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On the structure of some myeloma proteins
lmmunochemistry. Pergamon Press 1969. Vol.6, pp. 715-721. Printed in Great Britain
ON
THE STRUCTURE OF MYELOMA PROTEINS
SOME
E. AFONSO Laboratory of Health Services, Panjim, Goa, India and A. AFFONSO Goa College of Pharmacy, Panjim, Goa, India
(First received 2January 1969; in revisedform 20 March 1969) Abstract-Myeloma proteins are characterised by their electrophoretic homogeneity manifested by a sharp 'myeloma band' seen on pherograms. Extensive analytical work on amino acid sequence of these paraproteins has been reported [1-9]. However, there seems to be no clear basis for differentiation between proteins synthesised by normal plasma cells and those synthesised by abnormal or malignant ones. By using the methods of quantitative immunoelectrophoresis described previously[10-14] and correlating the results with a novel bidimensional analytical procedure, which combines electrophoresis on cellusose acetate followed by chromatography on set plaster of Paris, a good amount of information could be gathered on the papain cleavage products of normal IgG and myeloma proteins. It may be mentioned that the use of set plaster of Paris in chromatography was reported previously[15]. The results reported in the present work show, further, that it is possible to evolve a concept of malignancy of paraproteins in general. MATERIALS AND METHODS Materials (1) Serum. Samples of serum of normal individuals and of patients suffering from myeloma and other malignant diseases were stored at 4°C and used within a week. (2) Papain. For papain cleavage experiments, crystalline papain, in presence of cysteine and EDTA, was used. (3) Difco's bacto agar was used to prepare gel plates for immunoelectrophoresis. (4) Horse anti-human serum (Batch No. PHO-13-P2) obtained f r o m Netherlands Red Cross, was used for i m m u n o work. (5) Cellulose acetate. Special grade of cellulose acetate strips for electrophoresis was obtained from Messrs. Camag, Muttenz, Switzerland. T h e acetate strips are spongy in nature and have a large sample-holding capacity. (6) Plaster of Paris made by E. Merck, Darmstadt, W. G e r m a n y (Batch Nos. 2162, 162243) was used to prepare thin layer plates of set plaster for the c h r o m a t o g r a p h y work.
Methods (1) Immunoelectrophoresis. T h e principle of quantitative immunoelectrophoresis described previously[10-14] was used. Essentials of the m e t h o d are given below. Agar gel plates 9 cm x 11 cm were prepared in the usual way. T h e agar was allowed to set for 1 hr in perfectly horizontal position in a h u m i d 715
716
E. AFONSO and A. AFFONSO
chamber. Serum sample wells, 1.5 m m in diameter were bored by a special device described previously[13]. For experiments meant to study individual sera for the shape of precipitation lines, the distance between any two wells for serum sample is 15-20 mm. For experiments meant to establish tile antigenie nature of the fractions of two sera, the wells are situated at a transverse distance of 3-5 ram. By this placement the immunoprecipitation lines of tile patterns produced by each serum overlap on one side and enable one to study ttw antigenic identity of each fraction, quite easily. Electrophoresis was carried out at 6-8 V/cm for 90 min. T h e gel plates were next left in h u m i d chamber for a period of 5 hr. After this preliminary diffusions., 8 p,1 of horse anti-human serum were pipetted per cm 2 of the agar surface and spread by means of a bent glass tube spreader. T h e spreading is done by genIle to-and-fro movements until the anti-serum almost dries on the surface of agar T h e plates were placed in h u m i d chamber for a f u r t h e r period of 12-16 hr. After this period of diffusion the plates were placed in 1 per cent NaC1 solution for 6 hr in o r d e r to wash away excess of protein which was not precipitated by anti-serum. Clear precipitation lines, f o r m i n g the patterns shown in the photographs, were obtained by immersion of the plates in 1 per cent acetic acid solution for 2 min. T h e patterns were p h o t o g r a p h e d with slant illumination. (2) Bi-dimensional analysis using electrophoresisfollowed by chromatography. This m e t h o d of analysis of serum proteins is described in detail. Essentially it iravolves two steps. In the first step, electrophoresis of serum was p e r f o r m e d ill special cellulose strips and in the second step, the protein fractions thus separated were allowed to ascend a thin layer plate of set plaster of Paris where adsorption c h r o m a t o g r a p h y takes place, the solvent being diluted veronalveronal sodium buffer. (a) Electrophoresis on cellulose acetate. Special cellulose acetate papers 16 cm x 4 cm were used in the same way as normal paper strips. T h e volume of serum sample deposited was in the range of 10-30/xl. Due to weak electroendosmosis, the deposition line can be at about 3 cm away from the wick of the cathodic side. (b) Chromatography on set plaster of Paris. Glass plates 13 cm × 16 cm were used as backing for the layer of set plaster of Paris which was cast on them. Polystyrene strips exactly ½ram thick and 2 m m broad were struck with Araldite or any other water-proof cement to the four edges of each glass plate. T h e y served as regulators of the thickness of the layer of set plaster that would be cast on the plate. T h e pH of the plaster used is of great importance because adsorptive properties of the material d e p e n d on pH. Plaster of Paris made by E. Merck was f o u n d to be homogeneous and gave reproducihle results. To cast the plaster layer on the glass plates, plaster of Paris (10 g) were mixed with distilled water (12 ml). Air bubbles were broken with a rubber spatula. T h e mix, when slightly thick, was poured in the middle of the glass plate to form a mound. Immediately, a n o t h e r plain glass plate 20 cm × 30 cm was taken and pressed ~)i~ top so that the plaster was spread unifi)rmly on the bottom glass plate and was sandwiched between the plates in a layer thickness of ½ mm. T h e plaster was allowed to set for 30 min. At the end of the period, excess plaster was trimmed off and the plates immersed in water, when it was easy to separate the bottom plate with the adhering plaster layer anchored by the plastic ridges. T h e plates
