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Proteins in multiple myeloma. VI. Cryoglobulins
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 39-49 (1956)
Proteins in Multiple Myeloma. VI. Cryoglobulinsl Frank W. Putnam2 and Aiko Miyake From the Department
of Biochemistry and Argonne Cancer Research Hospital, University of Chicago, Chicago, Illinois
Received May 31, 1956
Cryoglobulins are a group of rare serum globulins that have in common the unique property of precipitating, crystallizing, or gelling upon cooling (1). They are only detectable in a few hyperproteinemic sera and are always associated with some disorder. Though infrequent in multiple myeloma, these abnormal proteins occur most often in the latter disease or in cryoglobulinemia, which is possibly a related disease that exhibits a syndrome of vascular and renal involvement, anemia, and hyperproteinemia. In cryoglobulinemia, some of the chief clinical symptoms result from the physical properties of the abnormal plasma protein, that is, cooling of the extremities produces temperature sensitivity, high blood viscosity, purpura, thromboses, and a strain on the circulatory system, and ultimately may lead to death becauseof the intravascular deposition of the abnormal protein (l-3). There is no uniformity in the sedimentation constant, isoelectric point, solubility, or other physical properties of the few cryoglobulins thus far characterized (3-10). Measurement of the physical constants is complicated by their dependence on temperature, and comparison of the available data on cryoglobulins is difficult becauseeach report deals with a single protein studied under separate conditions. In order to define 1 Aided by research grants of the National Cancer Institute, National Institutes of Health, United States Public Health Service (No. C-1331-C4) and the American Cancer Society. The N-terminal amino acid analyses were initiated by the senior author while on leave at the Department of Biochemistry at the University of Cambridge, and the preliminary data were presented at a seminar in the Carlsberg Laboratorium, Copenhagen in August, 1953. 2 Present address: Department of Biochemistry, College of Medicine, The J. Hillis Miller Health Center, University of Florida, Gainesville, Florida. 39
40
FRANK W. PUTNAM AND AIKO MIYAKE
more clearly the relationship of cryoglobulins to normal plasma components and to each other, eight highly purified specimens have been characterized both by physicochemical methods and by N-terminal amino acid analysis. All of the cryoglobulins differed from normal serum proteins in two or more of the following properties: crystal form, solubility, isoelectric point, electrophoretic mobility, sedimentation constant, ultracentrifugal or electrophoretic homogeneity, and N-terminal amino acid residues. The same properties differentiated all but two of the cryoglobulins from each other. It appears unlikely that at least seven different cryoglobulins occur naturally even in minute amount in normal serum. Thus, we have suggested that the cryoglobulins thus far investigated are unnatural proteins and that each may be individually specific (3). EXPERIMENTAL
Source of the Cryoglolndins Purified cryoglobulins (designated Ag, Th, etc.) or sera containing such proteins were obtained from various sources.8 The serum concentration of the cryoglobulins ranged from 3 g.% to as high as 12.7 g.%, the latter being about four times the usual total globulin concentration. Percentagewise, the distribution was from about 40 to 30% of the total serum protein. The normal globulins were diminished in both an absolute and a relative sense, and in one case (WK) the cryoglobulin comprised 98% of the total globulin. No attempt will be made to compare the electrophoretic serum patterns, for in a number of the cases the analysis could not be performed in the standard pH 8.6 Verona1 buffer because of the insolubility of the cryoglobulin at lo despite dilution of the aerum. a Sera containing cryoglobulins (designated Th, Ag, etc.) were kindly supplied by the following physicians: Dr. Charles B. Huggins, Ben May Laboratory for Cancer Research, University of Chicago (Th) ; Dr. Steven A. Schwartz, Hektoen Institute for Medical Research, Cook County Hospital, Chicago, 111.(Ag and Mi) ; Dr. Elliott F. Osserman, Dept. of Medicine, College of Physicians and Surgeons, Columbia University, New York City (Se); and Dr. F. W. Gunz, Pathology Department, Christchurch Hospital, Christchurch, New Zealand (Gu). Dr. Jan Waldenstrem, University of Lund, Lund, Sweden, supplied a cryoglobulinemia serum not fully reported on herein (Me). Purified cryoglobulins previously described in the literature (2, 9) were obtained in an amount of about 100 mg. each from Dr. David Barr and Miss Ella Russ, Dept. of Medicine, Cornell University Medical College (R and I), and from Dr. A. S. McFarlane, National Institute for Medical Research, Mill Hill, London, England (WK). A diagnosis of multiple myeloma was given in all cases except WK which resembled Waldenstr$m’s “macroglobulinemia” (10) and Gu for which a diagnosis has not been established although the complaint has persisted for some years.
PlZOTEINS
IN
MULTIPLE
MYELOMA.
41
VI
PuriJica tim The cryoglohulins could not be purified by a uniform procedure because their solubility and ionic properties varied considerably. Upon cooling, several specimens such as Th, Se, and Me formed gels, ot,hers such as Ag and Gu formed amorphous precipitates, and yet others such as Mi and It crystallized spontaneously. Generally, t,he purificat,ion was attempted by a modification of the method of Lerner and Greenberg (4). The serum was diluted with 0.9% IiaCl or with pH 8.6 Verona1 buffer and was cooled to ca. 2” for several days to yield a flocculent precipitate. The precipitate was removed by centrifugation in the cold and was washed with cold buffer or saline, the supernatant fluid and the washings being discarded. The precipitate was dissolved with a buffer or saiine at JO”, and an) insoluble prot,ein was removed by centrifugntion. The process of purification was repeated several times. With some proteins, the propert,y of precipitation on cooling was retained indefinitely; with others, it was lost after a few repetitions. Cryoglobulins Ag and Th were also isolated by precipit,ation with ammonium sulfate at pH. 6.5 at, a final salt concentration of 1.6 111.The mobility and sediment,at,ion constant, of the salt-fractionated protein were identical with the values for the globulin prepared as nhove. Prot,ein Mi crystallized in the serum on standing in the cold for several weeks (Fig. 1). Because only a small sample was available, the crystals were washed in t,he cold with saline and were studied wit,hout further pu-
FIG.
