PROTEINS IN MULTIPLE MYELOMA

I. PHYSICOCHEMICAL STUDY OF SERUM PROTEINS*

BY FRANK W. PUTNAM AND BERNARD UDINt

(From the Department of Biochemistry, University of Chicago, Chicago, Illinois)

(Received for publication, May 26, 1952)

Multiple myeloma, a tumor of the bone marrow, is frequently associated with change in the nature and amount of the serum proteins and is some- times accompanied by the excretion of a characteristic urinary protein. Most patients have an elevated globulin, and about 65 per cent are hyper- proteinemic (1). The increase in serum globulin is nearly always confined to one fraction (MM protein’) which appears homogeneous, but the electro- phoretic mobility of the MM component varies from case to case (24). The urinary protein (Bence-Jones protein)’ is identified by unusual heat- coagulation properties and may be a homogeneous substance. However, it is not identical in different cases since the isoelectric point, and possibly also the molecular weight (4~7), varies. Despite this variability in the nature of the proteins, multiple myeloma is the unique instance of a tumor in which the qualitative and quantitative distribution of serum proteins and the nature of the urinary protein are virtually pathognomonic of the disease. The frequent hyperproteinemia, the appearance of anomalous serum proteins, and the excretion of unusual urinary proteins are all symp- toms which indicate a disturbance of normal protein synthesis. The ob- ject of this series of investigations was to study this apparent metabolic derangement by use of the physicochemical methods of protein chemistry and by the isotopic tracer technique.

A number of electrophoretic surveys of multiple myeloma sera have been correlated with clinical findings (3, 4, 8). The interrelationships of the MM proteins and normal globulins have not yet been clearly defined by physicochemical methods. This paper presents quantitative studies of the

* Aided in part by research grants from the National Cancer Institute, National Institutes of Health, United States Public Health Service, and the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago.

t Present address, Department of Surgery, The Presbyterian Hospital of Chicago, Chicago, Illinois.

1 For brevity the serum protein increment will be designated “MM protein” without implying that it is identical in different cases or that it is unrelated to normal serum proteins. Similarly, “BJ protein” signifies a nearly homogeneous urinary protein with the classical heat-coagulation properties but does not imply physico- chemical identity.

727

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728 MYELOMA SERUM PROTEINS

electrophoretic homogeneity and mobility, and the sedimentation proper- ties of the isolated MM proteins. Later papers will describe the physical characterization of the BJ proteins and will consider the rate of protein synthesis as revealed by the incorporation of dietary isotopic glycine into the serum and urinary proteins (9).

Methods and Procedures

Electrophoretic AnalysisGSera were diluted to 2 per cent protein with Verona1 buffer, pH 8.6, ionic strength 0.1, and were dialyzed till equili- brated. Electrophoresis was carried out with the Klett or the Pearson apparatus with use of the Longsworth scanning method. Mobilities were calculated from the apparent centroidal ordinate of the descending bounda- ries and are corrected to 0”. They are expressed in units of lo+ cm.2 sec.-l volt-l designated u. The percentage distribution was obtained by planimetry of the ascending and descending patterns and by averaging the values. All concentrations given were estimated refractometrically but checked well with Kjeldahl N determinations on the sera. The relative concentration of the MM peak sometimes had to be estimated by difference by use of the optical constants of the apparatus.

The electrophoretic mobility curves of purified MM proteins were in- vestigated at a concentration of 0.6 per cent. Univalent buffers of ionic strength 0.1 were made up according to Alberty (10) in order to compare the mobility data with his results for purified normal serum r-globulins.

Isolation of Proteins-The MM protein was usually isolated by electro- phoretic separation at pH 8.6. For a y type protein, the product was about 95 per cent homogeneous in electrophoresis and ultracentrifugation. Even a p type protein was only slightly contaminated, because in this cir- cumstance y-globulin is usually a negligible electrophoretic component of the whole serum. However, some purified fi type MM proteins, though electrophoretically monodisperse, contain two or more sedimenting com- ponents.

In one instance the MM protein was a cryoglobulin, i.e. a globulin pre- cipitated by cooling a concentrated solution but dissolved by warming to room temperature. Advantage was taken of this property for the purifi- cation of this protein. However, at the dilutions employed, the cryoglobu- lin could be studied in electrophoresis at 1”. Salt fractionation was used to separate the MM protein in one case and isoelectric precipitation in another.

