IgG myeloma with closed tetrameric Bence Jones proteinemia

IgG Myeloma

with Closed

Bence Jones

Proteinemia*

V. CAGGIANO, M.D.,t #C. DOMINGLJEL, M.D., $ R. W. OPFELL, and L. R. WASSERMAN, M.D. New

Tetrameric

York, New

M.D.&

S. KOCHWA, PH.D.

York

A forty-one year old woman with multiple myeloma of sixteen months’ duration died with the clinical picture of plasma cell leukemia. During the course of her illness her serum contained h type IgG myeloma protein and h type Bence Jones protein, but concomitant Bence Jones proteinuria was absent. The serum Bence Jones protein was composed of two disulfide linked light chain dimers which were noncovalently bound to form a closed tetramer. The protein had a corrected sedimentation coefficient of 5.4 S and a molecular weight of about 88,000. The absence of Bence Jones proteinuria is explained by the high molecular weight of the tetramer.

are found in the serum and urine of patients with multiple myeloma and correspond to the light chains of the myeloma protein of these patients. Patients

B

ENCE JONES proteins

with multiple myeloma without detectable myeloma protein [1,2] in the serum often excrete Bence Jones protein [3], but detectable serum levels of Bence Jones protein without concomitant urinary Bence Jones protein is a most unusual occurrence. Described herein is a patient with multiple myeloma whose serum contained both IgG myeloma protein and Bence Jones protein of type x whereas the urine was consistently negative for Bence Jones protein. Physicochemical characterization of the serum Bence Jones protein revealed a closed tetramer of x type light chains. METHODS Serum electrophoresis was carried out on cellulose acetate membranes with a Beckman microzone apparatus. Immunoelectrophoresis and

immunodiffusion technics were performed by standard methods [4>]. Goat antiserum to human Fc and Fab fragments and rabbit antiserum to human IgG, IgA and IgM were either prepared Iocally or obtained from Hyland Laboratories, Los Angeles, California. Antiserum to kappa (K) and lambda (A) light chains were prepared in our laboratory by immunizing rabbits with suitable Bence Jones proteins emulsified in complete Freund’s adjuvant. Antiserum to IgD and IgE were kindly supplied by Doctors J. J. van Loghem of Amsterdam, the Netherlands, and K. Ishizaka of Denver, Colorado, respectively. Each antiserum was absorbed and rendered monospecific as tested by immunoelectrophoresis and immunodiffusion. Immunoglobulin quantitation was performed on Immunoplates@ (Hyland Laboratories) . Isolation from the serum of both the myeloma and the Bence Jones protein was first accomplished by starch block electrophoresis [6]. The fractions containing lambda chains predominantly (SB fraction IV) were concentrated by dialysis under positive pressure in collodion bags

*From the Department of Medicine (Hematology), Mount Sinai School of Medicine, New York, New York 10029. This study was supported in part by U.S. Public Health Service grants AM 12912, CA 04457, CA 05126 and PH.43-67-1359 from the National Institute of Arthritis and Metabolic Diseases, National Cancer Institute and the National Heart Institute and by the Albert A. List, Frederick Machlin and Anna Ruth Lowenberg Research Funds. Requests for reprints should be addressed to Dr. Shaul Kochwa, Department of Medicine (Hematology) Mount Sinai School of Medicine, New York, New York 10029. Manuscript received February 20, 1969. TPresent address: 2600 Capital Avenue, Sacramento, California. iSenior Clinical Trainee, U.S. Public Health Service Cancer Control Program Award CST 413B67. Present address: 1688 Meridian Avenue, Miami Beach, Florida. §Present address: Santa Anna, California.