On the Structure of some Myeloma Proteins
717
were left to dry in air overnight and used next day or stocked in air tight containers. The dry plaster plates were used for chromatography in horizontal position. Developing solvent was a dilute veronal buffer solution (veronal 0.1 g veronal sodium 0"9 g distilled water 1000.0 ml) contained in a rectangular flat tray. The edge of the cellulose acetate strip after electrophoresis was placed on the edge of the plaster plate, overlapping a few millimitres. A thick glass plate with two rubber bands hold in place, the cellulose acetate strip on one side, and a wad of filter paper on the other side, to form the chromatographic assembly as shown in the diagram of Fig. 1. The solvent front advanced into the plaster and after a run of 4 hr reached the filter paper, soaking it. During this period all the separated fractions were transported to the plaster. The assembly was dismantled and the plaster plate left to dry in air or in oven at 80°C. After drying it was immersed in a staining bath (bromophenol blue 0"02 per cent in water, filtered and acidified by adding 5 ml of glacial acetic acid). The pattern (which for convenience sake will be referred to as 'chromatophoretic pattern') stained instantly. The plate was washed in 1 per cent acetic acid and observed or photographed by transparency whilst moist. w
R
Fig. 1. Diagram of assembly for chromatography. PP set plaster plate. C, cellulose acetate strip after electrophoresis. W, wad of filter paper. R, rubber band. T, tray with solvent. (3) Preparative electrophoresis. Electrophoresis was done on special cellulose acetate thick strips which can take a sample of 0"1 ml/cm of deposition line. Sample was deposited from edge to edge, across the strip, close to the cathodic end. After electrophoresis a thin strip of the edge of the pad is cut by razor blade and stained. The region of IgG, slower than ~1, was located and divided by cutting into ten transverse strips. Each strip was squeezed by pressing with a small vice fitted with plastic jaws. The fluid thus pressed out was collected in glass capillaries. (4) Papain cleavage. Crystalline papain was used with cysteine and EDTA, st) that total protein/papain/cysteine/EDTA ratio was 200/1/0.8/33. Incubation temperature was of 2-6 hr at 37°C.
718
E. AFONSO and A. AFFONSO RESULTS
(1) Immunoelectrophoresis
Results obtained from immunoelectrophoresis of normal serum submitted to papain cleavage and of myeloma sera before and after cleavage, are reported below. Normal serum. Pattern B Fig. 2 shows the pattern of normal serum after cleavage. Precipitation lines corresponding to Fc and Fab fragments of IgG are clearly visible. Both lines are not circular showing that the fragments are not homogeneous in nature. Pattern shown in Fig. 3 was obtained by using less anti-serum than for pattern B Fig. 2. The amount of anti-serum was 4/zl for cm z of area of agar surface. By this method the Fab precipitation line was seen to consist actually of two components of identical shape one of which was much fainter than the other. Myeloma sera. Immunoelectrophoresis of myeloma proteins before and after papain cleavage both in whole sera as well as in isolated fractions, showed a set of results for the different sera analysed. The results could be classified as below: (a) All sera before cleavage showed a nearly circular precipitation line for the myeloma protein (Fig. 4 patterns A and B). The mobility of the precipitation line was variable in the different sera analysed. (b) Six myeloma sera gave, after papain cleavage, a single circular precipitation line (Fig. 5(A)). This line as can be seen (Fig. 5(B)) fuses with the Fc line of normal IgG and has complete identity with it. Although before cleavage, this precipitation line had varying mobility, after papain cleavage its mobility became constant in all the six cases. (c) One myeloma serum showed in addition to the above line, after cleavage, another faint precipitation line which had complete identity with Fab line of normal IgG. The diameter of this line was much smaller than that of the line having Fc identity. One myeloma serum showed two equally clear circular precipitation lines identical with Fab and Fc lines of normal IgG, respectively. (2) 'C hromatop hor esis ' As pointed out earlier, for convenience sake, the process of electrophoresis on cellulose acetate followed by adsorption chromatography on set plaster of Paris is designated as 'Chromatophoresis'. An inspection of the figures mentioned under this heading will show that the process of 'Chromatophoresis' yields a regular pattern which, at first sight, resembles a densitographic scan of a paper strip electrophoresis. But closer inspection will show that the pattern is not so simple and a lot of information can be gathered from it. Results obtained show that chromatophoresis can give useful information on: (a) concentration of protein fraction, (b) intrinsic quality of proteins, (c) electrophoretic homogeneity or heterogeneity of protein fractions. To illustrate these results Fig. 6 shows such patterns of native human serum. It was verified that all fractions get adsorbed to the plaster during development and spread in the direction of solvent flow until all the protein is sort of adsorbed or 'spent'. This gives uniform staining of the adsorbed area. As could
(--)
(A)
(+)
r"
(B)
(C)
Fig. 2. A, normal sexlun befl)re cleavage, B, alter cleavage and C, identity pattern of A and B. Intersection Y shown that Fc and Fab fragments have no c o m m o n antigenic determinants. Branching X shows that both the fragments have c o m m o n determinants with IgG.