Ultracentrifugal Analysis-The Spinco analytical ultracentrifuge model E described elsewhere (11) was used at room temperature with a rotor speed of 59,780 r.p.m. Since the chief error in determining the sedimenta- tion constant may involve the correction for temperature (12, 13), atten-

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F. W. PUTNAM AND B. UDIN 729

tion was given to this factor.2 Refrigeration was routinely used, and the mean of the initial and the final temperatures was taken. This figure averaged 1.3” higher than the initial temperature. All sedimentation con- stants given are corrected to a water basis at 20’ (~~0) and are expressed as Svedberg units of sedimentation, S = lo-l3 cm. per sec. per unit field of force. These constants were measured with the aid of a comparator and by the use of the interference fringes (11).

For ultracentrifugal analysis of whole sera, the samples were diluted to 3 per cent protein and dialyzed against 1 M NaCl. The isolated MM pro- teins were studied in the buffer at pH 8.6 except when s20 was determined as a function of protein concentration, in which case a buffer at pH 5.5 was employed (10).

Dijhsion Constants-Diffusion constants were determined in the electro- phoresis cell at lo with photographic recording by the scanning method over a period of at least 4 days. The values were calculated by the maxi- mal ordinate area method (15) from enlarged tracings of each photograph. The constants are corrected to a water basis at 20” (D20) and are expressed in units of lo+ cm.2 sec.-‘.

RESULTS AND DISCUSSION

Analysis of Sera

Electrophoretic Analysis of Sera-The results of electrophoretic analysis of twenty-five multiple myeloma sera are presented in Table I, which con- tains the mobilities of the proteins in twenty sera with MM components, and in Table II which gives the distribution of the proteins in the sera.3* 4 For comparison, average values for normal sera are given (16). Repre- sentative electrophoretic patterns are presented in Fig. 1. The case num- bers are assigned on the basis of increasing mobility of the MM protein.

The mobility data of Table I indicate that the MM proteins constitute

2 The uncertainty of the true temperature within the cell rotating at 60,000 r.p.m. in a vacuum attaches to all published sedimentation constant data. The recent report (14) that the rotor cools on acceleration is contrary to previous findings (11). In our work, no constant correction factor could be found which adjusted for the difference in “free” and “fixed” thermocouples, whether refrigeration was employed or not. Statistical analysis has revealed a standard error of 1 per cent in the meas- urement of the sedimentation constant with the Spinco machine in different labora- tories (12, 13). The consensus is that the error due to temperature measurement does not exceed 1 per cent.

3 Sera from these patients were kindly supplied chiefly by Dr. Charles Huggins and Dr. Matthew Block of the University of Chicago Clinics.

4 In cooperation with Dr. S. 0. Schwartz of Cook County Hospital, Chicago, Illinois, another fifteen sera have been studied in an attempt to correlate electro- phoretic analysis, ultracentrifuge data, and clinical findings.

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730 MYELOMA SERUM PROTEINS

a family of proteins of gradually varying ionic structure rather than a few discrete substances, as might be suggested by the patterns of Fig. 1. The almost continuous frequency distribution of our cases is illustrated by the

TABLE I Mob&ties of Multiple Myeloma Serum Proteins*

All the mobilities are given in units of 10m6 cm.2 sec.-r volt-r.

Case No. Albumin

1 5.9 2 5.9 3 6.1 4 6.0 5a 6.0 5b 6.0 6 6.5 7 6.0 8a 6.0 8b 6.0 9 5.9

10 6.1 lla 6.1 llb 6.1 12 6.0 13 6.0 14 6.0 15a 6.1 15b 5.9 16 5.8 17 5.9 18 6.0 19 5.9 20 6.0

Normal serat

6.1 f 0.1

4.9 5.0 5.2 5.3 5.1 5.1 5.5 4.8 5.1 5.2 4.6 5.0 5.2 5.2 5.2 4.9 5.0 5.2 5.0 5.0 4.9 4.9 5.0 5.2

~__ 5.0 f 0.1

Globulins

3.7 3.7 4.0 3.8 3.9 3.7 3.9 3.8 3.9 4.0 3.8 3.9 4.0 4.0 4.0 4.0 3.9 4.0 3.8 3.8 3.9 3.8 3.8

