Enzymic cleavage of human γM-globulins and their 8S subunits

lmmunochemistry. Pergamon Press 1969. Vol.6, pp. 587-607. Printed in Great Britain

ENZYMIC CLEAVAGE OF HUMAN yM-GLOBULINS AND T H E I R 8S SUBUNITS TSUNEO SUZUKI* The World Health Organization, International Reference Center for Immunoglobulins, Institut de Biochemie, Universit6 de Lausanne, Lausanne, Switzerland

(First received27July 1968; in revisedform 28 October 1968) A b s t r a c t - Two pathological 3' M-globulins and their 8S subunits were subjected to either

pepsin or papain digestion to study the location of an extra L-chain in these proteins [ 1]. Enzymic cleavage of yM-globulins led to the formation of three types of fragments. The heaviest fragments (12S) consisted of four different types of subunits. These include two molecules of 7S protein, one molecule of 4.5S protein, three molecules of a monomer of F'c like fragment and one molecule of a partially degraded L-chain dimer. The intermediate fragments (6S) had a molecular weight of 127,000 and consisted of the complete L-chain and a part of the H-chain. Reduction and alkylation of this fragment produced a monomer subunit(4S). The lightest fragments (3"5S) had a molecular weight near 55,000 and were composed of an L-chain and an Fd like fragment. The 8S subunits were cleaved with pepsin or papain to give rise to three distinct fragments which sedimented as 6S, 4S, and 3.5S, respectively. The 6S fragments resembled the intermediate fragment obtained from yM-globulins in physico-chemical and immunological properties. The 4S fragments were indistinguishable from the reduced-alkylated products of the 6S fragments in all of the characteristics studied and were considered to be a monomer of the 6S fragments. Monomer-dimer relationship between the 4S and the 6S fragments was directly indicated by molecular weight data and was supported by the results ofhexose analyses and immunological studies. The 3.5S fragments were found to be analogous to the lightest fragments obtained from y M-globulins. Findings were discussed from the viewpoint of yM-globulin structure of two heavy and three light chains. INTRODUCTION Structural studies o f y M - g l o b u l i n s a n d their subunits are o f particular i m p o r t a n c e for the elucidation o f the biological role o f such globulins in i m m u nity a n d in certain diseases. Miller a n d Metzger [2, 3] h a v e shown that a pathological y M - g l o b u l i n consisted o f five subunits o f m o l e c u l a r weight n e a r 180,000 a n d that each subunit was c o m p o s e d o f two heavy (H) a n d two light (L) chains. Results o f a m i n o acid analyses a n d p e p t i d e m a p p i n g led P u t n a m et al. [4] to a conclusion a b o u t the c h a i n c o m p o s i t i o n o f h u m a n y M - g l o b u l i n s similar to that p r o p o s e d by Miller a n d Metzger, a l t h o u g h this conclusion was m a d e on the a s s u m p t i o n o f the m o l e c u l a r weight o f 144,000 for y M - g l o b u l i n which was calculated on a basis o f the a s s u m e d m o l e c u l a r weight o f 50,000 a n d 22,000 for an H - a n d an L-chain, respectively. A two H- a n d two L-chain c o m p o s i t i o n for the subunits o f rabbit y M - g l o b u l i n has b e e n r e p o r t e d by L a m m a n d Small[5]. Suzuki a n d Deutsch[1], on the o t h e r h a n d , p r e s e n t e d evidence that the 8S *Present address: Department of Medical Biophysics, University of Toronto, and the Ontario Cancer Institute, Toronto, Canada. 587

588

T. SUZUKI

subunits produced by reduction of several pathological y M-globulins all had molecular weights near 200,000 and consisted of two H- and three L-chains. They further indicated that alkylation of the reduced proteins gave rise to two L-chains and a 'reduced-alkylated' 7S protein of two H- and one L-chain composition. The findings on the chain composition of the 8S subunits led us to the question as to the location and function of the three L-chains in this type of subunit. To solve this problem, it would be helpful if y M-globulins or their 8S subunits could be cleaved to produce homogeneous fragments in high yield, each of which contains one of the three L-chains. It is well known that either papain or pepsin cleaves the particular peptide bonds in the H-chains of yGglobulins to produce two Fab and an Fc, or an F(ab')2 fragments, respectively. Since both monovalent (Fab) and divalent (F(ab')2) fragments produced from yG-globulins consist of the intact L-chain and the H-chain fragment[6, 7], the cleavage of yM-globulins and their 8S subunits with either papain or pepsin may give us the answer to the question raised above. Although limited information on the fragmentation of these proteins with these enzymes [8, 9] and also with the other enzymes[10, 11] is available, all the data reported have dealt with the 'reduced-alkylated' protein. This paper will present the data relative to the process giving rise to various fragments from yM-globulins and their 8S subunits through pepsin or papain digestion, and some of the physico-chemical and immunological properties of the fragments. Results will be discussed from the viewpoint of yM-globulin subunit structure consisting of two H- and three L-chains. MATERIALS AND METHODS

Pathological y M-globulins Two Waldenstr6m's macroglobulinemic sera (LA and EL) of serological type K were studied, yM-globulins were isolated from sera by euglobulin precipitation at low ionic strength and were reprecipitated at least three more times [12]. Solutions of yM-globulins (4 per cent) were made up in 0.15M NaC1 and stored at - 2 0 ° until use. The purified y M-globulins contained approximately 82, 14 and 4 per cent of 19S, 26S and 32S components, respectively. No attempt was made to isolate the 19S component, since it has been well established that the 26S and 32S components are polymers of the 19S components.

Chemical methods Reduction, reaggregation and alkylation. These were carried out according to the method previously described[l]. ~/M-globulin (2 per cent) was routinely reduced for 3 hr with 0.1M ME 1. or with 0.1 M l-cysteine at pH 8 at 25 °. The various fragments obtained by enzymic cleavage were dissociated with 0.5 M ME. Reaggregation of the reduced proteins was carried out by removing the reducing reagents by exhaustive dialysis against Tris-HC1 buffer, 0.02 F/2, pH 8 containing 0.13M NaC1 and 1 × 10-3M NaN31. The alkylation of the reduced pro*Abbreviations used are: ME, 2-mercaptoethanol; IAM, iodoacetoamide: Tris-HCl buffer, 0.15F/2, pH 8, Tris-HCl buffer, 0-02F/2, pH 8.0, containing 0-13M NaCI and 1 × 10-a M NAN3.