FP.
Fig. 3, See text.
(Facingpage 718)
(-)
(+)
(A)
L IS1
(¢ Fig. 4. A and B show two dift~erent myeloma proteins (PP). C identity pattern of A and B. D identity pattern of a normal serum and the serum of pattern A, showing that PP has partial identity with IgG line (branching at X).
l-)
at
q~lO
Fig. 6. Above, chromatophoretic pattern of normal native serum. 1, tryptophan-rich prealbumin, 2, albumin, 3, alpha,A, 4, alpha1 antitrypsin, 5, not identified, 6, haptoglobin, 7, alpha2M, 8, not identified, 9, 10, common peak of transferrin and hemopexin, 11, betalc, 12, IgG and 13, IgM. Below, cellulose acetate pherogram of the same seturn.
t+}
(+)
(-I
(-)
; 5¸¢¸¸i¸¸¸¸:¸¸i¸¸¸ ¸¸ iii!ii ):?
F
• "~~ , ~ (A) (A)
_~_
Ii
Fig. 5. (A) A, myeloma sera after cleavage showing single precipitation line (PPFc). (B) identity pattern of A with normal serum after cleavage showing that P P F c has complete identity with Fc fragment of normal lgG.
(+)
On the Structure of some Myeloma Proteins
719
be expected from this mechanism, the area stained was found to be rigorously proportional to the concentration (Fig. 7). The use of the spent pattern for the quantitative estimation of serum protein fractions will be described elsewhere. Figure 6 illustrates clearly that albumin, for instance, stains more heavily than IgG. Measurements of optical density by photometry at 560 m/~ of the different accidents of the patterns showed that no two clearly separated fractions stain with the same intensity. It seems clear that the intensity of staining or 'color index' is useful in differentiating between types of proteins. Finally, electrophoretically homogeneous fractions get 'spent' with gaussian distribution which after completion of the adsorption chromatography process produces the shape of a sharp and elongated isosceles triangle. By finding the ratio between the height and base of the triangular areas produced by each type of protein, it was possible to establish the inference that, the more homogeneous the fraction, the higher is the value of the ratio. Fractions which are known to be homogeneous, like albumin, transferrin, hemopexin, gave highest ratio.
Chromatophoresis of normal serum Chromatophoretic patterns of IgG of native human serum (Fig. 6) illustrate its heterogeneous nature. Papain cleavage fragments Fab and Fc (Fig. 8) are also heterogeneous, Fc being less heterogeneous than Fab (ratio of height to base being higher for Fc). Sequence analysis and peptide mapping studies reported [1, 16-18] point to the deduction that the highly variable primary structures are mostly in the Fab fragment while the Fc fragment may be expected to have a more constant structure. Chromatophoretic patterns of various sub fractions of IgG after papain cleavage are shown in Fig. 9. The sub fractions of IgG were prepared by using the preparative method described above. It can be seen that there is no high degree of homogeneity of the fragments produced by cleavage (low ratio of height to base). Chromatophoresis of myeloma sera All types of myeloma sera before and after papain cleavage which were used for immunoelectrophoretic analysis were also studied by chromatophoresis. Results show that before cleavage, the patterns show a high degree of homogeneity of the paraprotein as evidenced by the peaked isosceles triangles corresponding to it (Fig. 10). After papain cleavage the following results were observed. (a) In one serum, the single paraprotein peak persisted after cleavage with no mobility change (Fig. 11(A)). (b) In six sera two highly homogeneous cleavage peaks were formed after papain cleavage. One had higher electrophoretic mobility and the other lower electrophoretic mobility than the peak present in original sera (Fig. l l(B)). (c) In one serum three highly homogeneous cleavage products were formed, one faster in mobility and two slower than the peak present in original serum (Fig. ll(C)). Conjugating the data obtained by immunoelectrophoresis and 'chromatophoresis', the following types of myeloma proteins could be distinguished.