1. Photomicrograph
of cryoglobulin crystals cooling of serum Mi.
that formed
on prolonged
42
FIlANK
u’.
PUTNAM
.4ND AIKO
TABLE Physical Protein
Constants of Human
Crystal
form
MIYAKE
I Plasma
Cryoglobulins” Mobility at 1°C. pH 8.6 pH 4.7
PI
10-a sp. cm./sec./v.
Normal Th Ag Mi R GU Se WK I
ye
7.2 7.5 >6.4
-1.3 -1.1 -1.1 -1.3 Insoluble Insolubls -0.8
Needles Hexagons
3.0 3.0
3.0 3.3
5.5 Insoluble
3.3
-ml,w s
6.6 6.6 6.6 6.75 7.6,” 11 6.0 6.4 7.18,” 28 7.6: 11
a Mobilities and sedimentation constants were determined in 0.1 ionic strength Verona1 buffer pH 8.6 or in 0.1 ionic strength acetate buffer, pH 4.7. ~~0.~~ given in Svedberg units (8) is extrapolated to infinite dilution except for ultracentrifugally heterogeneous proteins. pl signifies the isoelectric point. * Major component. All values so indicated are data of other authors obtained by analysis with turbine-driven ulbracentrifuges inst,ead of the Spinco apparatus. rification. Cryoglobulins R and I had been purified by cold ethanol fractionation by Miss Ella Russ. The cryoglobulin received from Dr. McFarlane had been isolated by salt fractionation. Some of the physical constants of cryoglobulins R, I, WK, Ag, and Th are cited from previous publications or were obtained by personal communication.
Electrophoretic
and Ultracentrifrlgal
Analysis
Electrophoresis was carried out at 1” with the Pearson apparatus or with the Model H Spinco apparatus.4 The area analysis and mobility calculations were per formed as previously described (6). Mobilities corrected to 0” are given for the descending boundary and are expressed in units of 10-S sq. cm./sec./v., designated U. Univalent buffers of 0.1 ionic strength were employed (11). With some globulins insoluble at 1” in either pH 8.6 Verona1 buffer or pH 6.5 cacodylate-NaCl buffer, electrophoresis could be done in pH 4.65 acetate buffer. Mobility data for pH 8.6 and pH 4.7 are given in Table I together with the isoelectric point where the latter value was obtained. Representative electrophoretic diagrams of purified cryoglobulins are reproduced in Fig. 2. The Spinco analytical ultracentrifuge was used for sedimentation velocit) analysis as before (6) except that the rotor was not refrigerated. All sedimentation constants are corrected to the water basis at 20” (~$0, ,) and are expressed in Svedberg units S (Table I). The sedimentation constants of proteins in the range of szO, w = 6.6 S are corrected to infinite dilution by a relationship derived from t,he 4 Some of the ultracentrifugal Gloria Rasch.
and electrophoretic
analyses
were done b.v Miss
PROTEINS
Ag I
IX
MULTIPLE
MYELOMA.
VI
43
r* -
Th
1 Gu
,,
L < -
FIG. 2. Electrophoretic diagrams of purified cryoglohulins Ag, Th, and Se which migrated at pH 8.6 at a current of 16 ma. for 273, 157, and 223 min., respectively. Gu was analyzed at pH 4.7 with photographs t,aken after 152 min. migration at the same current. Note the sharpness of t,he houndaries except for Gu, which was photographed after diffusion overnight. Although the mobility is low, homogeneity is indicated by the sharpness of the boundaries after electrophoresis for up t,o 4 hr.
slope of t,he sedimentation constant vs. protein concentration curve previously determined at room t,emperature for cryoglohulin Th and for normal y-globulin (6). This curve was independently verified hp Cann for fractions of normal human y-globulin separated by electrophoresis-convection (12). Representative ultracentrifugal patterns for purified cryoglobulins are shown in Fig. 3.
N-Terminal
Amino Acid Analysis
The amino t,erminal residues of the proteins were identified and e&mated by a series of procedures based on the fiuorodinitrobenzene (FDSB) method of Sanger (13). In principle, the dinitrophenyl (DNP) derivatives of the terminal amino acids were obtained hy ether est,raction of the acid hydrolyzate of the DNPprotein. In the first few cases undertaken, the DXP-amino acids were separated and tentatively identified by chromatography at pH 6.5 on buffered silica gel. In reparation was begun on buffered Celite and later work, the chromatographir
44
FR.4NH
\I-.
PUTNSM
AND
?\IIiO
MIYAKE
qgqjqqq $gqqqq seiI!Ll~~~B
diagrams of purified cryoglobulins. All photogr ‘al 3hs FIG. 3. Sedimentation taken at 16.min. intervals at a rotor spectl of 59,780 r .p.m.
completed on buffered silica gel. With silica gel columns, solvent systems w-erc employed that contained various mixtures of methyl ethyl ketone and chloroform (15:85, 3O:iO, G:55 V/V). Ethyl sret.at,e was used with Celite. The quantit.? of I)NP-amino acid was estimated from the light absorption measured in 1% NaHC& at 356 rnp in t,he Beckman spectrophotometer (13). In the calculation of molar ratios, literature values for the recovery of the various DNP-amino acids were assumed to apply, and the protein content of the DNP-cryoglobulins was taken as 75% (14). The identification of the terminal groups was confirmed b? paper chromatography using the tertiary smyl alcohol system of Blackburn and Loather (15) and the t,oluene and Decalin-glacial :tcet,ic acid systems of Biserte and Osteus (16). For verification, the DNP derivatives in some instances w-ere cleaved by hydrolysis in sealed tubes with hnrium hydroxide at 105”, and the free amino acids were identified by paper chromatography (17). The suitability of these procedures for N-terminal amino acid analysis of human serum globulins was established with fractions of normal human r-globulin and with preparations of other types of myeloma globulins for which much larger amounts were available. A subsequent communication will report on the amino end groups of 20 mveloma globulins and of various fractions of normal human globulins.6 Because not more than 100 mg. each of some of the cryoglobulins was availal)le, several of the analyses represent, a single determination. The presence of basic terminal residues such as lysine has not been rigorously excluded in all cases. The procedures employed do not permit a distinction hetween the presence of a dicarboxvlic acid or its amide form in the N-terminal position. Despite 5 Putnam,
F. W., iln~. .I. Jletl.
(in press)
PROTEINS
N-Terminal
IN MULTIPLE
MYELOMA.