978

AMERICAN

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Tetrameric

Bence Jones

Proteinemia-Caggiano

and (Scldeichcr and Schuell, Keene, N. H.) further purified by passage through a Sephadex@ G IO0 column equilibrated with phosphate bufin LKB fer, pH 7.5. followed b y electrofocusing .ipparatus I;] (purified SB fraction IV) . The H and I, chains of the reduced myeloma protein wcr(’ separated by column chromatography on Scph;tdex G-100 equilibrated with 1 R-1 propionic acitl iSI. Vertical starch gel electrophoresis was performed with 0.05 hl glycine buffer, pH 8.8, and a discOni iuuous buffer system [9] at 200 volts for sixtt.t.11 hours. Urea starch gel electrophoresis, pH 2.9 [ICI] and pH 8.8 [II], was performed for twenty-four hours at 200 volts. Starch gel immunoelectrophoresis was carried out according to the method of Poulik [IZ]. The heat test for Bcncc Jones protein was performed according to Putnam [I?]. Analytical ultracentrifugation of the patient’s serum, isolated myeloma protein and Bence Jones protein was carried out at 59,780 r.p.m. at 20°C. in a Spinco model E ultracentrifuge. The sedimentation coefficient of the Bence Jones protein was determined for the native protein and after treatment with 0.1 hl 2mercaptoethanol, 0.1 M glycine buffer, or both. Molecular weight determinations were made by the Archibald method [14]. Amino acid and peptide mapping analyses of the Bence Jones protein were kindly performed by I)octors K. \Valter of the Brookhaven National Laboratories, Lipton, New York, and Edward C. Franklin of tlie N.Y.U. School of Medicine, N.Y., respectively. CASE A

was

Joseph’s Hospiral, Orange County, <;alifnrnia, in January 1967 because of hone paitt of six montlia’ duration. On physical examiitatioii the patient appeared chronically ill a11tl p11c. Fusiform swelling and exquisite tenderric\s 01 tlie medial third of the right clavicle ;tr~tl tentleruess over the sternum and lower ribs postcrially were noted. The liver edge was felt L’ cm. I)~low 111~’ ri,ght costal margin, and a spleen tip W:IS pdpable. The remainder of the physical csamiuition was normal. The hemoglobin was 8.8 gtn. per 100 ml., hematocrit 25 per cent, leukocyte count G,300 per cu. mm. with 66 per cent neurophils, 31 per cent lymphocytes, 2 per cent rnonoc~tes and 1 per cent plasmablasts. The diagnosis of multiple myeloma was based upon (1) a sternal marrow aspiration which revealed almost complete replacement with plasmablasts and proplasmacytes, many of which contained Ku55ell bodies, (2) a monoclonal peak in the gamma region on the cellulose acetate elcctroplioretic pattern of serum, (3) anemia and (4) characteristic osteolytic lesions, visible on roenugenograms, of the clavicle, ribs, scapulas, humeri and lumbar spine. The heat test for Bence fones proteinuria was negative. Therapy was begun with 6-mercaptopllrine, 100 mg. daily, prednisone, 50 mg. daily, and oxymetholone, 40 mg. daily. One month later melphalan was substituted for 6-mercaptopurine at a dose of 4 mg. daily for two weeks and thereafter 2 mg. daily with adjustments according to the leukocyte and platelet counts. A clinical response

to treatment over the course of the next months was apparent with a rise in the hemoglobin level to 11.0 gm. per 100 ml., diminution of bone pain, improvement in 1)erformance swell

REPORT

forty-one year old white first seen by one of us

(179

et u/.

woman (C.S.W.) (K.W.O.) at St. TABLE LABORATORY

I

DATA OF PATIENT

C.S.W.

Results I)etermination

Hemoglobin (gm./ IO0 ml .) Hematocrit (%) Leukocyte count (1U‘/cu. mm.) Blood urea nitrogen (tng./luO ml.) Creatininc (mg./lOO ml.) Total serum protein (gm./lOO ml.) Alpha Monoclonal (myeloma) peak (gm./lOO ml.) Beta (Bence Jones) peak (gm./lOO ml.) IgG (mg./lOO ml.) IgA (mg./IOO ml.) IgM (tug./100 ml.) Urinary Bence Jones protein v-01..

47,

DECEMBER

1969

l/67

7167

4/6P,

8.8 25.5 6.3 15.0 1.3 12.1 6.0 0.8

11.4 35.0 3.8 18.0

i.3 22.0 3.0 37.0 2.2 I 1.9 5.8 0.8 6.600 35 25 Negative

Negative

11.3 5.1 0.6 5,000 17 17 Negative

Tetrameric

980

Bence Jones Proteinemia-Caggiano

et al.