IMM--Vol. 6 No. 5-F
720
E. AFONSO and A. AFFONSO
(1) Type 1 produces a single circular immuno-precipitation line of Fc antigenic type, which is not affected by papain. There is no change of electrophoretic mobility after cleavage. Chromatophoresis pattern shows a single homogeneous peak which is unaffected by papain. (2) Type II produces a single circular immuno precipitation line of Fc antigenic type. The electrophoretic mobility is faster after cleavage. Chromatophoresis pattern shows two highly homogenous fragments. One fragment has the Fc antigenic type while the other has no antigenic determinant in common with normal IgG. (3) Type III produces two circular immuno precipitation lines, one of which is of Fc antigenic type and the other of Fab antigenic type. Chromatophoresis pattern shows two highly homogeneous products. (4) Type IV produces two circular immuno precipitation lines, one of Fc antigenic type and the other of Fab antigenic type. The Fab type is weak showing that its concentration is much smaller than the other. Chromatophoresis pattern shows three homogeneous products. One corresponds to the Fc type, other to Fab type while the third has no antigenic determinant in common with normal IgG. Bone marrow examination of the patient having Type III myeloma protein showed that the histological structure of plasma cells was of the mature or normal type. Clinically, the case had a very benign course in contrast to the patients having the other three types of myeloma protein. DISCUSSION AND CONCLUSION The most striking result illustrated by 'chromatophoretic' patterns is: (a) the high degree of homogeneity of the native myeloma proteins and also of their papain cleavage products. (b) Lesser degree of homogeneity of families of normal IgG isolated by preparative electrophoresis and submitted to papain cleavage. Since it has been reported[19-24] that the immunoglobulins from a single cell contain one type of light chain, heavy chain and allotypic determinant, it can reasonably be expected that IgG produced by a single cell is homogeneous. Normal IgG can therefore be considered as a mixture produced by innumerable types of cells each synthesising homogeneous IgG molecules which differ in net charge. Abnormal proliferation of a clone of such cell could give rise to a myeloma like electrophoretic band and chromatophoretic pattern, but it would not mean malignancy as in the case illustrated by Fig. 12. Therefore malignancy cannot be attributed to excessive proliferation of cells of the same type which are capable of synthesising all the units of a homogeneous normal IgG. Typical malignant myeloma sera (Types I, lI and IV) all show the presence of antigenic determinant Fc. In Type I, the molecule is not affected by papain and has no Fab determinants. Type II has one Fc like fragment linked to fragments with no determinants in common with normal IgG. Type IV seems to be necessarily asymmetric because chromatophoresis showed three components, one Fc like, the other Fab like and the third having no determinants in common with normal IgG. Therefore a general concept of malignancy in myelomatosis may be defined as loss of ability of the malignant cell to synthesise normal
Fig, 7. Chromatographic patterns obtained by depositing 15/zl of albumin solutions containing 4, 2 and l gm per cent on a cellulose acetate strip and carrying the plaster chromatography without previou,~ electrophoretic migration.
Fig. 8. Chromatophoretic pattern of normal serum after papain cleavage.
(Facing page 720)
(+)f,~f~tl
I
(-)
Fig. 9. Chromatophoretic patterns of subfractions of IgG after papain cleavage.
:-)
Fig. 10. Above, chromatophoretic pattern of a myeloma serum. PP peak corresponds to the myeloma protein. Below, cellulose acetate pherogram of the same serum.
Fig. ll(A). Chromatophoretic pattern of a myeloma serum after papain treatment. No change of mobility was observed and the myeloma protein had antigenic determinants of the Fc fragment of IgG.
{+)
lea, (-)
Fig. ll(B). Chromatophoretic pattern of a myeloma serum after papain, showing two homogeneous cleavage fragments F1 and F2.
Fig. ll(C). Chromatophoretic pattern of myeloma serum after papain cleavage, showing three homogeneous cleavage fragments F,, F2 and F~. Immunoelectrophoresis showed that FI had complete identity with Fc fragment of normal IgG, F:I with the Fab fragment and F2 no identity with any of the fratments of normal IgG.
C+}
Fig. 12. Chromatophoretic pattern of the serum of an apparently healthy individual with no clinical or radiological evidence of myelomatosis. The highly homogeneous peak M gave rise to equally homogeneous peaks after papain cleavage, with Fc and Fab determinants.
