. Acad. Sci. U.S.A. 80 (1983) 539. [140] D. Cohen, M. Pras, E.C. Franklin, B. Frangione, Am. J. Med. 74 (1983) 513. [141] C.L. Masters, G. Simms, N. Weinman, G. Multhaup, B.L. McDonald, K. Beyreuther, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 4245. [142] H. Mori, K. Takio, M. Ogawara, D. Selkoe, J. Biol. Chem. 267 (1992) 17082. [143] A.E. Roher, J.D. Lowenson, S. Clarke, C. Wolkow, R. Wang, R.J. Cotter, I.M. Reardon, H.A. Zucher-Neely, R.L. Heinrikson, M.J. Ball, B. Greenberg, J. Biol. Chem. 268 (1993) 3072. [144] D.L. Miller, I.A. Papayannopoulos, J. Styles, S.A. Bobin, Y.Y. Lin, K. Biemann, K. Iqbal, Arch. Biochem. Biophys. 301 (1993) 41. [145] A.E. Roher, K.C. Palmer, E.C. Yurewicz, M.J. Ball, B. Greenberg, J. Neurochem. 61 (1993) 1916. [146] E. Gowing, A.E. Roher, A.S. Woods, R.J. Cotter, M. Chaney, S.P. Little, M.J. Ball, J. Biol. Chem. 269 (1994) 10987. [147] B. Kaplan, M. Pras, Clin. Chim. Acta 163 (1987) 199. [148] M. Baumann, M. Lauraeus, Anal. Biochem. 214 (1993) 142. [149] M. Baumann, A. Golabek, M. Lalowski, T. Wisniewski, Anal. Biochem. 236 (1996) 191. [150] G.A. Mathiesen, P.V. Syversen, K. Sletten, G. Husby, in: R. Kisilevski, M.D. Benson, B. Frangione, J. Gauldie, T.J. Muckle, I.D. Young (Eds.), Amyloid and Amyloidosis 1993, Parthenon Publishing, New York, 1993, p. 93.

[151] W.A. Gonnerman, E.S. Cathcart, J.D. Sipe, K.C. Hayes, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 547. [152] B. Kaplan, S. Yakar, Y. Balta, M. Pras, B. Martin, J. Chromatogr., 704 (1997) 69. [153] K.L. Stone, M.B. LoPresti, J.M. Crawford, R. DeAngelis, K.R. Williams, in: C. Mant, R.S. Hodges (Eds.), HPLC of Peptides and Proteins: Separation, Analysis and Conformation, CRC Press, Boca Rato, FL, 1991, p. 669. [154] K. Weber, D.J. Kuter, J. Biol. Chem. 246 (1971) 4505. [155] O.H. Kapp, S.N. Vinogradov, Anal. Biochem. 91 (1978) 230. [156] B. Kaplan, M. Pras, J. Chromatogr. 423 (1987) 376. [157] B. Kaplan, M. Pras, Biomed. Chromatogr. 5 (1992) 86. [158] P. Westermark, Biochim. Biophys. Acta 701 (1982) 19. [159] S. Shtrasburg, M. Pras, P. Langevitch, R. Gal, Am. J. Pathol. 106 (1982) 141. [160] R. Gal, S. Shtrasburg, M. Luria, B. Lifschitz Mercer, S. Viskin, S. Yakar, M. Pras, Amyloid: Int. J. Exp. Clin. Invest. 2 (1995) 119. [161] A.H. Forsberg, K. Sletten, L. Benson, P. Westermark, in: J.B. Natvig, O. Forre, G. Husby, A. Husebekk, B. Skogen, K. Sletten, P. Westermark (Eds.), Amyloid and Amyloidosis 1990, Kluwer Academic Publishers, Dordrecht, 1990, p. 797. [162] B. Kaplan, G. German, M. Ravid, M. Pras, Clin. Chim. Acta 224 (1994) 171. [163] B. Kaplan, B. Martin, S. Yakar, M. Pras, T. Wisniewski, J. Ghiso, B. Frangione, G. Gallo, in: A. Fisher, M. Yoshida, I. Hanin, Progress in Alzheimer's and Parkinson's Diseases, Plenum Press, New York, (1998) p. 823.