The Characterization of Soluble Amyloid Prepared in Water

M. PiRAS, M. SCHUBERT, D. ZUc7Mi-F&ANKLIN, A. RIMON, and E. C. FRANxIuNFrom the Department of Medicine, Rheumatic Diseases Study Group, NewYork University School of Medicine, NewYork 10016

AB ST RA C T Amyloid was extracted from thespleen of a patient with primary amyloidosis byhomogenizing it at high speed with water afterpreliminary treatments, first to remove proteinssoluble in saline, and then to remove salts. Theextracts containing amyloid appeared to be clearat concentrations up to 6 mg/ml of protein. Thematerial gave little sediment on being centrifugedup to 20,000 g for 1 hr, but the protein was sedi-mented at 100,000 g in 1 hr. The amyloid couldbe precipitated from the extracts by addition ofNaCl to 0.0075 mole/liter or of CaCl2 to 0.0025mole/liter. The protein-bound Congo red formeda red precipitate and this property was used toestimate recovery and purity of amyloid duringextraction.

On electronmicroscopy the isolated amyloidproved to be morphologically pure. It existedeither as single filaments measuring 60-80 A indiameter or as large aggregates of these filaments.

Freshly isolated amyloid in water sedimented asa single homogeneous peak with an s020, ofabout 45-50S. On standing, the solution becamecloudy and more rapidly sedimenting componentsappeared. On electrophoresis the material migratedas a homogeneous peak towards the anode. Theprotein had an amino acid composition differentfrom that of all known serum proteins. It was richin acidic amino acids and had little cysteine andmethionine and no hydroxyproline. The total con-tent of carbohydrate was less than 2%o.

Dr. Pras' permanent address is Tel Hashomer Hos-pital, Tel Aviv, Israel.

Dr. Rimon's permanent address is Tel-Aviv University,Tel Aviv, Israel.

Received for publication 29 July 1967 and in revisedform 25 November 1967.

INTRODUCTION

Investigations on amyloid have been hampered bythe fact that no solvent has been found in whichthis material can be quantitatively extracted andobtained in a reasonably pure and stable state.

Amyloid fibrils are known to be highly insolublein neutral aqueous media of ionic strengths com-monly used (1, 2). In recent years amyloid hasbeen dissolved in dilute alkali (3) and in concen-trated urea solutions (4, 5). However, the yieldsby these procedures are relatively poor, and theextracted amyloid is only partially brought intosolution. During the isolation procedures some ofthe material was degraded to particles of lowermolecular weight and some of it could not be fullyreconstituted to its original fibrillar form.

Reports on the composition of amyloid by differ-ent investigators have shown wide variation in thecontents of carbohydrate components (6-8) andamino acids (6, 9, 10). This variability can proba-bly be attributed to the difficulties of purifying asubstance that cannot be dissolved without decom-position. Cohen and Calkins (11) demonstratedthe fibrillar character of several types of amyloid.Since these fibrils have all the tinctorial character-istics of amyloid, it seems likely that they areamyloid, while the interfibrillar material is animpurity. The demonstration of fibrils as the mainconstituent in amyloid has resulted in a numberof electronmicroscope studies (5, 12-18). In gen-eral the amyloid fibrils appear to be 70-100 A wideand of lengths that range from 400 to 16,000 A.A detailed discussion of the structure of thesefibrils has recently been published by Shirahamaand Cohen (18).

In an effort to obtain a solution of amyloid in astable form, a method has been developed by which

924 The Journal of Clinical Investigation Volume 47 1968

a major fraction of amyloid was extracted fromthe spleen of a patient with primary amyloidosis indistilled water. In the course of this procedure, itwas possible to obtain a clear solution, containingamyloid in distilled water that lent itself readilyto physicochenmical, chemical, and ultrastructuralcharacterization.

METHODSTissue. The tissue used for extraction was a specimen

of human spleen obtained postmortem from a patient withprimary amyloidosis. The spleen was stored at - 20'Cuntil thawed and subjected to the procedures outlinedbelow. Histological examination showed classical amy-loidosis; the hematoxylin-eosin stain showed an ac-cumulation of extracellular eosinophilic material. Thematerial stained red with Congo red and showed greenbirefringence with a polarizing microscope (19). Crys-tal violet stained it metrachromatically. Electronmicro-scope examination of the "top layer," prepared by themethod of Cohen and Calkins (20), using negativestaining technique, demonstrated fine fibrils of 100 A + 20in diameter as well as collagen bundles.