Enzymic Cleavage of Human 7 M-Globulins

589

tein was carried out for 10 min at 25 ° with a two-fold excess of 1AM a. The pH during the alkylation was strictly maintained at 8.0 by simultaneous addition of solid tris. Purification of the 8S subunits and of the 'reduced-alkylated' 7S proteins. The details of the methods were described earlier[l, 13]. The 8S subunits were isolated from the reaggregated 7 M-globulins by passage over a column of Sephadex G 200 at pH 8. The purified 8S material will be referred to as the 8S subunit. The 7S materials were purified from the reduced-alkylated 7M-globulins by successive passage over columns of Sephadex G 200 and G 150 at pH 8, removing the aggregated material and slow shoulder components, respectively. The 7S material thus prepared will be denoted as the 'reduced-alkylated' 7S protein. Preparation of H- and L-chains. This was achieved by the method previously described[l]. Since it was found that the original method gave a poor yield of the L-chain protein from the 7 M-globulin (LA), separation of L- and H-chain of this 7M-globulin was carried out at pH 2.5. The other 7M-globulin (EL) was treated as described earlier. Hexose analysis. Hexose contents were analysed according to the Orcinol method [14]. Amino acid analysis. Amino acid analyses were carried out according to the method of Spackman, Stein and Moore [15]. Samples were exhaustively dialysed against double distilled water before hydrolysis. Hydrolysis was performed in 6 N HCI for 20 hr at 110° in thoroughly evacuated pyrex tubes.

Enzymic methods Papain digestion. Twice crystallized papain (Worthington Biochemistry Co.) was activated before use by incubation at 37 ° for 1 hr in the presence of 0.5 M ME and 5 x 10-2 M EDTA. The activated enzyme was added to 7 M-globulins or their 8S subunits in sodium acetate buffer, 0.02F/2, pH 5.0 so that its level was 2 per cent of the weight of the substrate. Unless otherwise stated, digestion was carried out for 3 hr at 37° and stopped by adding IAM at pH 8.0. Peptic digestion. Crystalline pepsin (Worthington Biochemistry Co.) was added to 7M-globulins or their 8S subunits in sodium acetate buffer, 0.1 F/2, pH 5.0 to provide its level of 2 per cent of the weight of the substrates. Unless otherwise stated, digestion was proceeded for 8 hr at 37° and stopped by adjusting pH to 8.0. Fractionation of the digests. The enzymic digestion products were fractionated by passage over a column (4.5 x 90 cm) of Sephadex G 100 which was previously equilibrated against Tris-HCl buffer, 0.15F/2, pH 8.0. Recovery of the materials was determined by the absorbancy at 280 m~.

Physical methods Ultracentrifugal analysis. Ultracentrifugal experiments were performed in a Spinco Model E Ultracentrifuge. Most of the experiments were carried out in 0.15 M NaCI at 20°. Molecular weight determination. Molecular weights of the various fragments were determined mostly in 0.15MNaCI by the approach to sedimentation

590

T. SUZUKI

equilibrium method of Archibald[16]. The partial specific volume of the fragments were assumed as 0-73. Vertical starch gel electrophoresis. This was carried out according to the method of Smithies [17]. Tris-borate-EDTA buffer, sodium formate buffer and glycineHCI buffer were used for the experiments at pH 8.6, 4.0, and 2"5, respectively. Immunological methods. Antisera against the 8S subunit, H-chain and L-chain proteins were prepared in rabbits. All antigens were obtained from LA yMglobulin. Four to five mg of the antigens were given intramuscularly twice to each rabbit with 30 days interval. Antisera were obtained from the marginal ear vein on the tenth day after the second injection of the antigens. Antibodies to H-chain were found to be slightly contaminated with those to L-chain and therefore absorbed with L-chain protein. Immunodiffusion employed Ouchterlony's technique[18] and microimmunoelectrophoresis Scheidegger's [19]. RESULTS

1. Peptic and papain digestion of yM-globulin~ Peptic digestion of yM-globulins. The time required to produce fragments from yM-globulin (LA) was determined by the ultracentrifugal analyses on the samples digested with pepsin for various lengths of time. The results shown in Fig. 1 indicate that the breakdown of yM-globulin into fragments occurred rapidly. A small amount of 19S protein persisted for the first 7 hr of the digestion but was no longer demonstrable after 8 hr. The material digested for 8 hr was seen to sediment as two distinct components with the sedimentation rates of approximately 12S and 3.5S, respectively. The yM-globulins digested for 8 h r could be separated into four major fractions by passing through a Sephadex G 100 column (Fig. 2(A)). Nearly 92 per cent of the digest applied to this column was recovered in four fractions (Pep I, II, III, IV), with the relative yield of 53, 8, 27 and 12 per cent, respectively. The last fraction consisted of dialyzable material and was not studied further. Papain digestion of yM-globulins. Preliminary experiments revealed that the bulk of a y M-globulin (LA) became dialyzable when it was digested for more than 4 hr with papain in the presence of ME and EDTA, but remained nondialyzable if the digestion was stopped within 3 hr. However, a small amount of residual 19S and/or faster sedimenting components were also found in the 3 hr digest. The y M-globulin digested for 3 hr with activated papain was fractionated by gel filtration over a column of Sephadex G 100 at pH 8 (Fig. 2(B)). More than 85 per cent of the digest applied to the column was recovered in four major fractions (Pap I, II, III and IV), with the relative yield of 40, 4, 30 and 26 per cent, respectively. The last fraction was dialyzable and was not studied further.

Physico-chemical properties of thefragments of LA y M-globulin Ultracentrifugal studies. The ultracentrifugal diagrams of the fragments obtained by peptic digestion are given in Fig. 3(A). The sedimentation rate of the main component of Pep I was determined at protein concentration of 10

Fig. 1. Peptic digestion of a yM-globulin (LA). Sedimentation is toward the left. Rotor speeds of 52,640 rev/min were used. The photographs were taken 48 min after reaching maximum speed. The leading peak observed in the photographs of 10 and 30 min digestion studies was 19s.

[Facing page 5901

Fig. 3. Sedimentation diagrams of the peptic fragments of a YM-globulin (LA) separated on Sephadex G 100 as illustrated in Fig. 2A (A) and of reduced-alkylated products (B). Rotor speeds of 52,640 rev/min were used. The photographs of peptic fragments (A) were taken 40 min, 96 min and 126 min for Pep I, Pep II and Pep III, respectively, after reaching maximum speed. The photographs of the reduced-alkylated products (B) were taken 128 min after reaching speed. Sedimentation is toward the left.