-

3.9 3.z 0.1

2.9 3.0 3.1 2.7 2.8 3.1 3.1 3.4 3.0 3.2 3.1 3.1 3.0 2.6 2.6 2.6

3.4 I 3.0 z.k 0.1

B

3.0 3.0 3.1 3.0 3.3 3.1

Y

1.6 2 .o 1.3 2.2

1.1 1.8

0.94 1 .o 1.1 1 .l 1 .a 1.2 1.2 1.3 1.4 1.6 1.4 1.6 1.1

1.3 1.5 1.3 1.5

1.25 f 0.1

0.64 0.66 0.71 0.77

* In Verona1 buffer, pH 8.6, at 0’. The bold-faced values refer to the MM pro- tein.

t Average value and standard deviation of the mobilities of components of thir- teen normal human sera, taken from Cooper et al. (16).

solid rectangles of the histogram of Fig. 2. Except for the work of Rundles et al. (4) undertaken concurrently with this investigation, mobility data are meager for multiple myeloma proteins in the buffer at pH 8.6 now in common use for human serum.6 However, all the available data have

6 Previous electrophoretic study of multiple myeloma sera either has been re-

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F. W. PUTNAM AND B. UDIN 731

TABLE II

Electrophoretic Distribution of Proteins of Multiple Myeloma Sera*

I I Globulins - Case No.

cc1 Cal

I I per cent per cent )W cent

1 4.3 0.5 2 2.3 0.3 3 1.8 0.3 5a 2.8 0.3 5b 2.6 0.3 5c 2.9 0.3 5d 2.7 0.3 5e 2.6 0.2 6 1.7 0.3 7 4.0 0.4 8.3 2.9 0.4 8b 2.4 0.2 9 2.8 0.6

10 5.0 0.4 lla 2.4 0.4 llb 1.9 0.4 12 2.1 0.6 13 3.3 0.6 14 2.2 0.7 15a 3.3 0.4 15b 2.1 0.4 16 3.2 0.4 17 3.1 0.2 18 3.0 0.4 19 2.2 0.2 20 3.2 0.4 21 4.2 0.6 22 3.3 0.4 23 4.4 0.4 24 3.1 0.7 25 3.0 0.7

i’J;;~i 14.3 f 0.2625l 0.196 f 0.

0.8 0.3 1.0 0.5 0.8 0.4 0.2 0.2 0.4 0.8 1.1 0.3 0.8 1.0 0.8 0.5 0.5 0.6 0.9 0.8 0.7 0.8 1.5 0.8 0.5

1.0 0.5 1.0 0.9 0.3 0.7 0.9 0.6 0.6 0.4 0.8 0.5 0.7 1.3 0.9 0.6 0.6 0.6 1.1 0.8 0.8 1.3 4.6 4.9 8.6

‘3 1.0 1.2 1.2 1.3 1.4

! .t

ifi .

i . !

L .I

-

1.0 0.2 6.6 7 .l .a .7 9.6 9.6 9.8 3.6 6.8 8.3 6.8 2.3 3.3 4.6

LO .o 6.9 0.2

0.3 0.1 0.4 0.6 0.4 0.6 0.6 1.0 0.8 0.3

ger cent

1.1 f 0.:

-7

i --

1, 4 tt ;

.3 1, ,l 1 .6

1 1 1 1 1 1

.9 ;: P p $4

--

z mo.9. :&

4.9 2.6 6.0 1.1 51 1.6 7.0 2 3.6 3.4 2.7 9.3 1.9 3t 1.8 0.6 0.0 7.9 18 8.0 3.7 1.1 8.0 0.4 6 9.3 9.9 9.5 9.6 1.9 .1.7 7.3 6.3 7.9 7.0 6.8 --

7.2

* In Verona1 buffer, pH 8.6. The bold-faced values refer to the MM protein. t Adrenocorticotropic hormone. 1 Average value and standard deviation of the per cent component distribution

in the ascending patterns of thirteen normal human sera, calculated from data of Cooper et al. (16).

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732 MYELQMA SERUM PROTEINS

been plotted in a frequency distribution diagram in Fig. 2, which includes unpublished data received from Dr. Henry Kunke16 and additional values from our laboratory.4 Although the preponderant grouping of MM pro- teins appears in the mobility range of the components of normal r-globulin, this protein was virtually absent in some sera. Moreover, the molecular and electrical homogeneity of the isolated proteins was striking compared to that of normal r-globulin.