Separation and purification. All procedures were car-ried out at 4°C. The Virtis-45 high speed homogenizerwas used for all homogenizations. A Spinco model L cen-trifuge with a No. 30 rotor was used for all centrifuga-tion.

The initial steps do not differ basically from the originalmethod of Cohen and Calkins (20). 20 g of the amyloid-laden spleen and 400 ml of 0.15 M NaCl were homoge-nized in a Virtis-45 high speed homogenizer for 5 min,and the mixture was centrifuged at 10,000 rpm for 30 min.The supernatant solution was discarded. The sedimentwas homogenized as before with 400 ml of 0.15 M NaCl,and the mixture centrifuged as before. These operationswere repeated six or seven times. The last supernatantsolution had an absorbance of less than 0.075 at 280 my.

At this point most soluble proteins and other solublematerials of the spleen had been removed. Salt was nowremoved from the residue by homogenizing the residuewith 300 ml of distilled water and centrifuging the sus-pension at 30,000 rpm for 1 hr. The supernatant (super-natant I) contained only small quantities of protein (0.1mg/ml) and was discarded. The residue was homoge-nized with 200 ml of distilled water and centrifuged at30,000 rpm for an hour. This yielded a supernatant (su-pernatant II) which contained a large quantity of pro-tein, about 2 mg/ml. The residue was homogenized with200 ml of distilled water, and after centrifugation at 30,-000 rpm for 1 hr, the supernatant (supernatant III)again contained about 2 mg of protein per ml. The fourthand last homogenization was done with 70 ml of distilledwater and the mixture was centrifuged at 10,000 rpm for3 hr. The supernatant (Supernatant IV) had a proteincontent of about 4 mg/ml. Supernatants II, III, and IVwere kept in the cold at 40C, and all showed similar prop-erties. The total yield of the soluble amyloid from 3.2 g

of dry tissue was 1100 mg. Thus, about 34%7 of the dryweight of the tissue was amyloid. Normal spleen, ex-tracted by the same procedure, did not contain measur-able amounts of protein in supernatants II, III, and IV.

Quantitative Congo red test. Congo red is bound byamyloid to form a stable product. Although this stain-ing property and its characteristic birefringence in thepolarization microscope have been used as a valuablequalitative identification method, the interaction betweenCongo red and amyloid has not been studied quantita-tively. Therefore, to compare quantitatively the relativepurities of different amyloid preparations, we developedthe procedure described below. The test depends on thefact that suspensions of amyloid in saline bind a fixedamount of Congo red and that the stained insoluble amy-loid is easily removed by centrifugation at low speeds.

The stock Congo red solution contains 1 g in 100 mlof distilled water. For use, a working solution is madeby diluting 1 ml of the stock solution with 0.15 M NaClto 100 ml. Generally, 2 ml of an unknown solution, con-taining no more than 2 mg of amyloid, is added to 10 mlof the Congo red working solution. As a control, 10 mlof Congo red is diluted with 2 ml of water. After incu-bating an hour, the mixture of dye and sample is cen-trifuged in a table top centrifuge (International ClinicalCentrifuge, International Equipment Co., NeedhamHeights, Mass.), and the absorbance of the supernatantsolution is measured at 490 m/A. The amount of Congored bound to and removed by the amyloid in the sampleis calculated by comparing the amount of Congo red leftin solution in the sample and the control. Fig. 1 showshow the absorbance of the supernatant solution decreasesafter addition of increasing amounts of amyloid. Theamyloid used to prepare the curve in Fig. 1 was a solu-tion in water similar to that called supernatant II. Incontrast, solutions of serum proteins or suspensions ofcollagen removed little if any Congo red from solution.The test has, therefore, been used as an assay procedureto estimate the amount of amyloid in the solution ofamyloid in water (supernatants II, III, and IV), and toestimate the total amount recovered. Because of the pres-ence of hemoglobin which interferes with the colorimetricreading and elastin and collagen which bind Congo red

Z- 2.4

° 2.0

Q 1.6

0 1.2za 0.80m 0.4

Or4

1.0 2.0 3.0 4.0 5.0mg AMYLOID ADDED

FIGURE 1 The linear relation between the amount ofCongo red left in solution, measured by the absorbanceat 490 m/A, and the amount of amyloid added.