Enzymic Cleavage of Human y M-Globulins

591

(A) :L 6 0 E ~j 4 . 0

,-I1~ 2-0

, ~ 50

3O

IV

,

70

90

I10

'

130

Froco ilnnumber

(B)

hi_,

6-0I u ~ --Cd hi

20

IV

,

30

50

70 90 FrGction number

HO

,

130

Fig. 2. Separation of fragments of LA yM-globulin (480 mg in 24 ml) digested with pepsin (A) and papain (B) on a Sephadex G 100 column (4.5 × 95cm) in Tris-HCl buffer, 0.15 F/2, pH 8. Each fraction contained 10 ml. Some points were omitted from the figure for simplication. mg/ml and was found to be 12.0S. The S20.wvalue at infinite dilution of Pep II was 5.9S, whereas that of PepIII was extrapolated to 3.5S (Fig. 4). The ultracentrifugal analyses of the fragments obtained by papain digestion revealed no difference in the sedimentation rates from those obtained by peptic digestion. Molecular weights of the various fragments are listed in Table 1. The value of 127,000 was obtained for Pep II. The molecular weights of Pep III and Pap III were 52,900 and 55,100, respectively. The Mm values for Pep II and for Pap III were in fair agreement with Mb values, respectively, reflecting the molecular homogeneity of these fragments. Molecular weights of Pep I and Pap I were not directly determined because of their apparent heterogeneity. However, an estimated value of 539,000 for Pep I is included in Table 1. This estimation is based on the studies of the subunit structure of this fragment which will be described later. Starch gel electrophoretic studies. The corresponding fragments obtained by peptic and by papan digestion were similar to one another not only in molecular size, but also in electrophoretic properties at pH 8"6 (Fig. 5(A)). Pep II slowly moved toward the anode as multiple bands, while Pep III and Pap III electrophoresed as several distinct bands. The mobility of Pap III was a little greater that that of Pep III.

592

T. SUZUKI 7.0[j--6.911 6S

%

Pl of

50

x

40

f - 3.48S

Peplll w

30 I

50

I

I00 Elcm 2BO m/~.

Fig. 4. Concentration dependencies of the sedimentation rate of yMglobulin fragments (Pep II and Pep III). This figure also includes concentration dependencies of one subunit of Pep I obtained by Sephadex G 100 gel filtration (Fig. 7), and of the 8S Pep I. Table 1. Molecular weights of the fragments of a yM-globulin (LA) obtained by either pepsin or papain digestion and separated as illustrated in Fig. 2. The molecular weights were determined in 0" 15 M NaC1 at 20° by the Archibald Method Molecular weights measurements

Fragments Pep I Pep II Pep III Pap III

Protein concentration S2owx 1013 (mg/ml) rev/min 12"01" 5.92~t 3"48:~ 3.5:~

. 10 14 14

.

M*

Mb*

M*w

. . 539,000§ 7000 126,100 128,000 127,000 8225 49,800 55,900 52,900 8225 54,300 56,400 55,100

*Mm,Mb, Mave:molecular weight at meniscus, at bottom and average respectively. tMeasured at 10 mg/ml. :~Extrapolated values. §Estimated value as described in the text. T h e reduced-alkylated products of Pep I and Pap I were resolved into two major zones in the starch. T h e electrophoretic pattern of the reduced-alkylated Pep II was entirely different f r o m those of either Pep III and Pap III or their reduced-alkylated products. T h e mobilities of the reduced-alkylated products of Pep III and Pap III differed very slightly f r o m those of the u n r e d u c e d Pep III and Pap III, respectively. Immunological studies of thefragments of LA y M-globulin. Figure 6 illustrates the results of Ouchterlony double diffusion experiments. Against antibody to H-

Fig. 5. (A) Starch gel electrophoretograms at pH 8.6 of the fragments of a yM-globulin (LA) obtained by either pepsin or papain digestion and of the reduced-alkylated products of each fragment. (B) Starch gel electrophoretic pattern of the subunits of Pep I separated on Sephadex G 100 after reduction and alkylation as illustrated in Fig. 7. The anodes were toward the right in each figure. Electrophoresis was carried out for 16 hr (A) and for 10 hr (B) at voltage gradient of approximately 10 V/cm.

Facing page 5921

Fig. 6. Ouchterlony experiments with various fragments of a YM-globulin produced by either pepsin or papain digestion. Contents of wells* Experiments

Centre 7 Anti H Anti L Anti H Anti L

1

2

3

4

Pep I

Pep II

Pep III

Pep III?

Pap I

Pap II

Pep III

Pap III

5

8S

Pep III?

6

19s

Pep I

* Antigen concentration was 1 mg/ml. t Pep III fragment obtained by digesting a YM-globulin (LA) in the presence of 0.01 M ME.

Fig. 8. Ultracentrifugal diagrams of fractions separated in Sephadex G 100 as illustrated in Fig. 7. Sedimentation is toward the left. Rotor speed of 52,640 rev/mm was usd for the analysis of P, protein. The other fractions were sedimented at 67,770 rev/min.’ The photographs of P, and Pz proteins were taken 80 min, and of P3 and P,, 98 min after reaching maximum speed.

Fig. 9. Starch gel electrophoretograms at pH 2.5 of Pep III and Pap III, and of their subunit components separated as illustrated in Fig. 10. Electrophoresis was carried out in Glycine-HCl buffer, pH 2.5 for 16 hr at a voltage gradient of approximately 10 V/cm. Cathode is toward the right.

Fig. 11. Sedimentation diagrams of a subunit (PI) of Pep III separated as illustrated in Fig. 10. Sedimentation is toward the left. Rotor speeds of 42,040 rev/min were used. The photographs were taken 160 min (left) and 10 hr (right) after reaching maximum speed.

Enzymic Cleavage of Human y M-Globulins

593

chain, both Pep I and Pap I showed a reaction of identity to the parent 19S molecules. Against the same antibody, Pep II seemed to show a reaction of partial identity to Pep I. It is, however, difficult to say whether the precipitin line between Pep II and the antiserum to H-chain may eventually cross that between Pep I and the antibody to H-chain. Pep III gave a reaction of identity to Pep II. Pap III failed to react with this antibody. Against antibody to L-chain, all the fragments gave a reaction of identity to the 19S protein. In the immunoelectrophoretic studies, Pep I and Pap I gave three precipitin lines with different mobilities against antibody to H-chain. Pep II, Pep III and Pap III gave a faint precipitin line against antibody to H-chain and a strong one against antibody to L-chain.

Subunit structure of thefragments of LA y M-globulin Subunit structure of Pep I and Pap I. As shown in Fig. 3(B), the reduced-alkylated Pep I sedimented as two distinct components with the sedimentation rates of 7S and 3"5S, respectively. As seen in Fig. 7, however, four fractions (P], P2, P3 and P4) were obtained from the reduced-alkylated Pep I by gel filtration over a column of Sephadex G 100. Nearly 99 per cent of the material applied to this ,-P,-. 6.0 ~L E ~: 0 4 . 0 bJ 20

5.0

I00

120

140

Fraction number

Fig. 7. Fractionation of the subunit proteins of Pep I on Sephadex G 100 (4.5 × 95cm) at pH 8. The reduced-alkylated Pep I (350 mg in 12.5 ml) was applied. Each fraction contained 7.5 ml. Some points were omitted from the figure for simplification. column was recovered in four fractions with relative yields of 69, 12, 14 and 5 per cent, respectively. A similar result was obtained also from the reducedalkylated Pap I. Figure 8 shows the ultracentrifugal diagrams of the four subunits obtained from the reduced-alkylated Pep I. The sedimentation rate of the P1 protein at infinite dilution was 7.0S (see Fig. 4). The S20,wvalues of P2, P3 and P4 proteins were determined at protein concentration of 10 mg/ml and were found to be 4.6S, 3"5S and 2"8S, respectively. The molecular weights of these subunit proteins were found to be 178,000 for P1, 70,000 for P2, 56,000 for Ps and 29, 000 for P4 • The result of a starch gel electrophoretic experiment is shown in Fig. 5(B). The P1 protein migrated with a mobility similar to the 'reduceti-alkylated' 7S protein. T h e P2 protein moved as several bands and resembled the reduced-