+

Anti-NHS

Anti-lg

G

Anti-h

IA

1B A, serum electrophoretic pattern. IgG and x tetramer peaks are indicated by arrows. B, immunoelectrophoresis of patients serum. Antiserum as indicated (NH&normal human serum). Note one characteristic precipitin arc with anti-IgG and two precipitin arcs with anti-h serum (arrows)

status and a slight decrease in the percentage of myeloma cells on bone marrow smear. However, the serum concentration of the myeloma protein, as estimated from cellulose acetate strips, did not diminish significantly. In July 1967 immunoelectrophoresis of the patient’s serum performed at The Mount Sinai Hospital, New York, revealed the presence of (1) an IgG myeloma protein having type A light chains and mid-gamma mobility, and (2) a lambda type Bence Jones protein in a more anodal position. Urine protein was 90 mg. per twenty-four hours but Bence Jones protein was not detected by either the heat test or immunoelectrophoresis of urine concentrated

twentyfold. Subsequent investigations during the course of the next nine months confirmed these findings. Pertinent laboratory data are recorded in Table I. Beginning in February 1968 blood transfusions were required at biweekly intervals, and in May 1968 the patient was admitted to St. Joseph’s Hospital for the last time with a clinical picture of plasma cell leukemia. She was cachectic and had marked gingival bleeding. The spleen edge was now felt 5 cm. below the left costal margin, and 1 by 1 cm. nodular infiltrates in the skin over the abdomen and buttocks were noted. The hemoglobin level was 2.8 gm. per 100 ml., hema-

FIG. 1.

I

I

I

2

I

I

I

4

I

6

I

I

I

8

I

I

I

IO

12

I

i,

14

,

I I

16

18

CENTIMETERS

1g.G -

+

J

Stained gel

2B

Anti-h

Stained gel

tt BJ TR

1, patient’s serum in 1:5 dilution; 2, isolated serum Bence Jones FIG. 2. A, starch gel electrophoresis. protein (SB Fraction IV); (BJ = Bence Jones protein, TR-transferrin) ; 3, isolated serum myeloma protein IgG. B, starch gel immunoelectrophoresis. Whole serum (1) and SB fraction IV (2) were separated on starch gel at pH 8.8. Half of the sliced gel was stained and the other imbedded in 1 per cent agar and the reaction with anti h serum in the center trough tested. Arrows indicate precipitin lines corresponding to IgG and Bence Jones proteins visualized on stained starch gel strips. AMERICAN

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Bence Jones Proteinemia-Caggiano

981

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8 per cent, leukocyte count 28,000 per cu. mm with 95 per cent plasma cells. The patient rlieti two days later despite blood transfusions .mtl p~ednisone therapy. infiltrations were At autopsy, rnyelomatous lou~~tl in the bone marrow, spleen, liver lymph Iocr-it

stomach, pancreas, kidney, esophagus, notlec, lungs. skin, adrenals and parathyroids. The spleen weixhetl 395 gm.: the right kidney 120 gm. and the left kidney 135 gm. On cut section the cortic~ometlullar-y junctions of the kidneys were indistint I, Imt the calices, pelves and ureters were not tlil;l ted. Histologic sections of the kidneys revealed nol-m;ll glomeruli with slight basement membrane thic kcning in some areas. The tubules were atIophic and focal areas of calcification were prcsent in both the distal and collectin,g tubules as well .IS in the interstitial tissue of the pyramids. ‘The interstitium of the cortex and medulla had infiltrates of myeloma cells and lymphocytes. The findings in the kidneys were those of chronic ~)\~(~lo~l(~pl~ritisand myelomatous infiltration. SPECIAL

STUDIES

Cell~~lose acetate elcctrophoresis of the serHIP ~~e~~~lecl ;I discrete monoclonal peak in the gamma region and a smaller though well defined peak in the p-globulin region (Fig. I.-I) These peaks represented 5.79 and 0.78 gm., respectively, of the total serum protein of 1 I.90 gm. per 100 ml. On immunoelectrophoI-esis of the serum a single precipitin arc with ,mti-IgG and two arcs with anti-X antiserum were apparent. The more anodal precipitin arc with anti-h was formed exclusively with anti-h antiserum and joined in a reaction of identity with the lambda type 1gG precipitin .Irc (Fig. IB) . Twenty-four hour urine colleclions revealed a protein excretion of 100 to 200 mg. daily consisting entirely of albumin. Rence Jones protein was not detected on repeated occasions by either the heat test or immunoelectrophoresis. These observations suggcs~etl the presence of either (1) Bence ,Jones proteinemia without Bence Jones proteinuria or (2) a biclonal gammopathy, the second protein having lambda light chains and antigenie determinants other than Y, a, P, 6 or C present on the heavy chains. On starch gel electrophoresis, a dense homogeneous band was observed a few millimeters to the cathodic side of the origin. ,4 narrower but equally intense band was observed in the region of transferrin (Fig. 2A) . Both

Y01..