Properties of Soluble Amyloid 925

in tissue and in the insoluble residues, the quantitativeCongo red binding could not be used to provide a pre-cise estimate of the amount of amyloid in the tissue orthe fraction of the total which was recovered. However,it appears likely that the bulk of the amyloid was ex-tracted from the spleen, since the final product con-stituted about 34% of the dry weight of the spleen.

Physical aned chemical studies. The sedimentationproperties of the supernatants of different preparationswere studied in the Spinco model E analytical ultra-centrifuge at 20'C and at speeds ranging from 20,400 to52,640 rpm. Because of the solubility properties of thematerial all studies were done in distilled water. Pro-tein concentrations of the samples ranged from 1 to 4mg/ml. Sedimentataion coefficients were calculated asdescribed by Trautman (21), and the sedimentation co-efficient at infinite dilution was determined by extrapola-tion from four values obtained at different concentrations.

Electrophoresis was carried out in a Perkin-Elmerapparatus, model 38A (22). Optical absorption spectrawere recorded with a Beckman model DU spectrophotom-eter. The nitrogen content was determined by micro-Kjeldahl procedure. Amino acid content was determinedby the automatic amino acid analyzer (Spinco model120) (23). Hydroxylproline was determined by theWoessner method (24). Neutral sugars were determinedby the anthrone method (25), hexosamines by the Elsonand Morgan method (26), uronic acid by Dische'scarbazol method (27), and sialic acid by Warren's method(28).

Electron microscopy. To characterize the structure ofthe isolated amyloid we subjected two preparations toelectron microscopy: (a) amyloid suspended in distilledwater referred to as soluble amyloid, and (b) amyloidsuspended in 0.15 M saline. In saline, purified amyloidformed a precipitate that was suitable for embedding ac-cording to standard methods used in electron microscopyas well as for direct examination with the aid of the nega-tive staining technique (29). The precipitate was fixedwith 3% glutaraldehyde (30) and (or) 2% osmium te-troxide, dehydrated in increasing concentrations of alco-hol and propylene oxide, and embedded in Epon 812(31). Thin sections were cut with an LKB ultrotome,stained with uranyl acetate (32) and (or) lead hydroxide(33), and viewed with a Siemens Elmiskop I electronmicroscope at instrument magnifications of 15,000-60,000.Negative staining was carried out either by allowinga drop of the suspension to dry on a grid and subse-quently contrasting it with 1% phosphotungstic acid(PTA) at pH 5-5.5 or by suspending the amyloid in anequal volume of 2% PTA or 0.5% uranyl oxalate (34)and subsequently applying the mixture to a carbon-coatedFormvar-covered grid. Soluble amyloid was fixed withan equal amount of unbuffered 6% glutaraldehyde in dis-tilled H20. The protein precipitated upon addition of thefixative and could thus be embedded in the same manneras the amyloid in saline. For negative staining a dropof amyloid in solution was allowed to dry on the gridafter which it was contrasted with 1% PTA or uranyloxalate.

RESULTS

General appearance. Supernatants II, III, andIV had a straw color and a clear serous appear-ance. They contained 2.1, 1.7, and 3.6 mg ofprotein per ml, respectively, checked by biuret andFolin methods. The protein could be sedimentedcompletely at 100,000 g in 1 hr in the form of abrown gel. After storage at 40C for a week ormore, an opalescence gradually appeared and muchof the material could now be sedimented at20,000 g. At room temperature the opalescenceappeared within 2 days. When small quantities ofdifferent salts were added (NaCl, CaCl9, or evenglycine buffer at various alkaline pH's) the clearsolution became opalescent within minutes and aprecipitate was formed within 1 hr.

