THE JOURNAL cm BIOLOGICAL CHEMISTRY Vol. 243, No. 22, Issue of November 25, PP. 5977-5984, 1968

Printed in U.S.A.

Binding Properties of a Waldenstriim Macroglobulin Antibody*

(Received for publication, May 28, 1968)

MARVIN J. STONES AND HENRY METZGER~

From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland Z?OOl4

SUMMARY

Further studies on the functional properties of a Walden- strom macroglobulin (yMLay) with antibody activity have been performed. Binding of the subunits, yM,, and tryptic fragments, FabF, to the antigen, human yG, has been examined by ultracentrifugation and equilibrium molecu- lar sieving. While the data have certain inevitable am- biguities, they are consistent with: (a) the antibody reacting with a single determinant on rG, (b) the subunits being potentially divalent although functionally univalent, and (c) all of the Fabp fragments being functionally equivalent.

Since the primary antigen-combining site interaction is energetically similar to that found in other systems, it is concluded that the cryoprecipitating property of the TM-yG complex is due to the univalence of the antigen as well as the solubility characteristics of the aggregates.

“Pamprotein” immunoglobulins synthesized under various pathological conditions are structurally similar to antibody immunoglobulins. Because they are markedly less heterogene- ous than typical preparations of antibodies they have been ex- tremely useful for detailed structural and genetic analyses. Recently, well defined antigen-combining activity has been discovered among paraprotein immunoglobulins of the three major classes yG (l), yA (2), and TM (3) .r With the possible exception of their mode of induction these proteins satisfy all of the criteria (3) for defining an “antibody.” Their homogeneity would seem to make them ideal subjects for studying structural- functional relationships of antibodies.

* Parts of this study were presented at the 52nd Annual Meeting of the Federation of American Societies for Experimental Biology Atlantic City, New Jersey, 1968.

$ Present address, Department of Internal Medicine, The University of Texas Southwestern Medical School, Dallas, Texas 75235.

§ To whom inquiries should be addressed. 1 The abbreviations used for the immunoglobulins, their chains,

and their fragments are in accord with those suggested by an international committee (4).

One such paraprotein antibody, a Waldenstrom macroglobulin, Y&.W, combines with the Fe portion of human rG (3). From recent studies involving the precipitation of various primate ~G’s by yMnay (5) we tentatively concluded that -yMLay reacted with a single determinant on yG and was functionally homo- geneous. The present investigation was undertaken to examine these points directly. Similarly, our previous studies on the valence of the subunit, TM,, were only indirect, involving both inhibition of precipitation assays (3) and agglutination reactions (6). By direct binding studies we wished to determine whether the two Fab fragments-which are active in their isolated form (6)-are both functional as they exist in the subunit.

In addition, we wished to examine the rather unusual proper- ties of the 7Mnay-yG precipitation reaction. Unlike ordinary protein-antiprotein precipitin reactions, the precipitation of -yMn,, with yG is extremely sensitive to temperature, ionic strength, and pH (3). The results of the present study provide a rational explanation for this phenomenon.

EXPERIMENTAL PROCEDURE

Source and Purijkation of Proteins

yMLaY is a K type macroglobulin obtained from the plasma of a 69-year-old male (NIH No. 06-71-60) with Waldenstrom’s macroglobulinemia and an atypical lymphoma. The method of purification has been described (5).

7Mhlar is a K type macroglobulin isolated by repeated euglobu- lin precipitations (7) from the serum of another patient (NIH No. 06-34-47) with Waldenstrom’s macroglobulinemia. This protein shows no anti-human yG activity.

yGwitr is a K-type, +yG immunoglubulin isolated from a patient (NIH No. 07-26-23) with typical multiple myeloma. Precipita- tion of serum with 0.8 M sodium sulfate was followed by DEAE- cellulose column chromatography (8). The monomer, isolated by Sephadex G-200 column chromatography, remained stable during the period of these experiments.

Mixed human rG immunoglobulins were purchased from Pentex, Inc., as Cohn Fraction II, precipitated from 40% saturated ammonium sulfate and then isolated in a manner similar to yGwar.