47,

DECEMBER

1969

1 -A 4

Anti

bands were shown by starch gel imml~noelectrophoresis to react with anti-h serum (upper gel Fig. 2B) . Electrophoresis of patient’s serum on starch block yielded two fractions (designated SB fraction I and SK fraction Iv) which wet-e itlentified by immunochemical methods. When tested by the immunodiffusion method, SB fraction 1 reacted with a precipitin arc against anti-IgG and anti-h serum, whereas SR fraction IV reacted with antiserum directetl against either A chains or Fab fragments but not with antiserum against 7, a p, 8 and E chains, OI- Fc fragments. Ky both starch gel electrophot-esis (Fig. 2A) and immunoelectrophoresis (Kg. hchaln

Transferrln

I S B fraction 50

55 60 65 TUBE NUMBERS

70

IV

-

3 0

75

FIG. 4. Purihcation of Bence Jones tetramer. G4.2 mg. of SB Fr. IV after passage through Sephadex G-100 were electrophoresed on an ampholite column in 5-8 pH gradient. The original solution contained about 20 per cent lransferrin. Fraction 65-72 were pooled, concentrated, and designated purified SB fraction IV.

Tetrameric

982 A-

Bence Jones Proteinemia-Caggiano

FIG. 5. Acid urea starch gel electrophoresis. All proteins at approximately 5 mg. per ml. concentration. 1, Cohn F II, reduced and alkylated; 2, isolated native myeloma propurified tein; 3, SB fraction rv; 4, reduced and alkylated myeloma proreduced tein; 5, and alkylated purified SB fraction Iv; 6, urinary Bence Jones protein of

another patient. Note the correspondence between the light chain of IgC (4) and one of the light chains of Bence Jones tetramer (5) : the urinary Bence Jones protein of another patient (6) has an intermediate mohility between the two light chains of (5) .

3) , the migration of isolated SB fractions I and IV was identical with that of the myeloma and the Bence Jones proteins, respectively, in the patient’s serum (Fig. 1B). Further purification of the SB fraction IV by electrofocusing in pH gradient between pH 5.0 and 8.0 (Fig. 4) resulted in removal of the contaminating transferrin. The results of reduction and alkylation followed by electrophoresis in urea starch gels are shown in Figure 5. In acid urea starch gel, unreduced purified SB fraction IV migrated in a position intermediate to that of the light and heavy chain regions of reduced-alkylated normal gamma globulin (Cohn Fr II) or SB fraction I, suggesting a molecular weight greater than light chains but less than gamma chains. Following reduction and alkylation, however, two distinct light chain bands were visible, the more cathodal corresponding in position to the light chain band of SB fraction I. Analytical ultracentrifugation of the purified SB fractions I and IV revealed homogeneous peaks with sedimentation coefficients of 6.2 and 5.0 Svedberg units, respectively (Fig. 6A). When corrected for concentration the IV was 5.4 S. S020a value of SB fraction TO test for the presence of noncovalent as well as covalent bonds, separate aliquots of SB fraction IV were reduced with 0.1 M 2-mercaptoethanol or treated with 0.1 M gIycine

et al.

buffer. The uncorrected Sosow values were found to be 4.4 S and 3.9 S, respectively, for the mercaptoethanol and glycine treated fractions. When SB fraction IV was reduced first with mercaptoethanol and later treated with glycine, a sedimentation coefficient of 2.3 S was obtained (Fig. 6B, 6C and Table II). However, when this fraction was dialyzed against phosphate-buffered saline solution at pH 8.0 an aggregate with a sedimentation coefficient of 4.5 S was obtained. The light chains of the myeloma protein, obtained following red’uction with mercaptoethanol and passage through a Sephatlex G100 column equilibrated with 1 M propionic acid, formed dimers (3.6 S) in vitro on removal of the dispersing agent. The molecular weight of purified SB fraction IV calculated from the meniscus and the bottom of the cell was determined to be 87,500 f 1000. Peptide mapping by two-dimensional chromatography and electrophoresis of purified SB fraction IV revealed a pattern consistent with that described in other lambda type Bence Jones proteins. Table III summarizes the amino acid analysis of purified SB fraction IV. Noteworthy is the presence of 1 M of methionine per 22,000 gm.