IMM Vol. 6 No. 4 - F

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T. SUZUKI

alkylated Pep II in the pattern. The electrophoretic pattern of the P3 protein was similar to that of the F'c fragment of yG-globulins [20]. The P4 protein moved as a few spots. The physico-chemical properties of the analogous subunits obtained from the reduced-alkylated Pap I were indistinguishable from those of the subunits of Pep I. Results of subunit structure studies of Pep I and Pap I thus suggest similarities between the P1 protein and the 'reduced-alkylated' 7S protein, between the P2 protein and the reduced-alkylated Pep II, between the P, protein and a dimer of F'c fragment, and between the P4 protein and a portion of L-chains. The P1 protein, however, may not be identical to the 'reduced-alkylated' 7S protein, since the sedimentation rate and molecular weight of the former were significantly higher than those of the latter. If the extinction coefficient of these subunits is assumed to be the same, the yield and the molecular weight data may allow us to conclude that both Pep I and Pap I consisted of two molecules of the PI, one molecule of each of the P2 and P4 and three molecules of a monomer of the P3 proteins. This subunit composition gives a molecular weight near 539,000 for Pep I or Pap I. Subunit structure of Pep H. Upon reduction and alkylation, this fragment was readily converted to somewhat heterogeneous material which sedimented as 4.5S (Fig. 3(B)). As illustrated by the immunological studies (Fig. 6), Pep II consisted of the intact L-chain and a fragment of the H-chain. Subunit structure of Pep III and Pap IlL Reduction and alkylation did not alter the sedimentation behavior of Pep III and Pap III at neutral pH (Fig. 3(B)) and very slightly changed their electrophoretic mobilities at pH 8.6 in starch gel (Fig. 5(A)). As shown in Fig. 9, however, the reduced-alkylated products of Pep III and Pap III were clearly separated into two components during the electrophoresis in starch gel at pH 2"5. These components could be separated by a Sephadex G 100 gel filtration using glycine-HC1 buffer, 0"02F/2, pH 2"5. Figure 10 shows the resulting elution pattern. Over 95 per cent of the material ~ .4p s t . ~

~

0

/

0.5

*

J

20

,

I

40 60 Fraction number

80

I00

Fig. 10. Elution pattern of reduced-alkylated Pep III in Giycine-HCl buffer, pH 2"5, 0-02 F/2 on a column of Sephadex G 100 (4 × 95 cm). The reducedalkylated Pep III (150 mg in 7.5 ml) was exhaustively dialyzed against GlycineHCI buffer, 0-02F/2, pH 2"5 before applying to the column. Each fraction contained 8-5 ml. Some points were removed from the figure for simplification. Similar results were obtained with the reduced-alkylated Pap III.

Enzymic Cleavage of Human y M-Globulins

595

applied to the column was recovered in three fractions (P1, Pz and P3) with the relative yield of 40.5 and 55 per cent, respectively. The ultracentrifugal analyses of the P1 protein revealed a single homogeneous peak in 5 M guanidine-HCl, but showed some heterogeneity in glycineHCI buffer, 0.1 F/2, pH 2"5 (Fig. 11). In Tris-HC1 buffer, 0.15 F/2, pH 8, the heavy aggregation of this protein was observed. The molecular weight of the P1 protein was determined in 5 M guanidine-HCl (pH 6.0) and was found to be 32,000. In a starch gel electrophoretic experiment at pH 2"5 (Fig. 9), the P1 proteins obtained from either Pep III or Pap III moved toward cathode and were free from the other components. The P3 protein obtained from Pep III was separated into two spots. One of them had the same mobility as the L-chain, and the other corresponded in mobility to the P3 protein obtained from Pap III. Both P3 proteins appeared to be slighly contaminated with the corresponding P~ proteins. The P1 proteins derived from either Pep III or Pap III failed to react with antibodies to H-chain and to L-chain. The P3 proteins gave a reaction of identity with the L-chain against antibody to L-chain but did not react with antibody to H-chain. Thus the results of the subunit structure studies of Pep III and Pap III indicate that these fragments consist of a fragment of molecular weight near 32,000 and an L-chain.

2. Peptic and papain digestion of the 8S subunits In the preceding section it has been shown that the limited hydrolytic cleavage of yM-globulins catalysed by either pepsin or papain gave rise to three distinct major fragments and to relatively small amount of dialyzable materials. However, it became difficult to interpret the results in terms of the whole structure of yM-globulins, particularly because of the molecular heterogeneity of the largest fragments (12S). We have previously shown that the 8S subunit produced by the reduction of y M-globulins has a molecular weight near 200,000, while the purified 'reducedalkylated' 7S protein has a molecular weight of 160,000. Since the molecular weight of yM-globulins was found to be near 1,000,000, although a relatively wide range of molecular weights of these globulins has been reported in the literature[2, 25-27], it was thought that the 8S subunit represents the basic monomer unit of yM-globulins[1]. The physico-chemical and amino acid analytical data of the y M-globulins, 8S subunit, 'reduced-alkylated' 7S protein, H-chain and L-chain supported this contention. In addition to these, the yield of H- and L-chain obtained by Sephadex G 100 gel filtration in 5 M guanidineHCI indicated that the 8S subunit is the fundamental monomer unit of y Mglobulins [Fig. 17 and Table 3 of Ref. 1]. Therefore, the 8S subunit was subjected to either peptic or papain digestion in attempting to obtain further information about the structure of ",/M-globulins. Peptic digestion of the 8S subunits. The LA 8S subunit fraction (10 ml of 2 per cent solution) was digested for 8 hr at pH 5.0 at 37° with 4 mg of pepsin. A slight precipitation occured during the digestion. The precipitates were, however, readily dissolved by adjusting the pH of the suspension to 8.0. The digest thus

596

T. SUZUKI

prepared was then applied to a column of Sephadex G 100 which was previously equilibrated against Tris-HC1 buffer, 0.15 F/2, pH 8.0. The resulting elution pattern is shown in Fig. 12(A). Over 96 per cent of the material applied to this column was recovered in four fractions (8S Pep I, 8S Pep II, 8S Pep III and 8S Pep IV) with the relative yield of 54, 4, 16 and 26 per cent, respectively. The small fraction which was eluted in front of the 8S Pep I consisted of undigested

(A)

30

~L2 C E

~iii,4

--0,1 W 2"0

IV

,-11~~ 5O

150 Fraction number

I00

200

o-l~

o 250

(B)