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5978 Waldenstriim Macroglobulin Antibody Vol. 243, No. 22

Chemicals

Carrier-free 1251 and 1311 were purchased from Union Carbide. Sephadex gels and blue dextran 2000 were obtained from Phar- macia. Trypsin reacted with L-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone to inhibit contaminating chymotrypic activity and soybean trypsin inhibitor were Worthington prod- ucts. Dithiothreitol was purchased from Calbiochem and used as such. Cysteine hydrochloride, obtained from Nu- tritional Biochemicals, was twice recrystallized from water. Iodoacetamide (K and K Laboratories) was recrystallized four times from water. All other chemicaLs were reagent grade; glass-distilled water and toluene-saturated buffers were used throughout.

Preparation of Subunits and Fragments

The subunits, TM,, were obtained from yMLsv either by re- duction with 0.005 M dithiothreitol for 45 min (interchain di- sulfides broken) or by reduction with 0.02 M cysteine hydrochlo- ride for 24 hours (interchain disulfides re-formed) (9). In both instances reduction was carried out at 25” at pH 8.6 and alkyla- tion with a 10% excess of iodoacetamide was done for 15 min at pH 8.0 (25”). The subunits were isolated by Sephadex G-200 chromatography.

The Fabp fragments were prepared by the tryptic digestion of yM (37” for 21 hours) at pH 8.0 in the presence of 0.01 M calcium chloride and isolated by Sephadex G-200 chromatography (10). For several experiments Fabp reduced fragments were obtained by a similar digestion of the reduced and alkylated subunit, yM,. The unreduced fragments were iodinated (11) and re- chromatographed on Sephadex G-100. Elution volumes for 129-Fabr,ay and rSII-FabMs, were identical. Ninety nine per cent of the counts (lo6 dpm per mg of protein) were precipitable by 10% trichloracetic acid.

Fey fragments were prepared from human rG (Pentex). The purified protein was reduced with 0.005 M dithiothreitol at 25” for 25 min at pH 8.6. After alkylation with a 10% excess of iodo- aceta.mide (25”, 15 min, pH 8.0), the protein was dialyzed against a Tris-HCl buffer, pH 8.0, and then digested with 1% (w/w) trypsin at 37” for 1 hour in the presence of 0.01 M CaCls. Di- gestion was stopped by the addition of 1% (w/w) soybean tryp- sin inhibitor. The peak tubes from a Sephadex G-200 elution pattern (containing a mixture of Fey and Faby fragments) were pooled and the concentrated sample was dialyzed against a Tris- HCl, 0.02 M, pH 8.6, buffer. Chromatography on DEAE- cellulose utilized a linear gradient with the Tris buffer supple- mented with 0.3 M NaCl (12). With rabbit anti-r and anti-y chain sera, Ouchterlony analysis revealed that the first (break- through) peak from the DEAE-cellulose column reacted only with the anti+ antiserum while the second bifid peak reacted very weakly with the ant& serum but gave an intense precipitin line with the anti-y chain serum. By quantitative radial im- munodiffusion (13) in which the anti-K serum was incorporated into the gel, protein from the second peak contained only 7% contaminating Faby fragments. This preparation of Fey frag- ments was used in binding studies with the -yMLay cysteine sub- unit.

All spectrophotometric analyses were performed with a Zeiss PM& II spectrophotometer with quartz cells with a l-cm path length. Preparative chromatography for the purification of the tryptic fragments and yM subunits utilized Sephadex columns equilibrated with borate buffer, pH 8.0, at 25”, unless stated

otherwise. Proteins were concentrated in collodion bags (Schleicher and Schuell) under vacuum. The preparation and characterization of antisera used in this study have been de- scribed (5).

Binding Studies

Fractional binding of Fabp to 7Gwar and Fey to yMs,nay was measured by (a) analytical ultracentrifuge experiments and (b) the method of equilibrium molecular sieving. Calculations were based on molecular weights and extinction coefficients pre- viously reported (3). For Fey, a molecular weight of 5.0 x

lo4 was used. The extinction coefficient for Fey was determined with ovalbumin as a standard. Two times recrystallized oval- bumin (Worthington) was alkylated with 0.011 M iodoacetamide and monomer isolated by Sephadex G-200 chromatography. Schlieren peaks of solutions of ovalbumin and PC? of known optical density were compared in the ultracentrifuge. With et& mC = 7.36 for ovalbumin (14), the extinction at 280 rnp of a 1% solution of Fey was determined to be 13.1.