45
VI
TABLE II Groups and Immunological Classification of Human Plasma CryoglobulinP N-tnmiml grouts Moks/l6O,OOO b.
Normal ye Th & Mi R Gu Se I WK
I I I I I
1.18
1.76
1.80
0.16
0.11
0.29
2.04 8.06 2.00 9.29 0.15 0.12
0.16 2.64 8.21 2.66 2.77 3.10
(i The major end groups are in italics. Blank spaces indicate the absence of detectable end groups. the reported lability of a terminal peptide bond involving aspartic acid (18), no evidence was found for the cleavage of N-terminal amino acids of either normal or abnormal serum globulins during the reaction with FDNB. This confirms the experience of Thompson with serum r-globulins from various species (19). The results of N-terminal amino acid analysis of eight cryoglobulins and of normal ra-globulins are given in Table II.
Immunological
Studies
Immunological analysis according to the agar diffusion method of Ouchterlony was kindly performed by Dr. Leonhard Korngold of the Sloan-Kettering Institute for Cancer Research. Dr. Korngold (17a) has found that myeloma globulins can be classified according to antigenic structure into three major groupa, all of which cross-react with an antiserum against normal r-globulin. However, the nature of the cross-reaction suggested that the myeloma globulins all lacked antigenic determinants present in the y-globulins of the human serum Fraction II. Of 23 myeloma globulin8 studied by Dr. Korngold (11 specimens from our laboratory), 14 fell in group I, 4 in group II, and 5 in group III. There was little correlation with electrophoretic mobility at pH 8.6, but all the cryoglobulins studied were in Group I (see Table II). The precipitin lines due to yz formed a faint spur at the site of fusion with the precipitin lines due to cryoglobulins Ag and Th. The spur results from a&body against those antigenic determinant8 of the homologous protein which are absent in the cross-reacting proteins, that is to say, the cryoglobulins are antigenically more deficient than ya-globulin. DISCUSSION
Since the first report of the reversible precipitation of plasma protein on cooling (1)) about 20 caseshave been described in some detail (l-lo).
46
FRANK
W.
PUTNAM
AND
AIKO
MIYAKE
Cryoglobulins occur occasionally at a concentration below 25 mg. % in pathological sera, their presence being regarded as a nonspecific indication of a disease process (1). However, cryoglobulinemia at the concentrations reported herein is extremely rare. About a dozen cryoglobulins have previously been characterized by physicochemical methods, each the subject of a separate paper and with study under individual conditions. The object of the present work has been to complete a systematic physical and chemical characterization of a number of cryoglobulins. Although we have reported an isotopic study of the biosynthesis of a cryoglobulin (20), no attempt will be made at this time to speculate either on the origin of these unusual proteins or the causesof their characteristic thermal solubility coefficient. Although the crystallization of normal serum globulins has never been observed, there have been previous reports of the spontaneous crystallization of pathological globulins in the cold similar to our cases Mi and R. Crystallizability may be taken as a qualitative index of homogeneity which is borne out by the electrophoretic and ultracentrifugal diagrams of Figs. 2 and 3. Where electrophoretic analysis of serum was feasible, the diagrams likewise exhibited sharp peaks in the mobility region of the cryoglobulin. In several instances, the patterns suggested the virtual absence of other globulins. Hence, by resort to a purification procedure based on the characteristic property of gelation in the cold, it was readily possible to prepare highly purified cryoglobulins suitable for the physicochemical study and end-group analysis. The physical constants of Table I do not permit classification of the eight cryoglobulins into a single group or into several closely related types. In the caseswhere complete pH-mobility curves were obtained, the isoelectric point varied from pH 5.5 to 7.5. However, all the cryoglobulins appeared to migrate in the general range of -y-globulin, either at pH 4.7 or 8.6 or at both pH’s if the two analyses were feasible. All the specimens migrated with a single sharp boundary upon electrophoresis (see Fig. 2), giving evidence of greater electrical homogeneity than normal human rz-globulin. Five of the proteins sedimented with a single sharp boundary with no indication of heavier components (cf. Fig. 3). Two specimens had a small proportion of the Be-11component regularly seenin normal y-globulins, and one protein was a macroglobulin with a sharp major peak and an SK,of 18s. This diversity of physical properties is in accord with literature data on the molecular constants of
PROTEINS
IN MULTIPLE
MYELOMA.