!5~k 1.0 _

L~

~"ll'l°'lll'°

0"5

50

I00 150 Fraction number

200

2,50

Fig. 12. Separation of fragments of LA 8S subunit produced with either pepsin or papain digestion on a column of Sephadex G 100 (4.5 x 95 cm), at pH 8.0. (A) Peptic digested 8S (280 mg in 20 ml); (B) Papain digested 8S (260 mg in 20 ml). Each fraction contained 7 ml. material and represented about 2 per cent of the recovered material. The 8S Pep IV was dialyzable and was not studied in detail. A similar result was obtained with EL 8S subunit. Papain digestion of the 8S subunits. The LA 8S subunit fraction (10 ml of 2 per cent solution) was digested with 4 mg of activated papain under similar condition to that used in the digestion of yM-globulins. The digestion mixture was fractionated by Sephadex G 100 gel filtration. Figure 12B illustrates one of the typical elution patterns obtained. Four main fractions were denoted as the 8S Pap I, 8S Pap II, 8S Pap III and 8S Pap IV, respectively. Their relative yields were 25, 16, 16 and 33 per cent, respectively. The undigested proteins which eluted ahead of the 8S Pap I consisted of approximately 10 per cent of the

Enzymic Cleavage of Human y M-Globulins

597

recovered material. The 8S Pap IV was dialyzable and was not studied further. The EL 8S Preparation gave similar results upon digestion with papain.

Physico-chemicalproperties of thefragments of the LA 8S subunit Uitracentrifugal studies. As seen in Fig. 13, the heaviest fragments (8S Pep I and 8S Pap I) and the lightest fragments (8S Pep III and 8S Pap III) both were fairly homogeneous. The intermediate fragments (8S Pap II), on the other hand, showed heterogeneity as apparent as asymmetry of the sedimenting peak. Figure 14 illustrates the concentration dependencies of S2o,wvalues of the fragments produced by papain digestion. The sedimentation rate at infinite 6"0

5"0

_o

X 4"0 o

o0 3-69S 3.0

I 50

I 10.0 Elcm 280 m,u.

15.0

Fig. 14. Concentration dependencies of the sedimentation rates of various fragments produced by papain digestion of LA 8S subunits. ~0

0 - - 8 S Pap I; ~Q)

®-- 8S Pap II; - - 0

O ~ 8S Pap III.

dilution of the 8S Pap I was 6.0S and agreed well with that of the Pep II obtained from yM-globulins. The S~o,wvalues of the 8S Pap II increased with protein concentration and were extrapolated to 4-0S, thus suggesting dissociation of this fragment to a monomer at low protein concentration. The S20.wvalue of the 8S Pap III was 3.7S at infinite dilution. The sedimentation rates of the fragments produced by peptic digestion were in good agreement with those of the corresponding fragments obtained by papain digestion. The molecular weights of the fragments obtained from LA 8S subunit are listed in Table 2. Values of 133,500 and 130,800 were obtained for the 8S Pep I and 8S Pap I, respectively. These values are in good agreement with that obtained for Pep II derived from yM-globulin. Values of 72,700 and 70,000 were found for the 8S Pep II and 8S Pap II, respectively. These values correspond to that of the P2 (4.5S) protein derived from the reduced-alkylated Pep or Pap I. A value of 55,000 was obtained for the 8S Pep III. Starch gel electrophoretic studies. Marked similarities of the electrophoretic patterns were observed between the heaviest fragments, obtained from the 8S subunit (8S Pep I and 8S Pap I) and Pep II derived from yM-globulin (Fig. 15). The electrophoretic pattern of the 8S Pap II resembled that of the reduced alkylated Pep II. The 8S Pep III electrophoresed in an identical manner to Pep

598

T. SUZUKI Table 2. Molecular weights of fragments of an 8S subunit protein produced by either pepsin or papain digestion and separated as illustrated in Fig. 12(A) and (B). The molecular weights were determined in 0.15M NaCI at 20° by the Archibald Method Molecular weights measurements Fragments 8S Pep I 8S Pap I 8S Pep II 8S Pap II 8S Pep III 8S Pap III

$20~x 10TM 5.95* 6.06* 4.0? 3-99* 3.51~ 3.69*

Protein concentrations§ rev/min Mm 16.8 13.0 4.7 4.9 12.4

Mb

Mave

7026 133,700 133,600 133,500 8225 134,400 127,200 130,800 6995 73,000 72,400 72,700 6995 70,400 71,000 70,700 10,589 54,700 55,300 55,000

*Extrapolated values. tDetermined at protein concentration of 4 mg/ml. SDetermined at protein concentration of 8 mg/ml. 1 em §E28om.

III derived fromTM-globulin. The main components of the 8S Pap III moved a little faster than those of the 8S Pep III. Immunological properties of the various fragments of LA 8S subunit. Against antibody to H-chain, as shown in Fig. 16, a reaction of identity was obtained between the heaviest (8S Pep I and 8S Pap I) and the intermediate (8S Pep II and 8S Pap II) fragments. The faint second precipitin line formed by the 8S Pap I suggests the possible presence of the undigested 8S molecules in this preparation. A partial identity observed between the heaviest fragments and the H-chains indicates that these fragments contain only a part of the H-chain. The lightest fragments (8S Pep III and 8S Pap III) did not react with the antibody to H-chain. Against antibody to L-chain, all fragments but the 8S Pep III and 8S Pap III gave a reaction of identity to L-chain. The precipitin lines of the 8S Pep III and of the 8S Pap III appeared to form a little spur against those of L-chain. Subunit structure of the 8S Pep I and 8S Pap I. Upon reduction and alkylation, 8S Pep I and 8S Pap I both were readily converted to the materials which sedimented as 4.0S and electrophoresed at pH 8.6 in starch gel with the same mobilities as the 8S Pep II and 8S Pap II. These findings suggested that the 8S Pep II and 8S Pap II may be the monomer of the 8S Pep I and 8S Pap I, respectively. The molecular weight data and the immunological findings are consistent with this contention. The hydrolytic cleavage of the 8S subunit with either pepsin or papain thus led to the formation of two physico-chemically and immunologically distinct fragments, the 6S fragments (8S Pep I and 8S Pap I) and the 3.5S fragments (8S Pep III and 8S Pap III). Since both types of fragments were found to contain L-chain, these fragments might be formed by cleavage of the peptic bond(s) in the H-chains. To examine this possibility, the hexose contents in these fragments as well as in their parent molecules were analysed.

Fig. 13. Sedimentation diagrams of fragments of 8s subunits separated as illustrated in Fig. 12. Sedimentation is toward the left. Rotor speeds of 47,660 rev/min were used for the analysis of 8s Pep I and 8s Pep III. Photographs of these fragments were taken 160 min after reaching maximum speed. Rotor speeds of 67,770 rev/min were used for the analysis of 8s Pap I and 8s Pap II. Pictures were taken 80 min after reaching speed. The photograph of 8s Pap III was taken 80 min after reaching maximum rotor speed of 59,780 rev/min. The top pattern was obtained at protein concentration of 8 mg/ml and the bottom 10 mg/ml.