Xtracentrijuge Experiments-All studies used a Spinco model E analytical ultracentrifuge equipped with overspeed and RTIC temperature controls. Sedimentation runs were per- formed at 56,000 rpm and 20-25” with two double sector cells with 0.01 p of P04-0.05 M NaCl, pH 6.0, as the solvent. Identical concentrations of the 3.5 S component (Fabpn,, or Fey) were used in both cells while the 7 S component (*/Gwar or yM,, nay, respectively) was present in the standard cell only. The cells gave schlieren peaks of identical area when loaded with identical protein solutions. Photographs were taken at 8-min intervals beginning 50 to 60 min after speed had been attained. Areas of the enlarged schlieren peaks were measured by planimetry. Duplicate measurements agreed to within 5 y0 and at least three frames were measured in each experiment. Care was taken ho compare peaks from comparable positions in the cells. Frac- tional binding was calculated by comparing the 3.5 S peak areas in the wedge and standard cells. The standard deviation of the calculated percentage of unbound 3.5 S component ranged from 1.1 to 3.7 (average 2.4) for the Fabp experiments and from 0.3 to 2.8 (average 1.8) for the Fey-TM, studies. No correction was made for the Johnston-Ogston effect, since it was computed to produce only a 1% error in the slow component area measure- ments

Equilibrium Molecular Sieving Experiments--The theory of the method and its ability to show specific binding have been described in a previous paper (15). For the FabpLLay-yGWar system, Sephadex G-75 was used as the semipermeable “dialysis membrane.” Starting solutions (0.2 ml) containing known amounts of reactants and markers were added to small siliconized tubes containing 1.7 ml of Sephadex. After equilibration, 50-~1 aliquots of the supernatant were removed and diluted appro- priately for spectrophotometric and radioactivity measurements. After reading the optical density of the blue dextran in 300+1 cuvettes, duplicate 50-~1 aliquots were removed from each cuvette for counting. 1251 and Ia11 radioactivity was determined on each aliquot in a Nuclear-Chicago dual channel y counter with 35% efficiency. A minimum of 10,000 counts was ob- tained for each isotope in each specimen. Specific activities (radioactivity/A& always agreed to within &5% and only rarely varied by more than 2 to 3%.

The starting solutions were handled in a similar manner. Each experimental point was run in triplicate and starting solu-

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Issue of November 25, 1968 M. J. Stone and H. Metzger 5979

tions were analyzed in duplicate or triplicate (see “Results”). The assays were routinely carried out at 25” in a 0.01 /.L of Pod, pH 6.0, buffer with either 0.05 M or 0.1 M NaCl. Six different binding points were usually determined in one experiment.

Precipitation Assays

Precipitation of ‘251-~M~ay by ~Gwar was studied under conditions similar to those used for the direct binding studies (3). The proteins were mixed in a total volume of 0.2 ml and then incubated for 4 or 24 hours. Duplicate determinations were made for each molar ratio studied. After centrifugation for 30 min, the supernatant radioactivity was determined. In the studies at different temperatures, all manipulations prior to the actual counting were carried out at the appropriate temperature.

For some of the precipitation studies, both reactants were iodinated.

RESULTS

Valence of Antigen

The number of effective antigenic determinants on yGwar with respect to 7MLBy was established by examining the binding of Fabpn,, to yGwar in the ultracentrifuge. The area under the peak representing the unbound FabLay was compared with that of an equivalent concentration of the fragment sedimenting in

FIG. 2. Scatchard plot of ultracentrifugal data for the interac- tion of FabpL.. with &war. Data plotted so as to give valence of the yG antigen: T equals moles of Fabp bound per mole rG; c equals unbound Fabp concentration. Solvent: 0.01 p of P04-0.05 M

NaCl, pH 6.0. Temperature: 20-25”. 0, Fabr unreduced; 0, Fabp reduced. Line drawn by method of least squares.

5-

4-

3-

2-

I-

o-

I I I I I

0.2 0.4 0.6 0.8 I.U I.2

r

the absence of rG (Fig. 1). Since the moles of Fabn,, and rG in the cell were known the moles of Fab bound could be directly calculated. Molar ratios of FabLay :YGwar ranged between 0.44 and 5.0. The concentration products (FabnltY x 7Gwar) varied between 1.3 x 10e8 and 1.8 X 1O-g M.