Table II gives the protein distribution of these sera expressed in terms of

FIG. 1. Representative electrophoretic patterns of multiple myeloma sera in Verona1 buffer, pH 8.6. The numbers refer to cases listed in Tables I and II. As- cending boundaries on the left; descending on the right. A schlieren photograph of the starting boundary is superimposed on the patterns.

actual concentration rather than in per cent. When reported in this man- ner, there is much less difference in the albumin, a1 and 0~~ concentrations, although to be sure the albumin is below normal in most instances. The striking difference is in the greatly increased concentration of the abnormal component which usually is a y type protein, but in a few instances is of the p type. Only a few patients (Cases 21 to 24) failed to exhibit this symptom, and these all excreted BJ proteins. Case 25 had an unusual cryoglobulin. No significant information on the rate of accumulation of

stricted to a few cases analyzed at pH 7.4 or 8.0 (1, 2, 17) or consists of extensive surveys which do not list mobilities (3, 8).

6 Personal communication from Dr. Henry Kunkel of the Hospital of The Rocke- feller Institute for Medical Research.

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F. W. PUTNAM AKD B. UDIiX 733

the MM protein was gained by electrophoretic study before and after blood transfusion in Case 5.

Ultracentrifugal Study of Xera-Although significant differences in the sedimentation diagram can be demonstrated for multiple myeloma sera, as shown in Fig. 3, B and C, the interpretation is obscured by the lipoprotein effect described by Gofman et al. (18) and ill&rated for normal human serum in Fig. 3, ,4. Thus the globulins are poorly resolved in Fig. 3, A. They appear as a major sharp peak in Fig. 3, B (Case l), and in Fig. 3, C (Case 17) t,here is a major new component with a higher sedimentation rate. Although t,he latter protein had an electrical mobility similar to that of

0 KUNKEL’

@%ii RUNDLES et al (4)

PUTNAM and SCHWARTZ 4

THIS PAPER

0.5 1.0 1.5 2.0 2.5 3.0 3.5

MOBILITY OF MM PROTEIN

(lG5 cfn2 voli’ sei’)

FIG. 2. Frcquenc~ distribution of the mobilit,)- of MLlhI proteins in 69 sera. The dat,a of the histogram were compiled from the sources indicated in the legend.

p-globulins (Fig. I), it is not a typical lipoprot,ein because of its rapid sedimentation at a density of 1.04 (1 M SaCl). Moreover, P-lipoprotein appeared to be abnormally lo\\- upon flotation analysis of a similar serum in which the /3 type MM component comprised 60 per cent of the total by electrophoretic analysis.? Although the lipoprotein effect is normal or di- minished in this study, T,ewis et al. (19) report considerable variation of the szO of the components of myeloma sera examined in 0.9 per cent iYaC1 in the ultracentrifuge.

Electrophoretic Analysis of Isolated MM Proteins

Heterogeneity Constant-The most characteristic feature of the MM peaks is t.he sharpness indicative of electrical homogeneity. Since t.he patterns

7 Flotation patterns in IiHr-Sac1 solution of density 1.21 were kindl?- made for us by Dr. Richard Jones.

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FIG. 3. Representative ultracentrifuge diagrams of multiple myeloma sera and of the isolated proteins. Sedimentation proceeds to the right with all photographs taken at 16 minute intervals. A, normal serum; B, serum of Case 1; C, serum of Case 17; D, electrophoretically isolated p type globulin from Case 17; E, purified cryoglobulin of Case 5; F, electrophoretically isolated y type globulin of Case 4; and G, normal pooled r-globulin obtained by alcohol fractionation.