6A

6B

6C

FIG. 6. Analytica ultracentrifugation at 1 per cent protein concentration, solvent 0.15 M sodium chloride. Direction of sedimentation is to the right. A, isolated serum Bence Jones protein (purified SB fraction IV) in upper frame; isolated myeloma protein (SB fraction I) in lower fr,me. Photographs taken at twenty minutes after attaining full rotor speed. B, reduced purified SB fraction rv in upper frame; glytine treated purified SB fraction IV in lower frame. Photographs taken at thirty-two minutes. C, reduced, alkylated and glycine treated purified SB fraction IV in upper frame; reduced and glycine treated purified SB fraction IV after dialysis against buffered saline solution pH 8.0. in lower frame. Photographs taken at forty minutes. Numbers indicate the sedimentation rates. AMERICAN

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Bence Jones

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TABLE

CO~l~lIs?‘S

proteins are structuralIy and antigenically identical with the light chains of the myeloma protein found in patients with multil)le n~yeloma and were demonstrated in i ii10 !I i,,l/~] :Intl iv 7~it7.0 [17,18] to represent excess synthesis de WXIO of light chains rather til;cn l~reaktlown product5 of the immunoglobuThe Bence Jones proteins exist 1111molecule. ;IZ niotlonier5 will1 a molecular weight of apIIt-osiinately 22,500 or as dimers linked by (c~alcnt or noncovalent bonds and having a molecula~~ weight of about 45,000. In addition. l)olymeric forms of Bence Jones proteins and low molecular weight fragments of Hence Jono proteins have been found in the urine of patients with multiple myeloma [f 9,201. Ln 1943 Moore, Kabat and Gutman [ZI] ticscril)etl ;I patient (Case 4 1) with multiple myeloma who had Bence ,Jones protein protcrincmia without Bence Jones proteinuria. IIe kerurn Bence Jones protein had a SaOm \aluc of 3.0 mtl the aurhors suggested that the absent c of Bence Jones proteinuria might have been due to complex formation in the circuLring fluids. Osserman, Takatsuki and TalaI [??I. ~II 1964, describetl a patient (Case 22) 1f.ith amyloiclo~i~ ant1 lambda type Bence Jones proteinemia in the absence of urinary I’,~lcc [ones protein. These investigators speculatt~d that either the protein had a molecular u ci$t greatrr than that of the usual Bence Belice

,]011es

TABLE SEDI~FNT*TION

COEFFICIENTS

PROTEIN

AND ISOLATED I@-MYELOXIA

II 0~ THE SERUM BENCE JONES LIGHT CHAINS

OF THE

PROTEIN

Data

Coefficient -__

Native fraction, uncorrected for concentration Native fraction, corrected for concentration After reduction with mercaptoethanol and alkylation with iodoacetamide After treatment with glycine, pH3.0 After reduction with mercaptoethanol, alkylation with iodoacetamide and treatment with glycinc, pH3.0 After reduction with mcrcaptoethanol, treatment with glpcinc, pH3.0, and dialysis against buffered saline solution pH8.0 Light chains separated from the IgG-h myeloma protein and dialyzed against buffered saline solution, pH8.0

VOL.