A Scatchard plot of the data is shown in Fig. 2 where r indi- cates the moles of Fabp bound per mole of yG and c represents the unbound Fabp concentration. At the larger values of r where high Fabp :yG molar ratios were used, there is some scatter to the data but no discernible trend is evident. The slope of the line drawn by the method of least squares ( =&) is 6.8 X lo4 M-1. The extrapolated valence of rG is 1.16. Fabp reduced and unreduced were indistinguishable in regard to their bind- ing affinities for the antigen.

Valen,ce of vMLay Cysteine Subunit

This was also assessed in the ultracentrifuge by attempting to saturate TM, with increasing amounts of Fcr. The same con- centration of TM, was used in all experiments. The results given in Fig. 3 show a defmite plateau at a level of about 1 mole of Fey bound per mole of yM,.

Determination of A.ssociation Constant

for FabpLay-TGWar Interaction

Centrifuge Experiments-Since the ultracentrifugal data per- mitted direct computation of the proportion of Fabp bound and the total concentrations of reactants were known, association constants could be calculated for each point shown in Fig. 2.

FIG. 1. Photograph of schlieren patterns obtained by sedi- At levels of 24, 45, 49, 71, 88, and 90% binding, the KA ranged menting 1.38 X 10T4 M Fabp (reduced) fragments in the presence between 5.4 and 6.9 x 104 cl. All variation outside of this (lower cell) and absence (upper cell) of 3.60 X 10-E M human rG (~Gw.~). Solvent: 0.01 p of PO4-0.05 M NaCl, pH 6.0. Tempera-

range (Ka =1.9 x 104, 3.3 x 104, and 2.0 X lo5 M?) occurred ture: 22.3”. Bar angle 60”. Picture taken 75 min after speed at low levels of binding ( <30%) where accurate measurements (56,000 rpm) reached. Area measurements indicated 30yo binding. are difficult.

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Waldenstr6m Macroglobulin Antibody Vol. 243, No. 22 5980

1.4

1.2

oL+--d-- 0 2 3 4 5 6

MOLES Fc, ADDED / MOLE YM, 7

FIG. 3. Saturation of constant amount of TM., r.sY (cysteine subunit) with increasing amounts of human Fcr calculated from ultracentrifugal data. Solvent: 0.01 p of Pod-O.05 M NaCl, pH 6.0. Temperature: 20-25”. Vertical lines indicate range of bind- ing for each point, as calculated from three separate exposures made at 8-min intervals during the centrifugation run (see “Methods”).

3.0

2.5

2.0 e ‘k x

2 1.5

1.0

0.5

I I I I I

A

0 0

0

0

A A

----- A-----O--- ----*-----P---

0 0 l

0

0

0

0 8 0

0

I I I I I IO 20 30 40 50

PERCENT BOUND

_-

60

FIG. 4. Plot of association constant for the FabpLay-YGwar interaction at various levels of site saturation as determined by method of equilibrium molecular sieving. Solvent: 0.01 p of Pod, pH 6.0 with added salt as indicated. Temperature: 24-25”. A, experiments in which duplicate aliquots of starting solutions were assayed; 0.1 M NaCl. 0, starting solutions assayed in triplicate; 0.1 M NaCl. 0, starting solutions assayed in triplicate; 0.05 M NaCl. - - -, mean KA for all points.

It should be noted, however, that although the average

standard deviation for the calculated percentage binding at any

one point was nominally small (2.4) it is sufficient to introduce a

considerable ambiguity (approximately a factor of 2) into the

calculated average KA at particular points. Thus, although the

centrifuge data show no consistent trend, heterogeneity in the range of a a-fold difference in Ka cannot be excluded.

Equilibrium Molecular Sie&ng Experiments-Association constants were also determined from the equilibrium molecular sieving data (15). KA could be calculated over a range of 5 to 56% site saturation. In the initial studies the starting solutions were only assayed in duplicate and occasional erratic values of Ka were obtained. When the starting solutions were assayed in triplicate, these discrepancies disappeared. The mean Ka for the entire group was 1.53 x lo4 M-‘. The standard error of this mean was 0.11 x lo4 M-I. For the 10 points in the 0 to 28% range of site saturation the mean Ka was 1.51 x 104; for the 12 points in the 29 to 56’% range of saturation the mean KA was 1.54 X 104. No consistent variation in KA was observed whether the solvent was 0.05 or 0.10 M in NaCl. The results of the Sephadex experiments are plotted in Fig. 4.