734

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F. W. PUTNAM AND B. UDIN 735

might result from an artifact due to high viscosity, etc., it was decided to measure the heterogeneity constant by the reversible boundary-spreading method of Alberty (10, 20). For this, the cryoglobulin of Case 5 was cho- sen because it could be readily separated from the small amount of contam- inating normal globulins by precipitation in the cold. The preparation studied exhibited a single symmetrical peak upon electrophoresis from pH 4 to 10 and also appeared homogeneous in the ultracentrifuge (Fig. 3, E). The diffusion constant was measured in a Verona1 buffer at pH 7.5,0.1 ionic strength, at the isoelectric point of the protein (see below), and the hetero- geneity constant was determined for a 0.6 per cent protein solution in the same buffer with adherence to the precautions prescribed by Alberty (10, 20). The patterns of the ascending and descending limbs were enantio- graphic. The apparent diffusion constant rose only slightly during electro- phoresis, and at the end of the period of reversal of the current returned to essentially the same value as before. For the cryoglobulin, the hetero- geneity constant h = 0.1 X 1O-K cm.2 sec.-l volt-* where h is the standard deviation of the mobility distribution. Under our conditions of measure- ment this figure is in the range which Alberty considers to be the minimal which can be measured. Thus, this cryoglobulin was as homogeneous elec- trically as any protein yet subjected to this criterion. For comparison, h = 0.52 X 10e6 cm.2 sec.-l volt? for yz-globulin in a buffer of ionic strength 0.1 (10). The electrophoretic and sedimentation patterns of a number of other MM proteins were as sharp as for this cryoglobulin. It can be con- cluded that the protein increment in multiple myeloma represents an in- crease in a unique species of globulin, although the species selected may vary from patient to patient.

pH-Mobility Curves-Because of the variation in the mobility of the MM components, the complete pH-mobility curves were determined for two of the proteins and compared with the data of Alberty (10) for human yl-globulin and yz-globulin. The rz-globulin represents the bulk of the r-globulin fraction and is practically identical with the sum of Fractions II-l,2 and II-3 obtained on alcohol fractionation; rl-globulin is a less soluble non-fibrinogen globulin migrating with the mobility of fibrinogen in the Verona1 buffer at pH 8.6 and is sometimes denoted as &,-globulin. These fractionated globulins are more heterogeneous than the MM proteins, e.g. -rz-globulin has an isoelectric point distribution from pH 6.2 to 8.6 with a mean isoelectric point of pH 7.3 (10).

From Fig. 4 it can be seen that the pH-mobility curve for MM protein of Case 5 follows that of rn-globulin, but is shifted about 0.2 pH unit to- wards the alkaline side so that the isoelectric point of the MM protein is about pH 7.5. MM protein of Case 11 migrated considerably more slowly than rz-globulin in the acid region, exhibiting an isoelectric point of about

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736 MYELOMA SERUM PROTEINS

pH 6.6 but closely approximating the normal rz-globulin curve in the alkaline range. Both of the MM globulins exhibited a single component over the range of study, but yl-globulin separates into two components below pH 5.

It is significant that the range in mobility at pH 8.6 of all but one of the MM proteins listed in Table I is within that given in Fig. 4 for the pre- ponderant components of rl-globulin and yz-globulin. This is compatible with the mobility distribution illustrated in Fig. 2 and the view that the MM proteins may represent individual species of a family of proteins re- lated to normal -r-globulins.

t4 O-CASE -5

@-CASE *I I T-

0 2.1 /a,- GLOBULIN

L I I I I I I I

4.0 5.0 6.0 7.0 0.0 9.0

PH

FIG. 4. pH-mobility curves of two MM proteins and of two fractions of normal r-globulin. The dash curves for yl- and rz-globulins are taken from the data of Alberty (10).

Ultracentrifugation of MM Proteins

Sedimentation Constants-The MM proteins encountered in this study fall into two main classes with respect to their sedimentation constants: (1) proteins with a single or a major component with s20 = about 6.6 S, and (2) proteins with a major component of about 9.5 S. The MM com- ponent was isolated electrophoretically from all but three of the twenty sera listed in Table I and was analyzed in the ultracentrifuge. None of the proteins was of the BJ type. In four instances the sedimentation constant was studied as a function of concentration. For calculations, a partial specific volume of 0.739, reported by Oncley et al. (21) for r-globu- lin, was assumed.