AMlNO

47,

DECEMBER

1969

5.0 S 5.4 s 4.4 s 3.9 s

2.3 S

4.5 s

3.6 S

983

et al. III

ACID COMPOSITION

OF THE SERlrM

BENCE JONFS PROTEIN (PURIFIED

SB FRACTION

IV)

Inoles.‘~,0~K)

~-__

Amino Acid

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

gm. protvilr 10 4 44 16 14 21 21 13 15 17 5 10 1 3 15 7 5

Jones protein and therefore could not be eliminated in the urine or that it had a rapid tissue clearance with accumulation in the extravascular tissues as amyloid. Grey and Kohler [Z?], in 1968, were the first to tlocumenl; a case of tetramer Bence Jones proteinemia. The protein described in thkir report consisted of a pair of climers which were noncovalently bonded to form the tetramer and had a molecular weight of 84,000. Our Iyatient had Bence Jones proteinemia without concomitant Bence Jones proteinuria. Furthermore, the serum Bence Jones protein was a polymer of lambda light chains (two disulfide-linked light chain dimers noncovalently bound to form a closed tetramer) , These conclusions were based on the following data: (1) the p urified SB fraction IV which reacted only with anti h and anti-Fab serum, corresponded to the fast monoclonal arc noted on immunoelectrophoresis of the p;ltient’s serum; (2) the purified SB fraction IV Ilad a corrected sedimentation coefficient of 5.4 S, higher than the usual 2.5 to 4.2 S values for Bence Jones proteins and yet lower than tiile 6.6 S value of the IgG myeloma protein also present in the patient’s serum; (3) treatment of SB fraction IV with mercaptoethanol and glycine indicated the presence of only light

Tetrameric

984

Bence Jones Proteinemia-Cuggiano

2-ME

I”

Glycine buffer pH3.0

I -+H

\

2-ME

FIG. 7. Schematic representation of the structure of the tetrameric Bence Jones protein. Bonds susceptible to dissociation by different agents indicated by arrows (ZME = 2 mercaptoethanol) .

chains linked by both covalent and noncovalent bonds (the S20w value of 2.30 obtained after treatment with both reagents is similar to that of monomer chains) ; (4) the tryptic peptide map, amino acid analysis and molecular weight determinations were all consistent with a tetramer of Bence Jones protein. The amino acid composition of this Bence Jones protein may give a clue to its structure. Methionine is present in K and absent in most x Bence Jones proteins [24]. Kappa light chains form noncovalently bound dimers whereas lambda light chains are found as COThe presence of valently linked dimers. methionine may confer on h dimers the aggregating property of K chains facilitating the formation of a tetramer. It is of interest that the Bence Jones proteins described by Osserman et al. [22] and by Grey and Kohler [23] were also of the x type. Figure 7 is a schematic illustration of the probable arrangement of the polypeptide chains of a tetrameric Bence Jones protein based on the subunits obtained after treatment with mercaptoethanol and glycine. Previous studies have indicated that the development of significant Bence Jones proteinemia depends on a balance of three rate-determining factors: synthesis, catabolism and excretion of Bence Jones proteins [25-271. Little is known about the synthesis rate of Bence Jones protein and, for the present, this can be estimated only indirectly from the rates of catabolism and excretion. Endogenous catabolism may account for 50 to 90 per cent. of the metabolism of Bence Jones proteins and is

et al.

dependent upon renal function [25]. Thus, patients with multiple myeloma who have renal disease may have a significantly reduced rate of catabolism of Bence Jones protein, thus contributing to the development of detectable serum levels of Bence Jones protein. elevated levels of free light Furthermore, chains in serum have been found in nonmyeloma patients with end-stage renal failure f28]. In these patients a rising urinary light chain concentration paralleled a falling glomerular filtration rate, suggesting that the rise in urinary light chain concentration was a function of the rising plasma concentration of these proteins and a decreasing catabolic rate. Our patient had a normal blood urea nitrogen and serum creatinine level for most of the clinical course of the disease. Only terminally was a rising blood urea nitrogen level observed. The myelomatous infiltration in the interstitium of the renal cortex and medulla, and the presence of atrophic tubules, may have resulted in decreased endogenous catabolism of light chains and contributed to the elevated serum concentration of the Bence Jones protein. However, myelomatous renal ;nvolvement does not readily account for the absence of Bence Jones proteinuria. A much more likely explanation for the presence of Bence Jones proteinemia without concomitant Bence Jones proteinuria is the high molecular weight of the tetrameric Bence Jones protein. As is well known, the renal glomerulus acts as a molecular sieve with respect to plasma proteins [29$0]. Harrison and colleagues [31] have demonstrated that in patients with multiple myeloma the renal clearance of protein is inversely related to molecular size. From this consideration tetrameric Bence Jones protein, with a molecular weight of about 88,000, would have difficuIty passing through the renal glomerulus. The finding of two light chain bands on acid urea starch gel electrophoresis of reduced and alkylated Bence Jones protein raises the possibility of two differently charged light chains, as might be expected from an amino acid substitution. A further observation of considerable interest was that the isolated light chains of the serum myeloma protein which formed only one band on acid starch gel electrophoresis failed to form tetramers in vitro (Table II) . Attempts to permit the reformation of tetramer Bence Jones protein AMERICAN

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‘l~ctrameric

Uetlce

Jones

et I!(.