Solubility and Congo red binding. To checkthe effect of salt on the solubility of this protein,we mixed 2 ml of supernatants II, III, or IV con-taining 2 mg of protein with an equal volume ofsolutions containing NaCl or CaCl. at concentra-tions ranging from 0.005 to 0.3 mole/liter. Afteran incubation period of 3 hr, the mixtures weresedimented at 500 g for 3 min and the supernatantswere then checked for protein content by measur-ing the absorbance at 280 m/A. The diagram in Fig.2 shows that the protein precipitates in the pres-ence of NaCl at concentrations as low as 0.0075-

100 -

U 80-zM 60-0U) 40 -

20 -

"An/CONGORED

----- o0 D 280 mP- OD 490 mp

0005 0010 0015 0.020 0.025 0.075 0.15SALT CONCENTRATION,mole/liter

FIGURE 2 Relation between the amount of amyloid leftin solution and the concentration of added salt, NaCl orCaCI2. The amount of amyloid left in solution was deter-mined in two ways: (a) by the per cent of the initial ab-sorbance at 280 m~t left in the supernatant solution afteradding salt and removing precipitated amyloid by cen-trifugation, and (b) by measuring the per cent of Congored in saline not precipitated when added to the samesupernatant solution. The solid Congo red line was ob-tained with the supernatant solutions after the additionof NaCI.

926 M. Pras, M. Schubert, D. Zucker-Franklin, A. Rimon, and E. C. Franklin

0.01 mole/liter and in solutions of CaCl2 at con-centrations as low as 0.0025 mole/liter, and thatprecipitation was virtually complete in either solu-tion at concentrations of salt greater than 0.01mole/liter.

Addition of Congo red in 0.15 M NaCl to super-natants II, III, and IV resulted in a red gelati-nous precipitate within 1 hr. This precipitateshowed the typical green birefringence of amyloidin the polarizing microscope (6). As shown inFig. 1, the amount of Congo red bound was pro-portional to the amount of protein added. Studieswith several preparations which appeared to bepure electron microscopically and sedimented asa single peak in the ultracentrifuge showed that1 mg of protein bound 0.32 mg of Congo red(Table I).

Another method of measuring the amount ofamyloid left in solution after the addition of salt(Fig. 2) was to add an equal amount of a Congored solution in 0.15 M NaCl to each of the super-natant solutions, incubate the mixture for 1 hr,centrifuge it again at 500 g for 30 min, and readthe optical density of the supernatants at 490 mfu.The solid line in Fig. 2 is the mirror image of thedashed line obtained with NaCl and clearly showsthat Congo red binding can be used as an alternatemethod for quantitating amyloid in solution.

Chemical analysis. The nitrogen content of theprotein in supernatant II was 14.3%. Comparablevalues were obtained by Cohen (6) on purifiedamyloid fibrils and by Benditt, Lagunoff, Eriksen,

TABLE IRelationship of the Amount of Congo Red Bound per

Milligram of Protein at Various Stages of thePurification of Amyloid

mg Congomg pro- mg Congo red red/mgtein/ml removed/ml protein

Spleen homogenate, 8.01 0.87 0.1120 g spleen in400 ml normalsaline

Residue left, after 3.4 0.82 0.247 washings insaline, 400 ml

Amyloid solution in 2.7 0.88 0.32H20 (supernatantsII, III, IV)

TABLE I IAmino Acid Composition of Soluble Amyloid

LysineHistidineAmmoniaArginineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineCystine (half)ValineMethionineIsoleucineLeucineTyrosinePhenylalanineHydroxyproline*

Amoles/100 mg

43.014.992.628.463.452.678.384.054.578.071.2

9.561.7

6.027.158.928.426.0

0

* Determined separately (24).

and Iseri (4) on urea separated amyloid and byPirani, Bestetti, Catchpole, and Meskauskas (35).

The amino acid composition, expressed as mi-cromoles per 100 mg of protein, is listed in TableII. There is a predominance of acidic amino acids,a low content of cysteine and methionine, and nohydroxyproline. These results are similar to thosefound by Cohen (6) and Benditt and Eriksen (5).

The values of total hexoses, hexosamines, uronicacid, and sialic acid expressed as per cent of dryweight are listed in Table III. The total carbo-hydrate was less than 2.0%o, and the amounts ineach of the three fractions studied were ratherconstant.

The ultraviolet spectral absorptions of the threesupernatants and of different preparations weresimilar, each having an absorption peak at 280 m~uand a small depression at 255 mp, (see Fig. 3).