VI
47
individual cryoglobulins studied by other authors (l-lo). It has its counterpart in the diversity in physical constants of other myeloma globulins that lack the cryoglobulin property (6, 10, 21). Two groups of workers simultaneously reported that normal human r-globulin contains both N-terminal aspartic and glutamic acids (22,23). At that time, evidence was given (22) for the existence of myeloma globulins containing only N-terminal aspartic acid. It was also suggested by one of us (22) that normal human y-globulin consists of at least two components, the one possessingonly N-terminal aspartic acid (an N-aspartylglobulin) and the other having only N-terminal glutamic acid (an N-glutamylglobulin) . Subsequently, five types of myeloma globulins have been described (24), i.e., those containing (a) only N-terminal aspartic acid (five specimens), (b) only N-terminal glutamic acid (five), (c) both N-terminal aspartic and glutamic acids (three), (d) only N-terminal leucine-or a related amino acid-(two), and (e) a heterogeneous group of proteins (six). The cryoglobulins listed in Table II fall into the first three categories just given. Three N-aspartylglobulins have about two moles of iv-terminal aspartic acid and are nearly devoid of N-terminal glutamic acid. Two contain both types of end groups in about the ratio of normal globulin fractions but in twice the normal amount. Three cryoglobulins have three N-terminal glutamyl groups and are essentially devoid of N-terminal aspartic acid. On this basis, the cryoglobulins, though comparable to other myeloma globulins, can be differentiated from the normal pooled human globulins. All the cryoglobulins also lacked the minor quantities of N-terminal serine and unidentified groups that are characteristic of the heterogeneous normal y-globulin. Thus, both in amino end groups and in physical properties, the cryoglobulins are more homogeneous than the customary ethanol fractions of r-globulin from normal pooled human sera. Although no systematic correlation among the physical constants, amino end groups, and antigenic type of the cryoglobulins is apparent, it is clear that all the specimenswith the possible exception of the macroglobulin WK are closely related to normal human r-globulin. Perhaps some of the differences in mobility are ascribable to the carbohydrate content as was the casefor certain myeloma globulins (21). Moreover, a difference in end groups owing to number, amidation, or type might lead to a charge difference of two or three groups per mole which is quite sufficient to produce appreciable mobility changes near the isoelectric
48
FRANK W. PUTNAM AND AIKO MIYAKE
point. None of these possibilities, however, adequately explains the origin of the cold insolubility which is the unique property of these proteins. The present observations demonstrate a far wider variety of amino end groups and of physical properties than can be accounted for readily by the hypothesis that cryoglobulins are normal constituents synthesized selectively and profusely in disease. All the cryoglobulins differed from the normal protein in two or more of the properties given in Tables I and II, and the same properties served to differentiate all the cryoglobulins from one another with the exception of the first two listed.6 It may be argued that at least seven cryoglobulins occur physiologically in trace amounts, each available to be elaborated singly in the disease at the expense of all other globulins. However, at the present stage of investigation, it seems to us more plausible to regard each cryoglobulin as an unnatural protein, the product of an abnormal cell. The hypothesis that cryoglobulins are truly abnormal proteins formed only in disease must receive due consideration in view of the fact that the selfsame plasmacytoma that produces cryoglobulins is involved in the formation of Bence-Jones proteins for which no physiological role has yet been established. The implications of the profuse and selective synthesis of abnormal globulins must be considered in relation to the unsolved problem of normal serum globulin formation. SUMMARY
Eight cryoglobulins (abnormal human plasma globulins that have diminished solubility in the cold) have been investigated by means of the analytical ultracentrifuge, electrophoresis, N-terminal amino acid analysis, and serological classification. Although no systematic correlation among the physical constants, amino end groups, and antigenic type was apparent, all the cryoglobulins but one were closely related to 6 In summary of earlier literature on the physicochemical properties of five cryoglobulins, Abrams, Cohen, and Meyer (5) concluded that these proteins differed widely in molecular size and shape. The electrophoretic properties of the five proteins also appeared to differ but could not be compared directly because of variation in the conditions of analysis. A cryoglobulin described by Smith et al. (21,23) had 1.5 moles each of N-terminal aspartic acid and of N-terminal glutamic acid and thus was dissimilar from the proteins listed in Table II. It would appear as if the number of physically or chemically different cryoglobulins to be encountered in multiple myeloma or related diseases seems limited only by the availability of specimens and the patience of the investigator.
PROTEINS
IN MULTIPLE
MYELOMA.
VI
49
normal human y-globulin. The cryoglobulins differed most notably from normal r-globulin and from each other in the nature of their amino end groups. In the N-terminal position, three proteins had essentially only aspartic acid, three essentially only glutamic acid, and two had both dicarboxylic acids. The hypothesis is suggested that cryoglobulins are truly unnatural proteins formed only in disease. REFERENCES 1. LERNER, A. B., AND WATSON, C. J., Am. J. Med. Sci. 214,410 (1947). 2. MCFARLANE, A. S., DOVEY, A., SLACK, H. G. B., AND PAPASTAMATIS, S. C., J. Pathol. Bacterial. 64, 335 (1952). PUTNAM, F. W., Science 122, 275 (1955). LERNER, A. B., AND GREENBERG, G. R., J. Biol. Chem. 162, 429 (1946). ABRAMS, A., COHEN, P. P., AND MEYER, F. O., J. Biol. Chem. 161, 237 (1949). PUTNAM, F. W., AND UDIN, B., J. Biol. Chem. 292, 727 (1953). PETERMANN, M. L., AND BRAUNSTEINER, H., ATch. Biochem. and Biophys. 63, 491 (1954). 8. MANDEMA, E., VAN DER SCHAAF, P. C., AND HUISMAN, T. H. J., J. Lab. CZin. Med. 46, 261 (1955). 9. BARR, D. P., READER, G. G., AND WHEELER, C. H., Am. 1nnt.Med. 32,6 (1950). 10. WALDENSTR~M, J., Advances in Internal Med. 6, 398 (1952). 11. ALBERTY, R. A., J. Phys. & Colloid Chem. 63, 114 (1949). 12. CANN, J. R., J. Am. Chem. Sot. 76, 4213 (1953). 13. SANGER, F., Biochem. J. 39, 507 (1945). 14. PORTER, R. R., Methods in Med. Research 3, 256 (1950). 15. BLACKBURN, S., AND LOWTHER, A. G., Biochem. J. 46, 126 (1951). 16. BISERTE, G., AND OSTEUX, R., Bull. sot. chim. biol. 33, 50 (1951). 17. LOWTHER, A. G., Nature 167, 767 (1951). 17a. KORNGOLD, L., AND LIPARI, R., Cancer 9, 183 (1956). 18. WEYGAND, F., AND JUNK, R., Naturwissenschajten 36, 433 (1951). 19. THOMPSON, E. 0. P., J. Biol. Chem. 200, 666 (1954). 20. HARDY, S., AND PUTNAM, F. W., J. Biol. Chem. 212, 371 (1956). 21. SMITH, E. L., BROWN, D. M., MCFADDEN, M. L., BUETTNER-JANUSCH, V., AND JAGER, B. V., J. Biol. Chem. 216, 601 (1955). 22. PUTNAM, F. W., J. Am. Chem. Sot. 76, 2785 (1953). 23. MCFADDEN, M. L., AND SMITH, E. L., J. Am. Chem. Sot. 76, 2784 (1953). 24. PUTNAM, F. W., J. Cellular Comp. Physiol. 47, Suppl. 1, p. 17 (1956). 3. 4. 5. 6. 7.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 39-49 (1956)