Proteirlenlia--(‘ugg/trno

f’(~f\ nwr or .Iggregate lormation of immunoyfol~~lli~ls is an intere5ting and clinically iml,ol t.ttlt I(>;I~IN: of monoclonal gammopathies. As e~;~nlj)le~ 01 ]dper formation, nne m:i) enwrrntci tlinlclic xitl tetrameric Bence /ones ant1 I,rot(Lills. pent:mleric. IgG [32], tlimeric trilnc,riL lg.1 / ii] 211~1pentameric IgiV 1341. Jn (:.I, 11 irlsC;lncv ;I monomer is a natLlrally OCCL~III~~ cnlity.

1X. Asho\ \s, Ii. ,\. and \\‘ri I.IAXION. \. K. l~alancetl hca\ \ and light chain synthcai\ ItI imtllune lisstie a11(1 ~lihulphitlc I)ontl formation in lg(, ;t\srml)lv. In: .X&cl Symposium 3, (iarnma Globulitis, p. 36X. Etlitetl by Killantler, ,J, \ew 5.(Irk. 1967. Illtel-science Publications, ln(,. 19. IsI-RN11 R, (;. ,\I. ;111d pUINA\I, p. 11’. ~‘U~\8111~~,‘ihfll, ],ol\lllc!r~~hislrl, plot(4lfs.

‘\., KIINK1.I , H.

IllC.

47,

22.

23.

24.

25.

26. 27

7. Slrussru, H. Tsocleclric fractionation, analysis, and characterization of ampholytes in natural pH gradients. Arch. Biorhem., supp. 1, p. 132, l9F2. 8. FIEISCHMAN, J. B., PAIN, R. H. and PORTER, R. R. Reduction of -,-globulins. _4rclz. Biochern., supp. 1, p. 171, 1962. 9. FUIE~, J. I,. Heterogeneity of myeloma proteins. J. C/in. Inufst., 42: 111, 1963. 10. Enr~,nr,\x, G. I%. and POULIK, M. D. Studies on structural mliis of the y-globulins. ,j. Exfxzr. ~%ffzd.. 113: 861, 1961. 11. COHEN, S. and PORTER, R. R. Heterogeneit) of the peptide chains of y-globulin. Biochem. J., 90: 278, 1964. 12. POI:IX, M. D. Starch gel immunoelcctrophorcsis. Inn. Sriu York Acad. SC., 121: 470, 1964. 13. PI INAM, F. XV., EASLEY, C. W., LYNN, L. T., RLTCHIE, A. E. and PHELPS, R. A. The heat precipitation of Bence-Jones proteins. I. Optimum conditions. Arch. Bioclzem., 83: 115, 1959. 14. ?+:I! XHMAN, H. K. IJltracentrifugation, diffusion and viscometry. In: Methods in Enzymology, vol. 4. p. 32. New York, 1957. Academic Press, Inc. 15. Pu.rN)\\\r, F. W. and HARDY, S. Proteins in multiple myeloma. III. Origin of Bence-Jones protein. J. Iliol. Chem., 212: 361, 1955. VOL.

21.

DECEMBER

1969

28.

29. 30. 31.

32.

33.