[Facing page 5981

Fig. 15. Starch gel electrophoretic studies LA 8S subunits obtained by either pepsin or and separated as illustrated in Figs. 12(A) electrophoresis was carried out at pH 8-6 for gradient of 10 V/cm. Anode is toward

of fragments of papain digestion and (B). The 12 hr at voltage the right.

Fig. 16. Ouchterlony experiments of the various fragments of LA 8S subunits. Contents of wells* Experiments

Centre 7

1

2

3

Anti Anti Anti Anti

8S 8S 8S 8S

H L H L

8s 8S 8S 8S

H L H L

* Antigen concentration was 1 mg/ml.

4 Pep Pep Pap Pap

I I I I

8S 8S 8S 8S

5 Pep Pep Pap Pap

II II II II

8S 8S 8S 8S

6 Pep Pep Pap Pap

III III III III

H L H L

599

Enzymic Cleavage of Human yM-Globulins

Hexose analyses. Table 3 presents the results o f hexose analyses. Some o f the c o m p a r a b l e data r e p o r t e d in the literature are also included in Table 3. T h e a m o u n t o f hexose f o u n d in LA yM-globulin was 4.8 per cent and a g r e e d well with those f o u n d in a n o t h e r pathological[2] and in n o r m a l yM-globulins[21]. T h e 8S subunit contained the same a m o u n t o f hexose as the p a r e n t molecules, thus indicating that no hexose was released d u r i n g reductive conversion o f yM-globulin to its m o n o m e r . Since no hexose was detected in the L-chain, the Table 3. Hexose contents of a yM-globulin and its subunit proteins. Hexose contents were determined by Orcinol Method H exose contents g/100 g protein Miller Present study Metzger (1966) Proteins yM-globulin 8S subunit H-chain L-chain Pep I Pap II Pep II Pap II Pep III Pap III 8S Pep I 8S Pap I 8S Pep II 8S Pap II 8S Pep III 8S Pap III

Pathological yM 4.8 4-8 7"1 0 4.7 4-8 3.8 ND* 1.8 1.8 3.9 3-8 ND* 3.9 1-8 1.8

Chaplin et al. (1965)

Pathological yM

Normal yM

4.9 4.9

4-8 7"0 0

MB 1-4 MC

MsI 1.4 MsII 0-8

*ND = not determined. theoretical a m o u n t o f hexose in an H-chain should be 7 per cent, when the molecular weight o f the H-chain is taken as 66,000[1]. A value o f 7.1 per cent obtained by Orcinol M e t h o d is in accordance with the prediction. T h e hexose moieties attached to an H-chain, which c o r r e s p o n d to molecules o f molecular weight n e a r 4600, a p p e a r e d to be distributed over at least two different parts o f the H-chain. T h e last contention is based on the findings that the 8S Pep I and 8S Pap I both contained hexose moieties o f molecular weight n e a r 2600, while the 8S Pep III and 8S Pap III had those o f molecular weight n e a r 1100. DISCUSSION Figure 17 summarizes the molecular transitions o f y M - g l o b u l i n s and their 8S subunits into various fragments and their subunits t h r o u g h the enzymic and chemical cleavages described above. T h e enzymic digestion o f y M-globulins

600

T. SUZUKI

FPI(7S) ......... Red. [-P2(4.5S) F r e p x or r a p l~lZ~) ..... ~t'l-k.- tP~(3"5S) 7M-globulins(19S) digestion-Enzymic _| o~.4 P4(2"8S) [-Pep II(6S) ................. A-I~.".... 4S

/

ed. Lpep III or Pap III(3.5Sj-~]]~-

P~(Fd like) [- P~(Lchain)

-8S Pep I or8S Pap I(6S} Red. Alk. Enzymic 8S subunits(8S) --']:]]gest]on -8S Pep II or 8S Pap II(4*S) -8S Pep III or Pap III(3.5S) Fig. 17. Molecular transitions of a M-globulins and their 8S subunits to various fragments and subunits through the enzymic digestion and chemical cleavages. led to the formation of three distinct types of fragments which sedimented as 12S, 6S, and 3.5S, respectively. The 8S subunits were digested to give rise to three distinct fragments which sedimented as 6S, 4S, and 3.5S, respectively. The 12S fragments (Pep I and Pap I) could be stoichiometrically converted to four different types of subunits by reduction and alkylation. The 6S fragments obtained from 7M-globulins (Pep II) and those derived from the 8S subunits (8S Pep I and 8S Pap I) both consisted of L-chain and fragment of H-chain and were readily converted to the 4S materials upon reduction and alkylation. The 3.5S fragments obtained from 7M-globulins (Pep III and Pap III) consisted of an L-chain and a fragment of molecular weight of 32,000. The 3.5S fragments derived from the 8S subunits (8S Pep III and 8S Pap III) were similar to those obtained from 7M-globulins in the physico-chemical properties. The low yield of small peptides (12 and 26 per cent in the peptic and papain digests of TMglobulin, respectively and 26 and 33 per cent in the peptic and papain digest of the 8S subunits) suggests that the enzymic digestion carried out in the present studies was less extensive than in the previously reported studies [8-10, 24]. The present studies, based" on physico-chemical and immunological data, indicate that the fragments obtained from either 7M-globulins or their 8S subunits by peptic digestion are similar to the corresponding fragments produced by papain digestion. The relative yield of each fragment, however, differed depending on the enzyme employed. Table 4 summarizes some of the physicochemical properties of the various fragments obtained in the present studies in comparison with those that have recently appeared in the literature[8, 10, 11]. Although it should be borne in mind that the data listed in Table 4 were obtained from pathological 7M-globulins of different sources, similarities in molecular size among the corresponding fragments can be clearly seen. More recently,

8S Pap I 6-06S 130,800 8S Pap II 3.99S 70,700 8S Pap III 3-69S

8S Pep I 5-95S 133,500 8S Pep II 4.0S 72,700 8S Pep II 3"51S 55,000

Heaviest

Intermediate

Lightest

Pap III 3"50S 55,100

Pep III 3-48S 52,900

Lightest

Pap I

Pap II 6.0S

12-0S 539,000

Papain

Pep II 5.92S 127,000

Pep I

Pepsin

Intermediate

Heaviest

fragments

Ms II 3-66S 47,000

Ms I 4.3S

C 3.7S

6.14S 114,000

B

A 12-5S

Trypsin

Miller & Metzger (1966)

EIII 3-8S

EII 6.6S

EI 16-5S 796,000 617,000

Esterase

Chen et al. (1967)

*Each row includes nomenclature, sedimentation rate, and molecular weight in this order.