Proteins in Multiple Myeloma. VI. Cryoglobulinsl Frank W. Putnam2 and Aiko Miyake From the Department
of Biochemistry and Argonne Cancer Research Hospital, University of Chicago, Chicago, Illinois
Received May 31, 1956
Cryoglobulins are a group of rare serum globulins that have in common the unique property of precipitating, crystallizing, or gelling upon cooling (1). They are only detectable in a few hyperproteinemic sera and are always associated with some disorder. Though infrequent in multiple myeloma, these abnormal proteins occur most often in the latter disease or in cryoglobulinemia, which is possibly a related disease that exhibits a syndrome of vascular and renal involvement, anemia, and hyperproteinemia. In cryoglobulinemia, some of the chief clinical symptoms result from the physical properties of the abnormal plasma protein, that is, cooling of the extremities produces temperature sensitivity, high blood viscosity, purpura, thromboses, and a strain on the circulatory system, and ultimately may lead to death becauseof the intravascular deposition of the abnormal protein (l-3). There is no uniformity in the sedimentation constant, isoelectric point, solubility, or other physical properties of the few cryoglobulins thus far characterized (3-10). Measurement of the physical constants is complicated by their dependence on temperature, and comparison of the available data on cryoglobulins is difficult becauseeach report deals with a single protein studied under separate conditions. In order to define 1 Aided by research grants of the National Cancer Institute, National Institutes of Health, United States Public Health Service (No. C-1331-C4) and the American Cancer Society. The N-terminal amino acid analyses were initiated by the senior author while on leave at the Department of Biochemistry at the University of Cambridge, and the preliminary data were presented at a seminar in the Carlsberg Laboratorium, Copenhagen in August, 1953. 2 Present address: Department of Biochemistry, College of Medicine, The J. Hillis Miller Health Center, University of Florida, Gainesville, Florida. 39
40
FRANK W. PUTNAM AND AIKO MIYAKE
more clearly the relationship of cryoglobulins to normal plasma components and to each other, eight highly purified specimens have been characterized both by physicochemical methods and by N-terminal amino acid analysis. All of the cryoglobulins differed from normal serum proteins in two or more of the following properties: crystal form, solubility, isoelectric point, electrophoretic mobility, sedimentation constant, ultracentrifugal or electrophoretic homogeneity, and N-terminal amino acid residues. The same properties differentiated all but two of the cryoglobulins from each other. It appears unlikely that at least seven different cryoglobulins occur naturally even in minute amount in normal serum. Thus, we have suggested that the cryoglobulins thus far investigated are unnatural proteins and that each may be individually specific (3). EXPERIMENTAL
Source of the Cryoglolndins Purified cryoglobulins (designated Ag, Th, etc.) or sera containing such proteins were obtained from various sources.8 The serum concentration of the cryoglobulins ranged from 3 g.% to as high as 12.7 g.%, the latter being about four times the usual total globulin concentration. Percentagewise, the distribution was from about 40 to 30% of the total serum protein. The normal globulins were diminished in both an absolute and a relative sense, and in one case (WK) the cryoglobulin comprised 98% of the total globulin. No attempt will be made to compare the electrophoretic serum patterns, for in a number of the cases the analysis could not be performed in the standard pH 8.6 Verona1 buffer because of the insolubility of the cryoglobulin at lo despite dilution of the aerum. a Sera containing cryoglobulins (designated Th, Ag, etc.) were kindly supplied by the following physicians: Dr. Charles B. Huggins, Ben May Laboratory for Cancer Research, University of Chicago (Th) ; Dr. Steven A. Schwartz, Hektoen Institute for Medical Research, Cook County Hospital, Chicago, 111.(Ag and Mi) ; Dr. Elliott F. Osserman, Dept. of Medicine, College of Physicians and Surgeons, Columbia University, New York City (Se); and Dr. F. W. Gunz, Pathology Department, Christchurch Hospital, Christchurch, New Zealand (Gu). Dr. Jan Waldenstrem, University of Lund, Lund, Sweden, supplied a cryoglobulinemia serum not fully reported on herein (Me). Purified cryoglobulins previously described in the literature (2, 9) were obtained in an amount of about 100 mg. each from Dr. David Barr and Miss Ella Russ, Dept. of Medicine, Cornell University Medical College (R and I), and from Dr. A. S. McFarlane, National Institute for Medical Research, Mill Hill, London, England (WK). A diagnosis of multiple myeloma was given in all cases except WK which resembled Waldenstr$m’s “macroglobulinemia” (10) and Gu for which a diagnosis has not been established although the complaint has persisted for some years.
PlZOTEINS
IN
MULTIPLE
MYELOMA.
41
VI
PuriJica tim The cryoglohulins could not be purified by a uniform procedure because their solubility and ionic properties varied considerably. Upon cooling, several specimens such as Th, Se, and Me formed gels, ot,hers such as Ag and Gu formed amorphous precipitates, and yet others such as Mi and It crystallized spontaneously. Generally, t,he purificat,ion was attempted by a modification of the method of Lerner and Greenberg (4). The serum was diluted with 0.9% IiaCl or with pH 8.6 Verona1 buffer and was cooled to ca. 2” for several days to yield a flocculent precipitate. The precipitate was removed by centrifugation in the cold and was washed with cold buffer or saline, the supernatant fluid and the washings being discarded. The precipitate was dissolved with a buffer or saiine at JO”, and an) insoluble prot,ein was removed by centrifugntion. The process of purification was repeated several times. With some proteins, the propert,y of precipitation on cooling was retained indefinitely; with others, it was lost after a few repetitions. Cryoglobulins Ag and Th were also isolated by precipit,ation with ammonium sulfate at pH. 6.5 at, a final salt concentration of 1.6 111.The mobility and sediment,at,ion constant, of the salt-fractionated protein were identical with the values for the globulin prepared as nhove. Prot,ein Mi crystallized in the serum on standing in the cold for several weeks (Fig. 1). Because only a small sample was available, the crystals were washed in t,he cold with saline and were studied wit,hout further pu-
FIG.