Since the ultracentrifuge and Sephadex experiments yielded different absolute association constants (see “Discussion”) the data could best be compared by examining the variation in Ka:Ko over the range of binding studied by each technique. The combined data are presented in Fig. 5, which presents data collected at various temperatures, ionic strengths, and pH values described below.

E$ect of Variation in Temperature, pH, and Ion.ic Strength on Association Constant

As is detailed in the following section the precipitation of yMnay with rG is very sensitive to variations in temperature

2.5 - I ’ (J/91

I I

d

2.0 - 0

2 l.5-

0 0,

0 \

YQ 0 0 a 1.0 - 5 “0. 00 O

q 0 0

8 m

0

0 boo

0.5- 0 0

I I I I

0 20 40 60 80 PERCENT BOUND

IO

FIG. 5. Composite of FabpL ay-~&ar binding data over site saturation range as determined in ultracentrifuge (0) and by method of equilibrium molecular sieving (circles). Association constant for each point expressed as deviation from mean value (Ko) for method, solvent, and temperature as indicated (see text). q , 0.01 p of PO4-0.05 M NaCl, pH 6.0, 20-25”; 0, 0.01 p of Pod-O.1 M NaCl, pH 6.0, 24-25”; 0, 0.01 p of Pod-O.05 M NaCl, pH 6.0, 24-25"; c>, 0.01 p of POb-0.05 M NaCl, pH 6.0, 3”; 0, 0.01 p of PO1- 0.15 M NaCl, pH 7.4, 3”.

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Issue of November 25, 1968 M. J. Bone and H. Metzger 5981

and to the pH and ionic strength of the solvent. To study TABLE 11

whether this sensitivitv reflects the primary antibody site inter- Thermodynamic parameters of protein antigen-antibody systems

action the following experiments were conducted. FabL,, binding to rG was assayed by equilibrium molecular system

seiving at two levels of binding (each determined in triplicate) at three different temperatures. The same solvent (0.01 /A of Dugan +:FabpLaya. PO1 buffer-O.05 M NaCl, pH 6.0) was used throughout. The Bovine serum albumin- results are shown in the upper portion of Table I. The values of rabbit anti-bovine se- Ka at 24” are in good agreement with earlier experiments. rum albuminb. Thermodynamic functions were evaluated. Fig. 6 is the van? Ovalbumin-rabbitanti- Hoff plot from which a standard enthalpy of 2 + 1 kcal per mole ovalbuminc.

AFQ I MO

kcal .mole-’

-5.8 f 0.2 2fl

-5.5 f 0.2 Of2

-5.6 f 0.2 j 0 f 2 i

was calculated. From this value and the standard free energy calculated from the Ka at each temperature, a standard entropy of 26 f 6 cal deg-1 mole-’ was derived. Table II lists these results and compares them with values obtained for two other

a This study. b Singer and Campbell (16). 0 Singer and Campbell (17).

protein-antiprotein systems (16, 17). To test the effect of pH and ionic strength on the value of KA,

binding was measured at 3” in 0.01 /.L of P04-0.05 M NaCl at pH 6.0 and in 0.01 Jo of PO&-O.15 M NaCl at pH 7.4. The values obtained at the lower pH and ionic strength (6.2 f 0.3 X 104) were in good agreement with those obtained previously in the

g c

same buffer at 3” (5.9 f 0.2 x 104). The association constant ‘E ; 60 LL t/

TABLE I

Effect of temperature and solvent on FabpLau-YGWar interaction

Solvent

0.01 p of P04-0.05 M

NaCI, pH 6.0

0.01 /A Of Pod-0.05 M

NaCl, pH 6.0

0.01 /.I of P04-0.15 M

NaCI, pH 7.4

Q Mean K.+. -

Temperature

3O 3

24 24 42 42

- F ‘ab,q,,,z bound

% 25.4 62.8 9.0

36.9 8.4

21.0

35.7 62.5

36.7 63.1

-

KA

:a1 &g-l mole-’

26 f 6

20 f 8

20 f 8

1.5

0.92 (1.1) MOLES yG/MOLE yM

FIG. 7. Effect of solvent and temperature on the precipitation 6.5 of W-~ML~~ by ~Gvar. Upper curve, 0.01 p of Pod-O.05 M NaCl, 5.9 (6.2) pH 6.0 solvent, 3”. Incubation for 4 or 24 hours gave identical

amounts of precipitation. Middle curve, 0.01 p of PO4-0.05 M

5.7 NaCl, pH 7.4 solvent, 3”, 24-hour incubation. Lower curve, 0.01

6.9 (6.3) p of PO*-0.05 M NaCl, pH 6.0 solvent, 25”, 4-hour incubation. Data obtained at 40” were indistinguishable.