On the Structure of some Myeloma Proteins
721
light chains and the amino terminal half o f the n o r m a l heavy chains, coupled with a variable ability to synthesise new or abnormal c o u n t e r parts o f the molecule. For practical purposes, t h e r e f o r e , a combination o f the p r o p o s e d methods o f i m m u n o e l e c t r o p h o r e s i s and 'Chromatophoresis' should be useful in detecting the characteristics o f malignant transformations o f those cells which are known sites o f IgG synthesis. Investigations are in progress to attempt to cover malignancy in general.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1l. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
REFERENCES Hilschmann N. and Craig L. C., Proc. natn. Acad. Sci. U.S.A. 53, 1403 (1965). Wikler M., Titani K., Shinoda T. and Putnam F., J. biol. Chem. 242, 1668 (1967). Millstein C., Biochem.J. 101,352 (1966). Gray W., Dreyer W.j. and Hood L., Science 155, 465 (1967). Perham R., Apella E. and Potter M., Science 154, 391 (1966). Press E. M., Piggot P.J. and Porter R. R., Biochem.J. 99,356 (1966). Waxdal M. J., Konigsberg W. H. and Edelman G. M., Cold Spring Harb. Symp. quant. Biol. 32 (1967). Lennox E. S. and Cohn M., A. Rev. Biochem. 36, 365 (1967). Cohen S. and Millstein C., Adv. Immunol. 6 (1968). Afonso E., Clinica chim. Acta 10, 114 (1964). Afonso E., Clinica chim. Acta 13, 107 (1966). Afonso E., Clinica chim. Acta 14, 63 (1966). Afonso E., Clinica chim. Acta 14, 567 (1966). Afonso E., Clinica chim. Acta 17, 131 (1967). Affonso A.,J. Chromat. 21,332 (1966). Titani K., Whitley E.,Jr., Avogardo L. and Patnam F. W., Science 149, 1090 (1965). Frangioni B. and Franklin E. C.,J. exp. Med. 122, 1 (1965). Frangioni B., Prelli F. and Franklin E. C., Immunochemistry 4, 95 (1967). Burtin P. and Buffe D., Proc. Soc. exp. Biol. Med. 114, 171 (1963). Mellors R. C. and Korngold L.,J. exp. Med. 118, 387 (1963). Bernier G. M. and CebraJ.J.,J. Immun. 95, 246 (1965). Pernis B., Chiapinno G., Kelus A. S. and Gel P. G. H.,J. exp. Med. 122, 853 (1965). Weiler E., Proc. natn. Acad Sci. U.S.A. 54, 1765 (1965). CebraJ. J., ColbergJ. E. and Dray S.,J. exp. Med. 123, 547 (1966).
ON
THE STRUCTURE OF MYELOMA PROTEINS
SOME
E. AFONSO Laboratory of Health Services, Panjim, Goa, India and A. AFFONSO Goa College of Pharmacy, Panjim, Goa, India
(First received 2January 1969; in revisedform 20 March 1969) Abstract-Myeloma proteins are characterised by their electrophoretic homogeneity manifested by a sharp 'myeloma band' seen on pherograms. Extensive analytical work on amino acid sequence of these paraproteins has been reported [1-9]. However, there seems to be no clear basis for differentiation between proteins synthesised by normal plasma cells and those synthesised by abnormal or malignant ones. By using the methods of quantitative immunoelectrophoresis described previously[10-14] and correlating the results with a novel bidimensional analytical procedure, which combines electrophoresis on cellusose acetate followed by chromatography on set plaster of Paris, a good amount of information could be gathered on the papain cleavage products of normal IgG and myeloma proteins. It may be mentioned that the use of set plaster of Paris in chromatography was reported previously[15]. The results reported in the present work show, further, that it is possible to evolve a concept of malignancy of paraproteins in general. MATERIALS AND METHODS Materials (1) Serum. Samples of serum of normal individuals and of patients suffering from myeloma and other malignant diseases were stored at 4°C and used within a week. (2) Papain. For papain cleavage experiments, crystalline papain, in presence of cysteine and EDTA, was used. (3) Difco's bacto agar was used to prepare gel plates for immunoelectrophoresis. (4) Horse anti-human serum (Batch No. PHO-13-P2) obtained f r o m Netherlands Red Cross, was used for i m m u n o work. (5) Cellulose acetate. Special grade of cellulose acetate strips for electrophoresis was obtained from Messrs. Camag, Muttenz, Switzerland. T h e acetate strips are spongy in nature and have a large sample-holding capacity. (6) Plaster of Paris made by E. Merck, Darmstadt, W. G e r m a n y (Batch Nos. 2162, 162243) was used to prepare thin layer plates of set plaster for the c h r o m a t o g r a p h y work.
Methods (1) Immunoelectrophoresis. T h e principle of quantitative immunoelectrophoresis described previously[10-14] was used. Essentials of the m e t h o d are given below. Agar gel plates 9 cm x 11 cm were prepared in the usual way. T h e agar was allowed to set for 1 hr in perfectly horizontal position in a h u m i d 715
716
E. AFONSO and A. AFFONSO