1. Photomicrograph
of cryoglobulin crystals cooling of serum Mi.
that formed
on prolonged
42
FIlANK
u’.
PUTNAM
.4ND AIKO
TABLE Physical Protein
Constants of Human
Crystal
form
MIYAKE
I Plasma
Cryoglobulins” Mobility at 1°C. pH 8.6 pH 4.7
PI
10-a sp. cm./sec./v.
Normal Th Ag Mi R GU Se WK I
ye
7.2 7.5 >6.4
-1.3 -1.1 -1.1 -1.3 Insoluble Insolubls -0.8
Needles Hexagons
3.0 3.0
3.0 3.3
5.5 Insoluble
3.3
-ml,w s
6.6 6.6 6.6 6.75 7.6,” 11 6.0 6.4 7.18,” 28 7.6: 11
a Mobilities and sedimentation constants were determined in 0.1 ionic strength Verona1 buffer pH 8.6 or in 0.1 ionic strength acetate buffer, pH 4.7. ~~0.~~ given in Svedberg units (8) is extrapolated to infinite dilution except for ultracentrifugally heterogeneous proteins. pl signifies the isoelectric point. * Major component. All values so indicated are data of other authors obtained by analysis with turbine-driven ulbracentrifuges inst,ead of the Spinco apparatus. rification. Cryoglobulins R and I had been purified by cold ethanol fractionation by Miss Ella Russ. The cryoglobulin received from Dr. McFarlane had been isolated by salt fractionation. Some of the physical constants of cryoglobulins R, I, WK, Ag, and Th are cited from previous publications or were obtained by personal communication.
Electrophoretic
and Ultracentrifrlgal
Analysis
Electrophoresis was carried out at 1” with the Pearson apparatus or with the Model H Spinco apparatus.4 The area analysis and mobility calculations were per formed as previously described (6). Mobilities corrected to 0” are given for the descending boundary and are expressed in units of 10-S sq. cm./sec./v., designated U. Univalent buffers of 0.1 ionic strength were employed (11). With some globulins insoluble at 1” in either pH 8.6 Verona1 buffer or pH 6.5 cacodylate-NaCl buffer, electrophoresis could be done in pH 4.65 acetate buffer. Mobility data for pH 8.6 and pH 4.7 are given in Table I together with the isoelectric point where the latter value was obtained. Representative electrophoretic diagrams of purified cryoglobulins are reproduced in Fig. 2. The Spinco analytical ultracentrifuge was used for sedimentation velocit) analysis as before (6) except that the rotor was not refrigerated. All sedimentation constants are corrected to the water basis at 20” (~$0, ,) and are expressed in Svedberg units S (Table I). The sedimentation constants of proteins in the range of szO, w = 6.6 S are corrected to infinite dilution by a relationship derived from t,he 4 Some of the ultracentrifugal Gloria Rasch.
and electrophoretic
analyses
were done b.v Miss
PROTEINS
Ag I
IX
MULTIPLE
MYELOMA.
VI
43
r* -
Th
1 Gu
,,
L < -
FIG. 2. Electrophoretic diagrams of purified cryoglohulins Ag, Th, and Se which migrated at pH 8.6 at a current of 16 ma. for 273, 157, and 223 min., respectively. Gu was analyzed at pH 4.7 with photographs t,aken after 152 min. migration at the same current. Note the sharpness of t,he houndaries except for Gu, which was photographed after diffusion overnight. Although the mobility is low, homogeneity is indicated by the sharpness of the boundaries after electrophoresis for up t,o 4 hr.
slope of t,he sedimentation constant vs. protein concentration curve previously determined at room t,emperature for cryoglohulin Th and for normal y-globulin (6). This curve was independently verified hp Cann for fractions of normal human y-globulin separated by electrophoresis-convection (12). Representative ultracentrifugal patterns for purified cryoglobulins are shown in Fig. 3.
N-Terminal
Amino Acid Analysis
The amino t,erminal residues of the proteins were identified and e&mated by a series of procedures based on the fiuorodinitrobenzene (FDSB) method of Sanger (13). In principle, the dinitrophenyl (DNP) derivatives of the terminal amino acids were obtained hy ether est,raction of the acid hydrolyzate of the DNPprotein. In the first few cases undertaken, the DXP-amino acids were separated and tentatively identified by chromatography at pH 6.5 on buffered silica gel. In reparation was begun on buffered Celite and later work, the chromatographir
44
FR.4NH
\I-.
PUTNSM
AND
?\IIiO
MIYAKE
qgqjqqq $gqqqq seiI!Ll~~~B
diagrams of purified cryoglobulins. All photogr ‘al 3hs FIG. 3. Sedimentation taken at 16.min. intervals at a rotor spectl of 59,780 r .p.m.
completed on buffered silica gel. With silica gel columns, solvent systems w-erc employed that contained various mixtures of methyl ethyl ketone and chloroform (15:85, 3O:iO, G:55 V/V). Ethyl sret.at,e was used with Celite. The quantit.? of I)NP-amino acid was estimated from the light absorption measured in 1% NaHC& at 356 rnp in t,he Beckman spectrophotometer (13). In the calculation of molar ratios, literature values for the recovery of the various DNP-amino acids were assumed to apply, and the protein content of the DNP-cryoglobulins was taken as 75% (14). The identification of the terminal groups was confirmed b? paper chromatography using the tertiary smyl alcohol system of Blackburn and Loather (15) and the t,oluene and Decalin-glacial :tcet,ic acid systems of Biserte and Osteus (16). For verification, the DNP derivatives in some instances w-ere cleaved by hydrolysis in sealed tubes with hnrium hydroxide at 105”, and the free amino acids were identified by paper chromatography (17). The suitability of these procedures for N-terminal amino acid analysis of human serum globulins was established with fractions of normal human r-globulin and with preparations of other types of myeloma globulins for which much larger amounts were available. A subsequent communication will report on the amino end groups of 20 mveloma globulins and of various fractions of normal human globulins.6 Because not more than 100 mg. each of some of the cryoglobulins was availal)le, several of the analyses represent, a single determination. The presence of basic terminal residues such as lysine has not been rigorously excluded in all cases. The procedures employed do not permit a distinction hetween the presence of a dicarboxvlic acid or its amide form in the N-terminal position. Despite 5 Putnam,
F. W., iln~. .I. Jletl.
(in press)
PROTEINS
N-Terminal
IN MULTIPLE
MYELOMA.