TABLE I IICarbohydrate Composition of Soluble Amyloid

Neutral Hexosa- Uronic Sialicsugars mines acid acid

%of dry weight

Supernatant II 1.17 0.26 0.23 0.12Supernatant III 1.31 0.18 0.30 0.10Supernatant IV 1.34 0.25 0.27 0.13

Properties of Soluble Amyloid 927

02mg AMYLOID /ml pH 6.4

240 260 280 300 320 340WAVELENGTHmy

FIGURE 3 Ultraviolet absorption spectrum of amyloid illwater.

Electrophoresis. In free electrophoresis in 0.1M unbuffered Tris, pH 10, supernatant IV mi-grated as a single sharp peak towards the anode.An accurate mobility could not be calculated sincethe low conductivity of the Tris solution did not

allow for more than 0.1 ma of current to be used.In this medium amyloid does not precipitate,whereas in salt solutions it does.

Sedimentation studies. Because of the solu-bility properties, all studies were done in distilledwater. Freshly prepared solutions of supernatantsII, III, or IV sedimented as a single, ratherhomogeneous peak in the analytical ultracentrifugeat speeds ranging from 20,000-52,000 rpm ('Fig.4 a). Occasionally, a small shoulder or a trace ofmore rapidly sedimenting components was noted.The concentration estimated from the area underthe peak corresponded very closely to the initialprotein concentration and indicated that more than95 %o of the material present was found in themajor peak. The sedimentation coefficient at in-finite dilution for several preparations was calcu-lated to be 45-50S (Fig. 5). More prolongedhomogenization, as in supernatants III and IV, or

treatment with ultrasound (80,000 cycles/sec, 15min) or with 6 M urea solution did not signifi-cantly change the sedimentation coefficient of theprotein.

Storage of the material at 40C for a week causedit to become opalescent. When the aged prepara-

tions were examined in the ultracentrifuge, theywere usually composed of two peaks (Fig. 4 b);the slow peak had the same sedimentation coeffi-cient as the fresh material, while the rapid peakhad a sedimentation coefficient of about 75S. This

FIGURE 4 a, Ultracentrifugal patterns of purified amy-

loid in distilled water. Bottom 3 mg/ml; top 2 mg/ml.Photograph after 24 min at 20,400 rpm. Phase plateangle - 45°. b1, Ultracentrifugal patterns of "aged" amy-

loid in distilled water. There are two components withsedimentation coefficients of 50S and 71S. Photographtaken after 36 min at 20,400 rpm. Phase plate angle - 45°.

801

S

40

30

| FRESH

0-e* "AGED"_-

1 2 3 4 5

CONCENTRATION (mg /mI)

FIGURE 5 Sedimentation coefficient of purified amyloidin distilled water plotted against protein concentration.Saoos ranged between 45 and 50S.

928 M. Pras, M. Schubert, D. Zucker-Franklin, A. Rimon, and E. C. Franklin

1.4

LLI

z

mcr0

mCr)

1.2

1.0

0.8

0.6

0.4

0.2

FIGURE 6 Appearance of fibrils in Epon-embedded sectioned amyloid. Fixation: glutaraldehyde and osmium te-troxide. Stain: uranyl acetate and lead hydroxide. Arrow points at a fibril which is seen at higher magnificationin inset. Here the fibril can be seen to consist of two filaments with a beaded structure. X 120,000. Inset X 330,000.

rapidly sedimenting material probably representsa dimer of the original protein. In one instanceonly the 75S peak was seen. After 5 wk an addi-tional faster peak that had a sedimentation coeffi-cient of about 100S appeared, while the sloworiginal 50S peak had disappeared. When thismaterial was centrifuged into a pellet, resuspendedin distilled water, and subjected to homogenizationin the Virtis-45 high speed homogenizer for 5min, it again became transparent and sedimentedas a single peak with a sedimentation coefficientof 45-50S.

Ultrastructure of fibrils. In thin positivelystained sections (Fig. 6), amyloid isolated by themethod described in this communication showedthe fibrillar structure found in electron micro-graphs of sectioned amyloid-laden tissues by otherinvestigators (11-18). The fibrils ranged from80 to 200 A in width, the majority measuringroughly 100 A. They crisscrossed into and out ofthe plane of section, making it difficult to deter-mine their length. One of the longest fibrils seenin one plane measured 0.5 ,u. In the salt-precipi-tated specimen, a large percentage of the fibrilsconsisted of two longitudinal subunits that wereequal in width and separated by a space of +25 A.