chamber. Serum sample wells, 1.5 m m in diameter were bored by a special device described previously[13]. For experiments meant to study individual sera for the shape of precipitation lines, the distance between any two wells for serum sample is 15-20 mm. For experiments meant to establish tile antigenie nature of the fractions of two sera, the wells are situated at a transverse distance of 3-5 ram. By this placement the immunoprecipitation lines of tile patterns produced by each serum overlap on one side and enable one to study ttw antigenic identity of each fraction, quite easily. Electrophoresis was carried out at 6-8 V/cm for 90 min. T h e gel plates were next left in h u m i d chamber for a period of 5 hr. After this preliminary diffusions., 8 p,1 of horse anti-human serum were pipetted per cm 2 of the agar surface and spread by means of a bent glass tube spreader. T h e spreading is done by genIle to-and-fro movements until the anti-serum almost dries on the surface of agar T h e plates were placed in h u m i d chamber for a f u r t h e r period of 12-16 hr. After this period of diffusion the plates were placed in 1 per cent NaC1 solution for 6 hr in o r d e r to wash away excess of protein which was not precipitated by anti-serum. Clear precipitation lines, f o r m i n g the patterns shown in the photographs, were obtained by immersion of the plates in 1 per cent acetic acid solution for 2 min. T h e patterns were p h o t o g r a p h e d with slant illumination. (2) Bi-dimensional analysis using electrophoresisfollowed by chromatography. This m e t h o d of analysis of serum proteins is described in detail. Essentially it iravolves two steps. In the first step, electrophoresis of serum was p e r f o r m e d ill special cellulose strips and in the second step, the protein fractions thus separated were allowed to ascend a thin layer plate of set plaster of Paris where adsorption c h r o m a t o g r a p h y takes place, the solvent being diluted veronalveronal sodium buffer. (a) Electrophoresis on cellulose acetate. Special cellulose acetate papers 16 cm x 4 cm were used in the same way as normal paper strips. T h e volume of serum sample deposited was in the range of 10-30/xl. Due to weak electroendosmosis, the deposition line can be at about 3 cm away from the wick of the cathodic side. (b) Chromatography on set plaster of Paris. Glass plates 13 cm × 16 cm were used as backing for the layer of set plaster of Paris which was cast on them. Polystyrene strips exactly ½ram thick and 2 m m broad were struck with Araldite or any other water-proof cement to the four edges of each glass plate. T h e y served as regulators of the thickness of the layer of set plaster that would be cast on the plate. T h e pH of the plaster used is of great importance because adsorptive properties of the material d e p e n d on pH. Plaster of Paris made by E. Merck was f o u n d to be homogeneous and gave reproducihle results. To cast the plaster layer on the glass plates, plaster of Paris (10 g) were mixed with distilled water (12 ml). Air bubbles were broken with a rubber spatula. T h e mix, when slightly thick, was poured in the middle of the glass plate to form a mound. Immediately, a n o t h e r plain glass plate 20 cm × 30 cm was taken and pressed ~)i~ top so that the plaster was spread unifi)rmly on the bottom glass plate and was sandwiched between the plates in a layer thickness of ½ mm. T h e plaster was allowed to set for 30 min. At the end of the period, excess plaster was trimmed off and the plates immersed in water, when it was easy to separate the bottom plate with the adhering plaster layer anchored by the plastic ridges. T h e plates
On the Structure of some Myeloma Proteins
717
were left to dry in air overnight and used next day or stocked in air tight containers. The dry plaster plates were used for chromatography in horizontal position. Developing solvent was a dilute veronal buffer solution (veronal 0.1 g veronal sodium 0"9 g distilled water 1000.0 ml) contained in a rectangular flat tray. The edge of the cellulose acetate strip after electrophoresis was placed on the edge of the plaster plate, overlapping a few millimitres. A thick glass plate with two rubber bands hold in place, the cellulose acetate strip on one side, and a wad of filter paper on the other side, to form the chromatographic assembly as shown in the diagram of Fig. 1. The solvent front advanced into the plaster and after a run of 4 hr reached the filter paper, soaking it. During this period all the separated fractions were transported to the plaster. The assembly was dismantled and the plaster plate left to dry in air or in oven at 80°C. After drying it was immersed in a staining bath (bromophenol blue 0"02 per cent in water, filtered and acidified by adding 5 ml of glacial acetic acid). The pattern (which for convenience sake will be referred to as 'chromatophoretic pattern') stained instantly. The plate was washed in 1 per cent acetic acid and observed or photographed by transparency whilst moist. w
R
Fig. 1. Diagram of assembly for chromatography. PP set plaster plate. C, cellulose acetate strip after electrophoresis. W, wad of filter paper. R, rubber band. T, tray with solvent. (3) Preparative electrophoresis. Electrophoresis was done on special cellulose acetate thick strips which can take a sample of 0"1 ml/cm of deposition line. Sample was deposited from edge to edge, across the strip, close to the cathodic end. After electrophoresis a thin strip of the edge of the pad is cut by razor blade and stained. The region of IgG, slower than ~1, was located and divided by cutting into ten transverse strips. Each strip was squeezed by pressing with a small vice fitted with plastic jaws. The fluid thus pressed out was collected in glass capillaries. (4) Papain cleavage. Crystalline papain was used with cysteine and EDTA, st) that total protein/papain/cysteine/EDTA ratio was 200/1/0.8/33. Incubation temperature was of 2-6 hr at 37°C.