45
VI
TABLE II Groups and Immunological Classification of Human Plasma CryoglobulinP N-tnmiml grouts Moks/l6O,OOO b.
Normal ye Th & Mi R Gu Se I WK
I I I I I
1.18
1.76
1.80
0.16
0.11
0.29
2.04 8.06 2.00 9.29 0.15 0.12
0.16 2.64 8.21 2.66 2.77 3.10
(i The major end groups are in italics. Blank spaces indicate the absence of detectable end groups. the reported lability of a terminal peptide bond involving aspartic acid (18), no evidence was found for the cleavage of N-terminal amino acids of either normal or abnormal serum globulins during the reaction with FDNB. This confirms the experience of Thompson with serum r-globulins from various species (19). The results of N-terminal amino acid analysis of eight cryoglobulins and of normal ra-globulins are given in Table II.
Immunological
Studies
Immunological analysis according to the agar diffusion method of Ouchterlony was kindly performed by Dr. Leonhard Korngold of the Sloan-Kettering Institute for Cancer Research. Dr. Korngold (17a) has found that myeloma globulins can be classified according to antigenic structure into three major groupa, all of which cross-react with an antiserum against normal r-globulin. However, the nature of the cross-reaction suggested that the myeloma globulins all lacked antigenic determinants present in the y-globulins of the human serum Fraction II. Of 23 myeloma globulin8 studied by Dr. Korngold (11 specimens from our laboratory), 14 fell in group I, 4 in group II, and 5 in group III. There was little correlation with electrophoretic mobility at pH 8.6, but all the cryoglobulins studied were in Group I (see Table II). The precipitin lines due to yz formed a faint spur at the site of fusion with the precipitin lines due to cryoglobulins Ag and Th. The spur results from a&body against those antigenic determinant8 of the homologous protein which are absent in the cross-reacting proteins, that is to say, the cryoglobulins are antigenically more deficient than ya-globulin. DISCUSSION
Since the first report of the reversible precipitation of plasma protein on cooling (1)) about 20 caseshave been described in some detail (l-lo).
46
FRANK
W.
PUTNAM
AND
AIKO
MIYAKE
Cryoglobulins occur occasionally at a concentration below 25 mg. % in pathological sera, their presence being regarded as a nonspecific indication of a disease process (1). However, cryoglobulinemia at the concentrations reported herein is extremely rare. About a dozen cryoglobulins have previously been characterized by physicochemical methods, each the subject of a separate paper and with study under individual conditions. The object of the present work has been to complete a systematic physical and chemical characterization of a number of cryoglobulins. Although we have reported an isotopic study of the biosynthesis of a cryoglobulin (20), no attempt will be made at this time to speculate either on the origin of these unusual proteins or the causesof their characteristic thermal solubility coefficient. Although the crystallization of normal serum globulins has never been observed, there have been previous reports of the spontaneous crystallization of pathological globulins in the cold similar to our cases Mi and R. Crystallizability may be taken as a qualitative index of homogeneity which is borne out by the electrophoretic and ultracentrifugal diagrams of Figs. 2 and 3. Where electrophoretic analysis of serum was feasible, the diagrams likewise exhibited sharp peaks in the mobility region of the cryoglobulin. In several instances, the patterns suggested the virtual absence of other globulins. Hence, by resort to a purification procedure based on the characteristic property of gelation in the cold, it was readily possible to prepare highly purified cryoglobulins suitable for the physicochemical study and end-group analysis. The physical constants of Table I do not permit classification of the eight cryoglobulins into a single group or into several closely related types. In the caseswhere complete pH-mobility curves were obtained, the isoelectric point varied from pH 5.5 to 7.5. However, all the cryoglobulins appeared to migrate in the general range of -y-globulin, either at pH 4.7 or 8.6 or at both pH’s if the two analyses were feasible. All the specimens migrated with a single sharp boundary upon electrophoresis (see Fig. 2), giving evidence of greater electrical homogeneity than normal human rz-globulin. Five of the proteins sedimented with a single sharp boundary with no indication of heavier components (cf. Fig. 3). Two specimens had a small proportion of the Be-11component regularly seenin normal y-globulins, and one protein was a macroglobulin with a sharp major peak and an SK,of 18s. This diversity of physical properties is in accord with literature data on the molecular constants of
PROTEINS
IN MULTIPLE
MYELOMA.