6.6 S Component-Fig. 5 shows s20 as a function of concentration for MM proteins having a sedimentation constant of about 6.6 S. A single sediienting boundary was obtained for nine of the thirteen proteins rep-

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F. W. PUTNAM AND 33. UDIN 737

resented; all these give values falling within f0.1 S of the curve drawn. Three of the proteins contained about 5 per cent of a more rapidly sedi- menting component; one had 16 per cent (Case 20). The concentration- dependence of szo was determined for proteins of Cases 5 and 11 because these preparations were electrophoretically as well as ultracentrifugally homogeneous over a wide pH range. For comparison, the results are given for a preparation of human y-globulin obtained by alcohol fractionation,8 which, however, contained only 90 per cent y2-globulin by electrophoretic analysis and sedimented in the ultracentrifuge with a main peak of 85 per cent and a minor peak of 15 per cent (Fig. 3, G). The extrapolated value of ~20 for the proteins represented in Fig. 5 is 6.6 S. Though the mobility

I’ $8.0- o CASE “5

6 @ CASE *I I

i 0 NORMAL ~-GLOBULIN

v 7.0-

5 x2 F n f’4- XI ,

F T3 :.

x x4 xl6 ,0- 8 *6

; 6.0- IO - 13 w x20

B

z

! ! I I I I _

0 .25 .50 .75 1.0 1.25 I.50

PER CENT PROTEIN FIG. 5. Concentration dependence of the sedimentation constant of multiple

myeloma serum proteins and normal r-globulin.

of these proteins varied over a 4-fold range at pH 8.6, all the values except for Case 20 were within the mobility distribution of normal r-globulin. MM protein of Case 20 migrated faster than the /3 anomaly (Fig. 1) ; it>s s20 falls below the curve. It is clear that with this exception the proteins represented in Fig. 5, and covering the majority of our cases, are closely similar to normal y-globulins in molecular properties as well as in electrical behavior.

The difference between the extrapolated value of 6.6 S found in this laboratory for MM proteins and y-globulin and the values of 6.9 to 7.3 S reported by others (1, 21-24) remains unexplained. Although normal hu- man r-globulin always contains a heavier component (21, 24) absent in most of the MM proteins, this does not explain the discrepancy in szo. The other values reported were obtained with air or oil turbine ultracen-

8 Lot No. 49, prepared by the Department of Physical Chemistry, Harvard Medi- cal School,

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738 MYELOMA SERUM PROTEINS

trifuges. Recent studies indicate that the discrepancies may be attributed to the method of maintaining and measuring temperature during the runs It is significant that Rundles et al. (4), employing the Spinco ultracentri- fuge, have reported values of szo for MM proteins in the range of 6.19 to 6.76 S, with an average of 6.46 S.

9.5 S Component-Fig. 6 presents data for the major components of four MM proteins sedimenting more rapidly than y-globulin, and for the minor component of normal r-globulin. The electrophoretic pattern of the MM protein of Case 15 was diffuse but was sharp for the other cases (see Fig. 1). Although Cases 17, 18, and 19 had MM components of similar mobility, they excreted BJ proteins of different mobility. All the proteins repre-

0 CASE’17 0 MINOR COMPONENT, NORMAL

. . . . . . . , . ,.I J

FIG. 6. Concentration dependence of the sedimentation constant

type XkfM proteins.

A CASE-IS I I 0 Cl50 1.00 I.50

PER CENT PROTEIN

of several 13

sented in Fig. 6 contained 20 to 30 per cent of a component with szo = about 6.6 S and all but that in Case 17 also contained minor components with szo = 11 to 12 S.

The occurrence of a major serum globulin component with $20 = about 9 S is rare, having previously been reported only by Kekwick (17). In his case also the protein migrated as a /3-globulin and contained minor com- ponents with s2o = 6.5, 11.3, and 13.3 S. We have recently found one similar case.

Normal y-globulin contains varying amounts of a heavy component

@ Kegeles and Gutter (ll), Miller and Golder (12), and Taylor (13) have all care- fully investigated the origin of the discrepancies in s@@ for serum albumin obtained with the Spinco machine and other ultracentrifuges. For two different lots of Armour bovine serum albumin at 1 per cent concentration, one of us (F. W. P.) ob- tained .a@@ = 4.00 and 4.05 S, which values fall within the range reported by the Ameri- can workers (11-13). We have likewise obtained good agreement with Kegeles’ precise measurements of s@@ for hog heart lactic dehydrogenase (11).