8S subunits

yM-globulins

Substrate

Present studies

Ms I Ms III 2"8S 3-2S 50,000

Papain

Onoue et al. (1967)

Table 4. Comparison of some of the results obtained by the digestion of yM-globulins and their subunits with various enzymes

O

6

e~

t~

~2

T. SUZUKI

Kishimoto et al.[9] r e p o r t e d that a pathological yM-globulin was digested with pepsin at p H 4.5 to give rise to 5.6S, 3.3S and 2.4S fragments. T h e a p p a r e n t discrepancy in the resulting molecular species between the present study and that o f Kishimoto et al. may be d u e to the difference in p H at which the digestion was carried out. T h e 12S fragments (Pep I and Pap I) consisted o f four different subunits (7S, 4"5S, 3.5S, and 2.8S) which a p p e a r e d to be held together t h r o u g h disulfide bridges. T h e stoichiometry based on the molecular weights and the yields f r o m Table 5. Amino acid contents of H-chain, L-chain and various fragments of LA ~/M-globulin and its 8S subunit Amino acid Lysine Histidine Arginine Aspartic acid Threonine* Serine* Glutamic acid Proline Glycine Alanine Half Cystine Valine Methionine Isoleucine Leucine Tyrosine* Phenylalanine S-CM Cysteinet

H-chain 27 12 27 47 52 60 60 43 39 36 12 51 6 20 39 20 25 4

L-chain

Pep II

Residues/mole 10 58 0 20 3 36 11 86 16 86 25 122 20 112 16 73 16 86 17 69 6 26 14 82 1 8 4 26 10 69 9 39 4 36 1 0

Pep III

8S Pep II

8S Pep III

24 1 13 40 35 67 46 37 44 37 11 42 5 12 35 21 15 0

30 7 16 43 41 76 58 38 43 35 12 41 6 15 38 19 20 trace

24 1 12 39 36 67 45 34 40 36 10 43 5 12 37 24 15 0

*Uncorrected values. tS-carboxymethylcysteine. the S e p h a d e x G 100 gel filtration o f these subunits allowed us to estimate a molecular weight o f 539,000 for the 12S fragments. As described above, each o f the subunits which compose the 12S fragments was u n i q u e in its physicochemical properties and amino acid composition (see Table 6). T h e slightly higher sedimentation rate (7.0S) and molecular weight (178,000) of the 7S subunit obtained f r o m the 12S fragments over those of the 'reduced-alkylated' 7S protein (6.8S and 160,000, respectively[l]) suggest that the 7S subunit which composes the 12S fragments may consist o f two H- and two L-chains in contrast to the two H- and one L-chain composition o f the 'reduced-alkylated' 7S protein. T h e amino acid composition o f the 7S subunit o f Pep I (12S) (Table 6) was f o u n d to be consistent with a two H- and two L-chain composition, although these data alone can not prove such chain composition. F u r t h e r study o f the structural relationship a m o n g the f o u r different types o f subunits o f the 12S fragments will help to u n d e r s t a n d the discrepancy in sedimentation rate and molecular weight

77 27 57 105 124 161 145 109 102 102 24 124 12 45 105 52 54 10

P1

P3

P4

Residues/mole 31 20 4 8 15 0 19 23 12 45 39 12 43 60 23 70 40 15 57 54 31 39 45 23 43 20 12 37 34 15 5 6 4 45 40 19 4 2 2 23 20 4 41 37 15 21 14 8 21 19 8 trace trace trace

P2

13 1 9 27 19 38 24 16 27 20 5 28 1 7 21 11 11 3

P1

12 0 4 12 16 28 22 16 16 17 4 14 1 4 10 9 4 1

P3

24 1 13 40 35 67 46 37 44 37 11 42 5 12 35 21 15 0

Pep I I I

Residues/mole 25 1 13 39 35 66 46 32 43 37 9 42 2 11 31 20 15 4

P~ + Pa

Subunits o f Pep I I I

Residues/mole 9 2 8 12 16 26 23 11 12 11 10 5 15 8 7 1

11 2 5 25 21 31 23 11 19 17 22 5 15 12 8 1

10 30 20 15 4

33

24 4 15 35 37 62 57 25 33 29

~-piece r - p i e c e Ms Pap Ia

Subunits o f Ms Pap IaT

*Based on molecular weights o f 178,000, 65,000, 56,000 and 29,000 for P1, P2, P3 a n d P4 subunits o f Pep I, and o f 32,000 a n d 22,500 for P1 a n d P3 subunits o f Pep l I I , respectively. ? T h e values for/z-piece, r - p i e c e and Ms Pap Ia are taken from the data o f O n o u e et al. [8]. :~Uncorrected values. §S-Carboxymethylcysteine.

Lysine Histidine Arginine Aspartic acid Threonine:~ Serine~: Glutamic acid Proline Glycine Alanine H a l f cystine Valine Methionine Isoleucine Leucine Tyrosine$ Phenylalanine S-CM cysteine§

Amino acid

Subunits o f Pep I

T a b l e 6. Amino acid contents of the various subunits of Pep 1 and of Pep I I I *

O

6_.

",d

t'~

~4

T. SUZUKI

between the 7S subunit of the 12S fragments and the 'reduced-alkylated' 7S protein and by extension, hopefully the whole structure of the 7 M-globulins. The physico-chemical and starch gel electrophoretical studies demonstrated a resemblance between the 3.5S subunit obtained from the 12S fragments and the F'c fragment of yG-globulins[20]. Chen et al.[ll] reported that a similar fragment was obtained from the reduced-alkylated product of the heaviest component of the esterase digested yM-globulin. As mentioned above, it was concluded from the molecular weight and yield data that three molecules of a monomer of the 3.5S subunit are included in the 12S fragment. This conclusion was made, however, on the assumption that each subunit of the 12S fragment has the same extinction coefficient at 280rn/z. Further experiments would be required to establish the exact number of the 3-5S subunit molecules in the 12S fragment. The 6S fragments could be derived from either 7M-globulins (Pep II) or the 8S subunits (8S Pep I and 8S Pap I) through the enzymic digestion. They were similar to each other in molecular size, hexose contents, and immunological properties. The stoichiometrical conversion of the 6S fragments to t h e 4S material by reduction and alkylation led us to the contention of the 4S fragments produced from the 8S subunits (8S Pep II and 8S Pap II) being a monomer of the 6S fragments. The data of molecular weight determinations (Table 1 and 2), starch gel electrophoresis (Figs. 5(A) and 15), and hexose analysis (Table 3) are consistent with the interpretation cited above. The results of amino acid analyses of the 6S fragment (Pep II) and of the 4S fragment (8S Pep II) also indicate a dimer-monomer relationship between these fragments (Table 5). Two subunit proteins (P1 and P3) which composed the 3.5S fragments obtained from yM-globulins (Pep III and Pap III) appeared to be held together noncovalently after reduction and alkylation and could be separated only in acid media. One of these subunits had a molecular weight of 32,000 and appeared to correspond to the Fd fragment of yG-globulins[22]. This contention was weakened by the finding that this subunit failed to react with antibody to Hchain, although this may be due to the strong tendency for aggregation in aqueous media near neutral pH. Since the other subunit appeared to be the Lchain, stoichiometry based on the molecular weight and yield data indicate that the 3.5S fragments obtained from yM-globulins consisted of an L-chain and an H-chain fragment of molecular weight near 32,000. This idea is further supported by the amino acid analysis data of Pep III and its subunits (Table 6), which show the complementarity of amino acid composition of the Fd like fragment (P,) and L-chain (P~) to form Pep III. Furthermore, the data in Table 6 show a general agreement of the amino acid composition of the Pep III and its subunits with those of the analogous fragment (Ms Pap Ia) and its subunits (/z-piece and Kpiece) reported by Onoue et a/.[ll], when the latter data were recalculated on the basis of the molecular weights of the fragment and its subunits used in this study. Although the present studies revealed that the 3"5S fragments obtained from the 8S subunits were similar to those derived from-/M-globulins in their physicochemical properties, hexose contents and amino acid composition (Table 5), certain differences between these fragments were demonstrated by the immuno-