These two subunits appeared to remain parallelthroughout their course (Figs. 6 and 7). However,when fibrils consisted of more than two longitudi-nal subunits, they pursued a parallel course onlyfor a short distance, after which they twisted,crossed, or diverged. Scattered among the fibrilswere irregularly shaped dots (Fig. 6) which prob-ably represented tangential and cross sections ofthe fibrils. Their shape would presumably dependon the number of longitudinal subunits and theangle of the plane of section to the long axis ofthe fibril. Occasional cross sections showed aradiolucent core. The only differences noted be-tween "soluble" and salt-precipitated amyloid inembedded and sectioned specimens were that in"soluble" amyloid the number of longitudinal sub-units was smaller, the fibrils were generallyshorter, and single units measuring only +40Ain diameter were often observed. As others havereported (5, 17, 18), such single units appearedto have a beaded or helical structure.

The ultrastructure of the negatively stainedpreparations closely resembled that recently re-ported by Shirahama and Cohen (18) (Figs.7-9). As these authors noted, the terminologyadopted to describe amyloid fibrils in tissue sec-

Properties of Soluble Amyloid 929

FIGURE 7 A clump of precipitated amyloid fibrils negatively stained with 1% phosphotungstate pH 5.5. At theperiphery of the clump, filaments run in pairs and end together (see half-circles). Arrows at bottom point to twotwists in same pair of filaments. X 120,000.

tions is difficult to apply to isolated negativelystained amyloid because of the greater resolutionobtained in the latter specimens. Therefore, indescribing the negatively stained material, we willavoid use of specific nomenclature as much aspossible. However. in order to forestall confusion,we will call the unit structure most commonlyseen at moderate magnifications amyloid filament,as Shirahama and Cohen have done (18). In allthe preparations examined, filaments measured40-75 A in diameter and ranged from severalmillimicrons in length to the smallest resolvablefragments. They clearly had a beaded or helical

substructure which will not be described in detailhere. The salt-precipitated amyloid showed thickbundles of filaments (sometimes as many as 30could be counted within one bundle) as well aslarge clumps of intertwining filaments (Fig. 7).Strikingly, at the periphery of such clumps, thestructures were commonly seen in pairs. In thatcase, they were separated by a dense area, pre-sumniably a space which varied from 30 to 60 Ain different pairs, but which seemed to be uniformthroughout the course of the same pair. Pairs offilaments remained parallel even when twistingoccurred (Figs. 7 and 8), and they usually termi-

FIGURE 8 Salt-precipitated amyloid shows paired filaments negatively stained with 2% PTA. Note beaded struc-ture and apparent break. X 200,000.

930 M. Pras, M. Schubert, D. Zucker-Franklin, A. Rimon, and E. C. Franklin

.I

W...

FIGURE 9 Negatively stained amyloid obtained from"soluble" preparation demonstrates that the bond be-tween pairs of filaments is not firm. Small arrow pointsto an incomplete pair. Large arrow indicates smallersubunit which may represent the protofibril described byShirahama and Cohen (18). White round objects arebelieved to be artifact of the preparation. X 200,000.

nated together as if they had been formed orbroken simultaneously. Binding of these lateralaggregates did not seem to be very firm, however,since incomplete pairs (Fig. 8) or splitting of apair was sometimes seen. In addition to the fila-ments, smaller longitudinal subunits which mea-sured only 20-25 A in width were seen singly oralongside the larger units (Fig. 8). A discussionof the relationship of these smaller structures tothe filaments is beyond the scope of this paper,especially since a hypothetical interpretation ofsimilar observations has been recently published(18).

Soluble amyloid, which had been allowed to dryon grids before the application of the contrastingagent, generally showed smaller clumps and thin-ner bundles of filaments. Moreover, single fila-ments and the small longitudinal units were morecommonly encountered than in the salt-precipitatedspecimens.

DISCUSSION

The present study presents clear evidence that aprotein having the known properties of amyloidhas been obtained in a water-soluble form from

the spleen of a patient with primary amyloidosis.Since the material recovered included almost allof the Congo red-binding substances present inthe spleen, it seems likely that this represents amajor fraction of the amyloid material. However,the possibility does remain that additional con-stituents of amyloid with different solubility prop-erties may have remained in the insoluble residue.Furthermore, although amyloid from patients withprimary, secondary, genetically determined amy-loid and experimental amyloid from differentspecies appear to have many common properties(6, 11-13), they may not be identical.