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F. W. PUTNAM AND B. UDIN 739

(820 = 9.5 S) which is absent or present only in negligible proportion in most of the y type MM proteins (compare Fig. 3, E, F, and G). Although a protein of similar szo is predominant in the /3 type MM proteins covered by Fig. 6, electrophoretic analysis of the sera indicated a greatly decreased concentration of y-globulin. A 9 S subcomponent has also been detected in yl-globulin (‘24), and Oncley et al. (21) have suggested that this “heavy” fraction of normal human y-globulin may be a dimer. Although the coin- cidence of the sm versus the concentration curves in Fig. 6 for several of the MM proteins and the minor component of human r-globulin is pro-

TABLE III Physical Constants of Globulins from Multiple Myeloma Sera and from Normal Human

Serum

sm, Swfisberg &a, lo-’ cm.* sec.- Mol. wt. f/f0

Multiple myeloma proteins

Case4 ........................ 6.6* 3.96 158,990 1.51 “ 5 ........................ 6.6* 3.70 167,066 1.59 “ 13. ...................... 6.6* 3.82 165,996 1.55

Normal r-globulins

Oncley et al. (21) 7.2* Bridgman (22). Pedersen (23). /

7.3 3.7 7.1 4.0

Deutsch et al. (24). j 7

156,960t 1.38$ 170,969 1.58 153,699 1 1.51

I

The figures in parentheses represent bibliographic references. * Extrapolated to zero protein concentration. t From szo and f/fo. $ From intrinsic viscosity measurements.

vocative, no relationship between the normal and pathological proteins can be deduced solely on the basis of sedimentation constants.

Diflusion Constants and, Molecular Weight and Shape

6.6 S Component-The results of the diffusion studies on three multiple myeloma proteins are presented in Table III. These proteins exhibited no trace of a second component during ultracentrifugation; that of Case 5 was the electrophoretically homogeneous cryoglobulin. The sedimenta- tion constants for these proteins closely fitted the curve of Fig. 5. The value given is sTo, the sedimentation constant at infinite dilution extra- polated from Fig. 5. Table III also contains the molecular weight calcu- lated from the Svedberg equation. The molecular weight may be slightly

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740 MYELOMA SERUM PROTEINS

in error because s was obtained by extrapolation to infinite dilution, whereas D was measured at 0.G per cent protein concentration. For com- parison the molecular constants of normal human y-globulin are included; the disparity in these values may result from the heterogeneity of this protein.

The diffusion constants for the three MM proteins and for normal y- globulin are the same within the error of the method which has been esti- mated to be about ~3 per cent (25, 26).‘O The molecular weights of the MM proteins recorded in Table III are also well within the range reported for normal human r-globulins. Thus the molecular weights and diien- sions of the predominant class of MM proteins are the same as those of normal human r-globulins.

Rundles et al. (4), who attempted to measure Dzo in ten instances without isolation of the MM proteins, have recorded a greater range in Dm and in molecular weight. However, the majority of their proteins had a molecu- lar weight of between 140,000 and 160,000. Further studies on the purified proteins are needed before significance can be attached to this variation.

Cryoglobul~ns-Cryoglobulins occur in a number of disease states but somewhat more often in multiple myeloma (27-30). In our study of al- most 50 myeloma patients, only two clear cases of cryoglobulinemia have been encountered. Of these, Case 5 was the most pronounced. In both instances the cryoglobulins had the molecular kinetic constants of normal human r-globulins when studied at room temperature, and the cryoglobu- lin in Case 5 was closely related immunologically to r-globulin or a portion thereof .6 Despite this instance of identity, cryoglobulins may differ from normal serum proteins, both electrophoretically and in molecular weight. In four other cases in which szo and Dzo are both available, the molecular weights were 200,000 (31) and 1,080,OOO (23), respectively, for two differ- ent myeloma proteins and 190,000 (28) and 600,000 (32) for proteins from cases of acre purpura and lymphosarcoma, respectively. In three cases (28-30) the mobility in acetate buffer, pH 4.7, was identical with that of protein in Case 5, but the mobility of the latter protein at pH 8.6 in Ver- onal buffer differs from that of a cryoglobulin obtained from a lympho- sarcoma serum (32).