VI
47
individual cryoglobulins studied by other authors (l-lo). It has its counterpart in the diversity in physical constants of other myeloma globulins that lack the cryoglobulin property (6, 10, 21). Two groups of workers simultaneously reported that normal human r-globulin contains both N-terminal aspartic and glutamic acids (22,23). At that time, evidence was given (22) for the existence of myeloma globulins containing only N-terminal aspartic acid. It was also suggested by one of us (22) that normal human y-globulin consists of at least two components, the one possessingonly N-terminal aspartic acid (an N-aspartylglobulin) and the other having only N-terminal glutamic acid (an N-glutamylglobulin) . Subsequently, five types of myeloma globulins have been described (24), i.e., those containing (a) only N-terminal aspartic acid (five specimens), (b) only N-terminal glutamic acid (five), (c) both N-terminal aspartic and glutamic acids (three), (d) only N-terminal leucine-or a related amino acid-(two), and (e) a heterogeneous group of proteins (six). The cryoglobulins listed in Table II fall into the first three categories just given. Three N-aspartylglobulins have about two moles of iv-terminal aspartic acid and are nearly devoid of N-terminal glutamic acid. Two contain both types of end groups in about the ratio of normal globulin fractions but in twice the normal amount. Three cryoglobulins have three N-terminal glutamyl groups and are essentially devoid of N-terminal aspartic acid. On this basis, the cryoglobulins, though comparable to other myeloma globulins, can be differentiated from the normal pooled human globulins. All the cryoglobulins also lacked the minor quantities of N-terminal serine and unidentified groups that are characteristic of the heterogeneous normal y-globulin. Thus, both in amino end groups and in physical properties, the cryoglobulins are more homogeneous than the customary ethanol fractions of r-globulin from normal pooled human sera. Although no systematic correlation among the physical constants, amino end groups, and antigenic type of the cryoglobulins is apparent, it is clear that all the specimenswith the possible exception of the macroglobulin WK are closely related to normal human r-globulin. Perhaps some of the differences in mobility are ascribable to the carbohydrate content as was the casefor certain myeloma globulins (21). Moreover, a difference in end groups owing to number, amidation, or type might lead to a charge difference of two or three groups per mole which is quite sufficient to produce appreciable mobility changes near the isoelectric
48
FRANK W. PUTNAM AND AIKO MIYAKE
point. None of these possibilities, however, adequately explains the origin of the cold insolubility which is the unique property of these proteins. The present observations demonstrate a far wider variety of amino end groups and of physical properties than can be accounted for readily by the hypothesis that cryoglobulins are normal constituents synthesized selectively and profusely in disease. All the cryoglobulins differed from the normal protein in two or more of the properties given in Tables I and II, and the same properties served to differentiate all the cryoglobulins from one another with the exception of the first two listed.6 It may be argued that at least seven cryoglobulins occur physiologically in trace amounts, each available to be elaborated singly in the disease at the expense of all other globulins. However, at the present stage of investigation, it seems to us more plausible to regard each cryoglobulin as an unnatural protein, the product of an abnormal cell. The hypothesis that cryoglobulins are truly abnormal proteins formed only in disease must receive due consideration in view of the fact that the selfsame plasmacytoma that produces cryoglobulins is involved in the formation of Bence-Jones proteins for which no physiological role has yet been established. The implications of the profuse and selective synthesis of abnormal globulins must be considered in relation to the unsolved problem of normal serum globulin formation. SUMMARY
Eight cryoglobulins (abnormal human plasma globulins that have diminished solubility in the cold) have been investigated by means of the analytical ultracentrifuge, electrophoresis, N-terminal amino acid analysis, and serological classification. Although no systematic correlation among the physical constants, amino end groups, and antigenic type was apparent, all the cryoglobulins but one were closely related to 6 In summary of earlier literature on the physicochemical properties of five cryoglobulins, Abrams, Cohen, and Meyer (5) concluded that these proteins differed widely in molecular size and shape. The electrophoretic properties of the five proteins also appeared to differ but could not be compared directly because of variation in the conditions of analysis. A cryoglobulin described by Smith et al. (21,23) had 1.5 moles each of N-terminal aspartic acid and of N-terminal glutamic acid and thus was dissimilar from the proteins listed in Table II. It would appear as if the number of physically or chemically different cryoglobulins to be encountered in multiple myeloma or related diseases seems limited only by the availability of specimens and the patience of the investigator.
PROTEINS
IN MULTIPLE
MYELOMA.
VI
49
normal human y-globulin. The cryoglobulins differed most notably from normal r-globulin and from each other in the nature of their amino end groups. In the N-terminal position, three proteins had essentially only aspartic acid, three essentially only glutamic acid, and two had both dicarboxylic acids. The hypothesis is suggested that cryoglobulins are truly unnatural proteins formed only in disease. REFERENCES 1. LERNER, A. B., AND WATSON, C. J., Am. J. Med. Sci. 214,410 (1947). 2. MCFARLANE, A. S., DOVEY, A., SLACK, H. G. B., AND PAPASTAMATIS, S. C., J. Pathol. Bacterial. 64, 335 (1952). PUTNAM, F. W., Science 122, 275 (1955). LERNER, A. B., AND GREENBERG, G. R., J. Biol. Chem. 162, 429 (1946). ABRAMS, A., COHEN, P. P., AND MEYER, F. O., J. Biol. Chem. 161, 237 (1949). PUTNAM, F. W., AND UDIN, B., J. Biol. Chem. 292, 727 (1953). PETERMANN, M. L., AND BRAUNSTEINER, H., ATch. Biochem. and Biophys. 63, 491 (1954). 8. MANDEMA, E., VAN DER SCHAAF, P. C., AND HUISMAN, T. H. J., J. Lab. CZin. Med. 46, 261 (1955). 9. BARR, D. P., READER, G. G., AND WHEELER, C. H., Am. 1nnt.Med. 32,6 (1950). 10. WALDENSTR~M, J., Advances in Internal Med. 6, 398 (1952). 11. ALBERTY, R. A., J. Phys. & Colloid Chem. 63, 114 (1949). 12. CANN, J. R., J. Am. Chem. Sot. 76, 4213 (1953). 13. SANGER, F., Biochem. J. 39, 507 (1945). 14. PORTER, R. R., Methods in Med. Research 3, 256 (1950). 15. BLACKBURN, S., AND LOWTHER, A. G., Biochem. J. 46, 126 (1951). 16. BISERTE, G., AND OSTEUX, R., Bull. sot. chim. biol. 33, 50 (1951). 17. LOWTHER, A. G., Nature 167, 767 (1951). 17a. KORNGOLD, L., AND LIPARI, R., Cancer 9, 183 (1956). 18. WEYGAND, F., AND JUNK, R., Naturwissenschajten 36, 433 (1951). 19. THOMPSON, E. 0. P., J. Biol. Chem. 200, 666 (1954). 20. HARDY, S., AND PUTNAM, F. W., J. Biol. Chem. 212, 371 (1956). 21. SMITH, E. L., BROWN, D. M., MCFADDEN, M. L., BUETTNER-JANUSCH, V., AND JAGER, B. V., J. Biol. Chem. 216, 601 (1955). 22. PUTNAM, F. W., J. Am. Chem. Sot. 76, 2785 (1953). 23. MCFADDEN, M. L., AND SMITH, E. L., J. Am. Chem. Sot. 76, 2784 (1953). 24. PUTNAM, F. W., J. Cellular Comp. Physiol. 47, Suppl. 1, p. 17 (1956). 3. 4. 5. 6. 7.