718
E. AFONSO and A. AFFONSO RESULTS
(1) Immunoelectrophoresis
Results obtained from immunoelectrophoresis of normal serum submitted to papain cleavage and of myeloma sera before and after cleavage, are reported below. Normal serum. Pattern B Fig. 2 shows the pattern of normal serum after cleavage. Precipitation lines corresponding to Fc and Fab fragments of IgG are clearly visible. Both lines are not circular showing that the fragments are not homogeneous in nature. Pattern shown in Fig. 3 was obtained by using less anti-serum than for pattern B Fig. 2. The amount of anti-serum was 4/zl for cm z of area of agar surface. By this method the Fab precipitation line was seen to consist actually of two components of identical shape one of which was much fainter than the other. Myeloma sera. Immunoelectrophoresis of myeloma proteins before and after papain cleavage both in whole sera as well as in isolated fractions, showed a set of results for the different sera analysed. The results could be classified as below: (a) All sera before cleavage showed a nearly circular precipitation line for the myeloma protein (Fig. 4 patterns A and B). The mobility of the precipitation line was variable in the different sera analysed. (b) Six myeloma sera gave, after papain cleavage, a single circular precipitation line (Fig. 5(A)). This line as can be seen (Fig. 5(B)) fuses with the Fc line of normal IgG and has complete identity with it. Although before cleavage, this precipitation line had varying mobility, after papain cleavage its mobility became constant in all the six cases. (c) One myeloma serum showed in addition to the above line, after cleavage, another faint precipitation line which had complete identity with Fab line of normal IgG. The diameter of this line was much smaller than that of the line having Fc identity. One myeloma serum showed two equally clear circular precipitation lines identical with Fab and Fc lines of normal IgG, respectively. (2) 'C hromatop hor esis ' As pointed out earlier, for convenience sake, the process of electrophoresis on cellulose acetate followed by adsorption chromatography on set plaster of Paris is designated as 'Chromatophoresis'. An inspection of the figures mentioned under this heading will show that the process of 'Chromatophoresis' yields a regular pattern which, at first sight, resembles a densitographic scan of a paper strip electrophoresis. But closer inspection will show that the pattern is not so simple and a lot of information can be gathered from it. Results obtained show that chromatophoresis can give useful information on: (a) concentration of protein fraction, (b) intrinsic quality of proteins, (c) electrophoretic homogeneity or heterogeneity of protein fractions. To illustrate these results Fig. 6 shows such patterns of native human serum. It was verified that all fractions get adsorbed to the plaster during development and spread in the direction of solvent flow until all the protein is sort of adsorbed or 'spent'. This gives uniform staining of the adsorbed area. As could
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(A)
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(B)
(C)
Fig. 2. A, normal sexlun befl)re cleavage, B, alter cleavage and C, identity pattern of A and B. Intersection Y shown that Fc and Fab fragments have no c o m m o n antigenic determinants. Branching X shows that both the fragments have c o m m o n determinants with IgG.
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Fig. 3, See text.
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(A)
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(¢ Fig. 4. A and B show two dift~erent myeloma proteins (PP). C identity pattern of A and B. D identity pattern of a normal serum and the serum of pattern A, showing that PP has partial identity with IgG line (branching at X).
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at
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Fig. 6. Above, chromatophoretic pattern of normal native serum. 1, tryptophan-rich prealbumin, 2, albumin, 3, alpha,A, 4, alpha1 antitrypsin, 5, not identified, 6, haptoglobin, 7, alpha2M, 8, not identified, 9, 10, common peak of transferrin and hemopexin, 11, betalc, 12, IgG and 13, IgM. Below, cellulose acetate pherogram of the same seturn.
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Fig. 5. (A) A, myeloma sera after cleavage showing single precipitation line (PPFc). (B) identity pattern of A with normal serum after cleavage showing that P P F c has complete identity with Fc fragment of normal lgG.
(+)
On the Structure of some Myeloma Proteins
719
be expected from this mechanism, the area stained was found to be rigorously proportional to the concentration (Fig. 7). The use of the spent pattern for the quantitative estimation of serum protein fractions will be described elsewhere. Figure 6 illustrates clearly that albumin, for instance, stains more heavily than IgG. Measurements of optical density by photometry at 560 m/~ of the different accidents of the patterns showed that no two clearly separated fractions stain with the same intensity. It seems clear that the intensity of staining or 'color index' is useful in differentiating between types of proteins. Finally, electrophoretically homogeneous fractions get 'spent' with gaussian distribution which after completion of the adsorption chromatography process produces the shape of a sharp and elongated isosceles triangle. By finding the ratio between the height and base of the triangular areas produced by each type of protein, it was possible to establish the inference that, the more homogeneous the fraction, the higher is the value of the ratio. Fractions which are known to be homogeneous, like albumin, transferrin, hemopexin, gave highest ratio.
Chromatophoresis of normal serum Chromatophoretic patterns of IgG of native human serum (Fig. 6) illustrate its heterogeneous nature. Papain cleavage fragments Fab and Fc (Fig. 8) are also heterogeneous, Fc being less heterogeneous than Fab (ratio of height to base being higher for Fc). Sequence analysis and peptide mapping studies reported [1, 16-18] point to the deduction that the highly variable primary structures are mostly in the Fab fragment while the Fc fragment may be expected to have a more constant structure. Chromatophoretic patterns of various sub fractions of IgG after papain cleavage are shown in Fig. 9. The sub fractions of IgG were prepared by using the preparative method described above. It can be seen that there is no high degree of homogeneity of the fragments produced by cleavage (low ratio of height to base). Chromatophoresis of myeloma sera All types of myeloma sera before and after papain cleavage which were used for immunoelectrophoretic analysis were also studied by chromatophoresis. Results show that before cleavage, the patterns show a high degree of homogeneity of the paraprotein as evidenced by the peaked isosceles triangles corresponding to it (Fig. 10). After papain cleavage the following results were observed. (a) In one serum, the single paraprotein peak persisted after cleavage with no mobility change (Fig. 11(A)). (b) In six sera two highly homogeneous cleavage peaks were formed after papain cleavage. One had higher electrophoretic mobility and the other lower electrophoretic mobility than the peak present in original sera (Fig. l l(B)). (c) In one serum three highly homogeneous cleavage products were formed, one faster in mobility and two slower than the peak present in original serum (Fig. ll(C)). Conjugating the data obtained by immunoelectrophoresis and 'chromatophoresis', the following types of myeloma proteins could be distinguished.