It is surprising that the physical constants of the cryoglobulin are so similar to those of the other globulins in Table III. The frictional ratio of 1.59 calculated for this protein at 20” gives no clue as to the cause of its property of gelation in the cold. Viscosity measurements confirm that the

lo As a check on apparatus constants and experimental method, DUO was deter- mined for an Armour preparation of bovine serum albumin which was electrophoreti- tally homogeneous at pH 8.6. The value obtained was 6.16 X 10m7 cm.2 sec.-l, in good agreement with data in the literature (4, 10).

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F. W. PUTNAM AND B. UDIN 741

molecule is not unusually elongated at room temperature. However, the phenomenon may be explained by an association and dissociation reaction regulated by the temperature and protein concentration such as that de- scribed by Pedersen for a cryoglobulin obtained from multiple myeloma serum (33).

9.5 S Component-The molecular constants for the /? type proteins of the 9.5 S class are incomplete because these proteins were heterogeneous in the ultracentrifuge. Moreover, electrophoretic and ultracentrifugal distribu- tions between p- and r-globulins and light and heavy components did not coincide. In one instance the diffusion constant of an MM protein (Case 17) migrating close to the p peak was determined after electrophoretic resolution and resharpening of the peak. Dzo was about 2.1 X lo-’ cm.2 sec.-l which, together with an szo of 9.5 S, yields an approximate molecular weight of 400,000.

In regard to the possible normal occurrence of similar proteins, it should be noted that four distinct pl-globulins have been identified in normal plasma, that only one is a lipoprotein, and that none have sedimentation constants close to 9 to 10 S (34). The sedimentation and flotation studies of whole sera and of the isolated p type proteins in buffers of varying den- sity rule out the possibility that the 9.5 S component is related to the & lipoprotein.7 The ~20 and the absence of color indicate that it is not the iron-binding globulin. Despite the similarity in ~20 of the p type MM proteins and the minor component of normal human r-globulin, the mo- bility differences appear to exclude a relationship. Accordingly, it must be concluded that the 9.5 S component is a unique protein of uncertain origin found in some cases of multiple myeloma. Its presence seems un- related to the phase of the disease.

Neither in this investigation nor in that of Rundles et al. (4), totaling 75 clinically verified cases, was any evidence found for a major serum protein component with ~20 = 20 S. Yet Pedersen and Waldenstrom (35) have reported five cases of a marked increase in a high molecular weight globu- lin with s20 = about 20 S and molecular weight about 1 million in an un- known disease of the bone marrow.

Interrelationships of Normal and Multiple Myeloma Globulins

All the above observations support the hypothesis that the serum pro- tein-synthesizing system goes awry in multiple myeloma, in each case pro- ducing an abundance of a highly homogeneous representative which is otherwise present only in small proportion in the family of normal globulins. The physicochemical dat,a presented are largely consistent with this view. There is immunochemical evidence that the y type MM proteins are related to normal y-globulins despite the variation in electrical mobility, but that

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742 MYELOMA SERUM PROTEINS

some /3 type MM proteins may be unrelated to normal globulins (36,37). Since these data cannot prove the above hypothesis, sequence analysis of the terminal part of the peptide chains of MM and normal globulins has been begun. Isotopic investigation of the rate of synthesis of myeloma globulins and Bence-Jones proteins is also in progress (9).

The assistance of Peter Stelos in some of the electrophoretic and ultra- centrifugal analyses is gratefully acknowledged.

SUMMARY

Sera from twenty-five patients with multiple myeloma were studied by electrophoresis. The concentration of the proteins was calculated, and the diversity in electrophoretic patterns is described. The anomalous glob- ulins were isolated by electrophoretic separation and were characterized by physicochemical methods. At pH 8.6, the mobility of individual myeloma proteins is distributed over a wide range but with a maximal incidence at the mobility of normal r-globulins. The myeloma globulins fit into two principal classes of molecular weight without respect to sedimentation constants of about 6.6 and 9.5 Svedberg units. Most of the proteins closely resemble normal y-globulin in molecular kinetic constants but are distinguished by a high degree of molecular and electrical homogeneity. The proteins of the other group are ultracentrifugally heterogeneous. It is suggested that the disturbance in protein synthesis in this disease usually results in the massive production of one globulin randomly selected from the family of normal globulins.

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Frank W. Putnam and Bernard UdinSERUM PROTEINS

PHYSICOCHEMICAL STUDY OF PROTEINS IN MULTIPLE MYELOMA: I.

1953, 202:727-743.J. Biol. Chem. 

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