Enzymic Cleavage of Human yM-Globulins

605

logical studies. All but Pep III did not react with the antibody to H-chain. The 3"5S fragments derived from the 8S subunits (8S Pep III and 8S Pap III) appeared to be slightly antigenically deficient in reaction with antibody against L-chain, while no such deficiency was obtained with the 3.5S fragments derived from y M-globulins. This discrepancy in immunological properties between the 3.5S fragments obtained from yM-globulins and those derived from the 8S subunits may be due to the structural difference between the 8S subunit and the pentameric form of the y M-globulin. This point should be clarified by further experimentation. We have previously presented evidence that the 8S subunit ofyM-globulins consists of two H- and three L-chains which are covalently linked together. Figure 18 illustrates the hypothetical schematic model for this type of subunit. 8S Subunit L-chain

H-chain

I

S S I

H-chain

I

S S 1

L-chain

L-chain

Enzymic cleavage I

S S I

8S Pep I or

8S Pap I

I S S I[ I !

l ................. Dialyzable material S S I I 8S Pep III S or S Ii 8S Pap III i z

Reduction and alkylation 8S Pep II

i I +

i X ,I,

SCM 1

SCM 1

I I I SCM SCM SCM

Fd like fragment

or

8S Pap II

L-chain

Fig. 18. Hypothetical schematic model of the 8S subunit of yM-globulin.

606

T. SUZUKI

T w o L-chains may be linked to two H-chains in an analogous m a n n e r to the f o u r chains o f y G-globulins [23]. A possible site to bind a n o t h e r L-chain may be on the part o f one o f H-chains opposite to the part where two L-chains are already attached. With the use o f this hypothetical model o f the 8S subunit, the results o f enzymic digestion o f this type o f subunit may be i n t e r p r e t e d as following. T h e cleavage o f petide bond(s) with enzymes may occur on the H-chains, possibly at the site(s) n e a r the inter H-chain disulfide b o n d on the side where an extra L-chain is linked. This cleavage will lead to the f o r m a t i o n o f a 6S (8S Pep I and 8S Pap I) and a 3'5S (8S Pep III and 8S Pap III) f r a g m e n t , each o f which consists o f the L-chain and a f r a g m e n t o f the H-chain. Reduction o f an inter H-chain disulfide b o n d will convert the 6S f r a g m e n t s to their 4S m o n o m e r s (8S Pep II and 8S Pap II). T h e distinctly high yield o f the 4S f r a g m e n t f r o m the papain digest over that f r o m the peptic digest may be d u e to the presence o f ME which was used for the activation o f papain. T h e part o f the H-chain, to which no L-chain is attached, may be d e g r a d e d rapidly and b e c o m e a dialyzable material. T h e results o f the enzymic digestion o f the 8S subunits based on physicochemical, hexose and amino acid analytical and immunological data are thus consistent with the suggested model o f the 8S subunit o f y M-globulins. In o r d e r to provide additional evidence to test the model, it would, however, be o f particular interest to d e t e r m i n e the amino and carboxyl terminal amino acids o f the 6S (and its m o n o m e r ) and the 3.5S fragments f r o m the 8S subunit. Acknowledgements-I wish to thank Dr. D. S. Rowe for generous supply of EL macroglobulinemic serum, Professor H. Isliker for encouragement during the course of this work, and Professor B. Cinader for criticism of the manuscript. I am grateful to Dr. J. J. Scheidegger of the Beckman Research Institute at Geneva and to Dr. J. Mauron of Research laboratories of Nestle's products for amino acid analyses. Thanks are also due to Miss D. Pasquier for excellent technical assistance.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

REFERENCES Suzuki T. and Deutsch H. F.,J. biol. Chem. 242, 2725 (1967). Miller F. and Metzger H.,J. biol. Chem. 240, 3325 (1965). Miller F. and Metzger H.,J. biol. Chem. 240, 4740 (1965). Putnam F. W., Kozuru M. and Easeley C. W.,J. biol. Chem. 242, 2435 (1967). Lamm M. E. and Small P. A.,Jr., Biochemistry 5,267 (1966). Porter R. R., Biochem.J. 73, 119 (1959). Nisonoff A., Markus G. and Wissler F. C., Nature, Lond. 189,293 (1961). Onoue K., Kishimoto T. and Yamamura Y.,J. Immun. 98, 303 (1967). Kishimoto T., Onoue K. and Yamamura Y.,J. Immun. 100, 1032 (1968). Miller F. and Metzger H.,J. biol. Chem. 241, 1732 (1966). ChenJ. P., Tomasi T. B. and Reichlin M., Fed. Proc. 529 (1967). Deutsch H. F. and MortonJ. I.,J. biol. Chem. 231, 1107 (1958). Suzuki, T. and Deutsch H. F.,J. exp. Med. 124, 819 (1966). Winzler R. J., Methods of Biochemical Analysis (Edited by Glick D.), Vol. 2, p. 279. I nterscience, New York (1955). Spackman D. H., Stein W. H. and Moore S., Analyt. Chem. 30, 1190 (1958). Archibald w . j . , j , phys. Colloid Chem. 51, 1204 (1947). Smithies O., Biochem.J. 71,585 (1959). Ouchterlony O., Actapath. microbiol, scand. 26,507 (1949). ScheideggerJ. J., Int. Archs Allergy appl. Immun. 7, 103 (1955). Poulik M. D., Nature, Lond. 207, 1092 (1965).

Enzymic Cleavage of Human yM-Globulins

607

21. Chaplin H., Cohen S. and Press E. M., Biochem.J. 95,256 (1965). 22. Heimer R., Immunochemistry 3, 81 (1966). 23. Porter R. R., In Basic Problems of Neoplastic Disease (Edited by Crellhorn A. and Hirshberg E.), p. 177. Columbia University Press, New York (1962). 24. Mihaesco C. and Seligmann M.,J. exp. Med. 127,431 (1968). 25. Kabat E. A.,J. exp. Med. 69, 103 (1939). 26. Kovacs A. M. and Daune M., Biochim. biophys. ,4cta 50, 249 (1961). 27. Caputo A. and Apella E., Archs Biochim. Biophys. 91,201 (1960).