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E. AFONSO and A. AFFONSO
(1) Type 1 produces a single circular immuno-precipitation line of Fc antigenic type, which is not affected by papain. There is no change of electrophoretic mobility after cleavage. Chromatophoresis pattern shows a single homogeneous peak which is unaffected by papain. (2) Type II produces a single circular immuno precipitation line of Fc antigenic type. The electrophoretic mobility is faster after cleavage. Chromatophoresis pattern shows two highly homogenous fragments. One fragment has the Fc antigenic type while the other has no antigenic determinant in common with normal IgG. (3) Type III produces two circular immuno precipitation lines, one of which is of Fc antigenic type and the other of Fab antigenic type. Chromatophoresis pattern shows two highly homogeneous products. (4) Type IV produces two circular immuno precipitation lines, one of Fc antigenic type and the other of Fab antigenic type. The Fab type is weak showing that its concentration is much smaller than the other. Chromatophoresis pattern shows three homogeneous products. One corresponds to the Fc type, other to Fab type while the third has no antigenic determinant in common with normal IgG. Bone marrow examination of the patient having Type III myeloma protein showed that the histological structure of plasma cells was of the mature or normal type. Clinically, the case had a very benign course in contrast to the patients having the other three types of myeloma protein. DISCUSSION AND CONCLUSION The most striking result illustrated by 'chromatophoretic' patterns is: (a) the high degree of homogeneity of the native myeloma proteins and also of their papain cleavage products. (b) Lesser degree of homogeneity of families of normal IgG isolated by preparative electrophoresis and submitted to papain cleavage. Since it has been reported[19-24] that the immunoglobulins from a single cell contain one type of light chain, heavy chain and allotypic determinant, it can reasonably be expected that IgG produced by a single cell is homogeneous. Normal IgG can therefore be considered as a mixture produced by innumerable types of cells each synthesising homogeneous IgG molecules which differ in net charge. Abnormal proliferation of a clone of such cell could give rise to a myeloma like electrophoretic band and chromatophoretic pattern, but it would not mean malignancy as in the case illustrated by Fig. 12. Therefore malignancy cannot be attributed to excessive proliferation of cells of the same type which are capable of synthesising all the units of a homogeneous normal IgG. Typical malignant myeloma sera (Types I, lI and IV) all show the presence of antigenic determinant Fc. In Type I, the molecule is not affected by papain and has no Fab determinants. Type II has one Fc like fragment linked to fragments with no determinants in common with normal IgG. Type IV seems to be necessarily asymmetric because chromatophoresis showed three components, one Fc like, the other Fab like and the third having no determinants in common with normal IgG. Therefore a general concept of malignancy in myelomatosis may be defined as loss of ability of the malignant cell to synthesise normal
Fig, 7. Chromatographic patterns obtained by depositing 15/zl of albumin solutions containing 4, 2 and l gm per cent on a cellulose acetate strip and carrying the plaster chromatography without previou,~ electrophoretic migration.
Fig. 8. Chromatophoretic pattern of normal serum after papain cleavage.
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I
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Fig. 9. Chromatophoretic patterns of subfractions of IgG after papain cleavage.
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Fig. 10. Above, chromatophoretic pattern of a myeloma serum. PP peak corresponds to the myeloma protein. Below, cellulose acetate pherogram of the same serum.
Fig. ll(A). Chromatophoretic pattern of a myeloma serum after papain treatment. No change of mobility was observed and the myeloma protein had antigenic determinants of the Fc fragment of IgG.
{+)
lea, (-)
Fig. ll(B). Chromatophoretic pattern of a myeloma serum after papain, showing two homogeneous cleavage fragments F1 and F2.
Fig. ll(C). Chromatophoretic pattern of myeloma serum after papain cleavage, showing three homogeneous cleavage fragments F,, F2 and F~. Immunoelectrophoresis showed that FI had complete identity with Fc fragment of normal IgG, F:I with the Fab fragment and F2 no identity with any of the fratments of normal IgG.
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Fig. 12. Chromatophoretic pattern of the serum of an apparently healthy individual with no clinical or radiological evidence of myelomatosis. The highly homogeneous peak M gave rise to equally homogeneous peaks after papain cleavage, with Fc and Fab determinants.
On the Structure of some Myeloma Proteins
721
light chains and the amino terminal half o f the n o r m a l heavy chains, coupled with a variable ability to synthesise new or abnormal c o u n t e r parts o f the molecule. For practical purposes, t h e r e f o r e , a combination o f the p r o p o s e d methods o f i m m u n o e l e c t r o p h o r e s i s and 'Chromatophoresis' should be useful in detecting the characteristics o f malignant transformations o f those cells which are known sites o f IgG synthesis. Investigations are in progress to attempt to cover malignancy in general.
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