34.

rl

impurities

in

/~io/ll/~.\.

cwtn,

Bcrlw-Jones

‘L95. 1964. ,J., (:RE>‘, 1I. hf. atld G. Low-molecular-weight protelus wlarctl IO Bcrrce ,Joues proteins in mlillipl(~ mvcloma. .Ycienc~, 1.59: 1237, 1966. MOORF, D. H., K;\nAr, E. A. and GUI ~1\.L, .‘i. 13. Benre Jones proteinemia in multiplr rn)clol~~a. ,I. Cliu. Irrw~t., 22: 67, 1943. OSSPRMAN, E. F., TAKArxw, K. ant1 ‘T..u 41 N. Multiple mycloma, Part I. ‘The pathogcnesls of “amyloidosis.” Sern. Hemat., 1 : 3, l%iZ. GREY, M. M. and KOHI.ER, P. F. A case 01 tctl,lmer Bence ]ones proteincmia. Clin. Ex/x?. /r~rfri~~nr~l., 3: 277, 1968. Pu.rN.<\l, E. \V. and E,zsr LY, C. \\‘. Stl-uctural ttrltlieb of the im~nunoglol~ulirls. I. The lr!l)tic pc’l’titles IIt Bence-Jones proteins. / f!i’iol. (:/rrrr?., 2-10: lli26, 1963. Soronlo;\, A., \\‘.~I.IIMANN, 7‘. A., F~rll;\, J. 1.. alltl A. S. Metabolism ot Beuce JOIICS .\fCF\R, ANI:, protcitts. 1. Clin. Imjest., 43: 103. 196 1. Sor.on~on, .A. and FAHEY, J. S. Bence jones protcinemia. Aln. J. Med., 37: 306, 1964. WOCHZILK, R. D., STROBLR, W. and U’AI.UMANN, T. .4. The role of the kidney in the catabolism of Rence Jones proteins and imn~ut~oglobt~lit~ fragments. 1. &per. Med., 126: 207. 19ti7. EI’S~EIS, IV. V., Gur.y~su, P. F., T!\.L. AI. antI R:\E, A. I. Effect of I-cnal homotl-atisl)l;rllt;Ition 01, the metabolism of the light chains of immunoglohulins. Ann. Int. hfed., 68: 48, 1968. P?\PPENHEIMER, J. R. Passage of mo1ecul~a threough capillary walls. Plzysiol. Ren.. 33: 387, 1953. CHINARD, F. P. Rate of formation of glomerular fluid. .4nl. J. Physiol., 171: 578. 1953. HARRISON, J. F., BLAIUE~, J. 11.. ~IARI~WICKI:, J., ROINII, D. S. and Soorlm.r., J. F. Protcinuria in multiple mycloma. Clirz. SC., 31 : 95, 1966. KOCHWA. S., Snrrr~, E., BRO~XIXI.. M. al111 WA.SSEPAIAN, 1.. R. .-~ggP$ptiOn of Ig<; globulill in vivo. II. Physicochemical propertics of I hc isolated _ . protein. Biochemistry, 5: 277, 1966. HEREMANS, J. F., HEREMANS. 11. 7‘. .md SCHUIZX, H. E. Isolation and descriptiou of a few propertics of the ~,A-globulin of human sc‘wm. Clin. chim. acta, 4: 96, 1959. SOLOMON, A. and KUNKTI., H. G. ,\ “monocIona1” type, low molecular weight protein related to TM-macroglobulin. Am. 1. .Iled., 42: 958, 1967.

20. SOI.O\IO\. F. \V. Stlllctural relationships among I. PI IX\\,, I~uman y-globulin, mycloma globulins, autl Bencc joncs proteins. Biot /rim. bioj/hyc. crcln, 63: .5X), I !lG2. 2. SCIIWARI‘Z, J, 1). anti EDELMAN, G. M. Comparison of Benrc Jones proteins and L polypeptidc chains of myeloma after hydrolysis with trypsin. 1. I:‘spr. Med., 118: 41, 1963. 3. KL YK~I H. (;. The “ahnormality” of mycloma l”orc.ina. Cil,,rr,r- Rrs., 28: 1351, l9F8. 4. GK~HAR, P. a1111 Bt RTIN, P. Immunoel~clropl~oretic 1964. Elscvicr Publishing ;tllal+\. .~\mstertlanl. (‘0. 0. Antigen-antibody reactions in 5. OCCHTI~RLON~, gels. IV. Types of reaction in coordinated systems of diffusion. Actn pnth. et microbial. .scandinav., 32: “31, 1953. 6. Konsl;r., H. C. Zone: rlectrophoresis. In: Methods of Biochemical Analysis, vol. 1, p. 141. Edited by (;lick, D. New York. 19.54, Interscience Publishers,

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