The unusual ability of amyloid to precipitate atlow salt concentration, while many contaminatingtissue constituents are soluble, was extremely use-ful in its purification. To extract this material, itwas necessary first to homogenize the tissue andthen to wash out the electrolytes present in thesuspension. It would appear that once the fibrilsare broken up, the electrolytes are responsible forthe precipitation of the fibrils. A suspension ofpure amyloid in saline or buffer will not dissolveto give a clear solution even on prolonged dialysisagainst distilled water. Only by repeated homoge-nization with distilled water, after removal of allsalts, can a clear aqueous solution be obtained.The reason why solubility in water cannot beachieved by extensive and prolonged dialysisagainst water remains to be explained.

The method used here yields a product whoseelectronmicroscope appearance and chemical com-position do not differ significantly from that pre-pared by Shirahama and Cohen's method (6, 15).An additional observation which proved of valuein following the process of purification of theamyloid was the stoichiometric interaction withCongo red. Several preparations which appearedpure on ultracentrifugal analysis bound 0.32 mgof dye per 1 mg of amyloid.

Electronmicroscope studies of the fibrils undera variety of conditions in general confirmed thefindings of Shirahama and Cohen (18). Individualfilaments had a diameter of 40-75 A and rangedfrom a few angstroms to many angstroms inlength. They were seen as single filaments, pairs.or large intertwining bundles containing as manyas 30 or more filaments.

Although a precise relation of the material insolution and the fibrils has not been determined,

Properties of Soluble Amyloid 931

it seems likely that the molecules in solution withsedimentation coefficient of 45-50S in waterare much smaller than the fibrils seen in the elec-tron microscope. It seems possible that the fibrilsmay represent polymers of these soluble mole-cules or, alternatively, a different physical state ofthe soluble amyloid. The fact that these moleculesare highly reactive and tend to polymerize readilyis indicated by the fact that the sedimentationcoefficients rose from 50S to lOOS with age, andthat the solution became more and more opalescenton standing. It is not known whether amyloidreacts also with other proteins. If this were thecase, this reactivity, taken together with its in-solubility in salt, may be responsible for thedeposition of amyloid in a variety of tissues.

A number of questions remain to be answeredbefore the nature of amyloid can be definitivelydetermined and its pathogenesis elucidated. Amongthese are the identity or nonidentity of amyloid indifferent disease states, its homogeneity or hetero-geneity in any one disease, the nature of the bind-ing of this highly reactive molecule to tissueconstituents, and the factors that determine itssites of deposition. The similarity of electron mi-crographs of amyloid from a variety of tissues anddifferent disease states (11-15) as well as the iso-lation of soluble amyloid with a sedimentationcoefficient of about 45 S from a patient withfamilial Mediterranean fever (36) suggests thatthere may be many similarities in the structure ofdifferent preparations of amyloid. Additional stud-ies of its primary, secondary, and tertiary struc-tures and a more sophisticated search for serumproteins, such a y-globulin and its fragments, inthe purified preparations will shed much light onits precise nature. The availability of a pure.water-soluble preparation will help to provideanswers to many of these questions in the nearfuture.

ACKNOWLEDGMENTSThese studies were supported by U. S. Public HealthService grants AM02594, AM01431, AM05064, and AM00028. Dr. Pras is a U. S. Public Health Service Traineein Rheumatology (Fulbright Fellow). Dr. Schubert isa recipient of U. S. Public Health Service Career AwardAM 18434. Dr. Zucker-Franklin is a recipient of U. S.Public Health Service Career Development Award Al9572. Dr. Rimon is a U. S. Public Health Service Re-search Fellow (TW 1065). Dr. Franklin is a N. Y. C.Health Research Council Career Scientist (I 274).

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9. Letterer, E., W. Gerok, and G. Schneider. 1955. Ver-gleichende Untersuchungen uiber den Aminosfiuren-bestand von Serum-Eiweiss, Amyloid, Hyalin, Leber-eiweiss und Kollagen. Arch. Pathol. Anat. Physiol.327: 327.

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Properties of -Soluble Amyloid 933