I I I I I I I

at high ionic strength and pH was not significantly different (6.3 f 0.6 x 104) from that in the lower ionic strength and pH buffer. Results of the pH and ionic strength studies are given in the lower portion in Table I.

I I I I I I I 3.0 3.1 3.2 3.3 3.4 3.5 36 3.7

+ x I03

FIG. 6. van? Hoff plot of FabbLay-YGWar interaction as deter- mined by method of equilibrium molecular sieving. Line drawn by method of least squares. Standard enthalpy of 1.9 kcal mole-1 was calculated from the slope.

Precipitation of lz51-yMLay by rG; Efect of

Temperature and Solvent

7MLay can be quantitatively precipitated at low molar ratios of human rG:rM (3). Unlike many precipitin reactions, addition of more antigen does not lead to inhibition of precipita- tion. The marked sensitivity of the precipitation to temperature and to solvent pH and ionic strength is documented in Fig. 7. The upper curve shows the precipitation of 4.9 x 10’ M 1251- 7MLBY in a 0.1 /J of Pod-O.05 M NaCl buffer, pH 6.0, at 3” incu- bated for 4 hours while the lower curve gives the result of a similar experiment conducted at 25”. An experiment conducted at 40” yielded results indistinguishable from those obtained at 25”. The loss in precipitability is out of proportion to that expected from the change in association constant (see Table I).

The effect of increasing the pH to 7.4 and the salt concentra-

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5982 Waldenstrtim Mac~oglobdin Antibody Vol. 243, No. 22

TABLE III

Effect of solvent on composition of 1251-yMLay-1311-yG~~~ precipitate5

Solvent

0.01 p Of P04-0.05 M

NaCl, pH 6.0

0.01 /A Of Po&.lj M

NaCI, pH 7.2

yM precipitated

%

19.6 f 0.76 29.4 f 1.3 42.2 zt 1.5 50.6 f 0.5

65.6 f 0.8

21.0 f 0.3 36.8 f 1.3

71.6 f 3.4 81.6 f 0.9 88.4 * 0.5

yG:yM molar ratio

Added In precipitate

0.25 0.91 f 0.02b 0.50 1.20 f 0.01 0.75 1.31 f 0.04 1.00 1.52 f 0.02

1.50 1.81 f 0.02

0.98 1.95

3.90 5.85

7.80

2.20 f 0.05 2.42 f 0.13

2.72 z!z 0.31 3.01 f 0.23

3.21 + 0.10 i

a The concentration of 12%yM~,,y was 5.0 X 1W7 M; tubes were incubated for 24 hours at 3”.

b Mean and range of quadruplicate determinations.

tion to 0.15 M NaCl is shown by the middle curve in Fig. 7. IT-

?MLay concentration was 4.0 x 10d7 ~1 and tubes were incubated for 24 hours at 3”. A separate experimental group in which the 0.01 p of Pod-O.05 M NaCl, pH 6.0, solvent was used yielded re- sults indistinguishable from those shown in the upper curve in Fig. 7. Despite the fact that no effect of pH and ionic strength on the association constant was demonstrable (Table I), there was an 8- to 15-fold decrease in precipitation compared to the reaction at the lower pH and salt concentration (Fig. 7, middle to upper curve).

The complexes precipitating at low pH and ionic strength were compared to those precipitating at a higher pH and ionic strength (Table III) at comparable levels of yM precipitation. The precipitated complexes formed at pH 7.2 and 0.15 M NaCl show a higher yG:yM ratio than those formed at pH 6.0 and 0.05 M

NaCl.

DISCUSSION

Functional Homogeneity of yM Lay-Our studies are consistent with the combining sites of yML,? being functionally equivalent. Two lines of evidence are pertinent.

The data in Fig. 2 indicate that at saturation only 1 molecule of Fab is bound per molecule of +yG. Such data can never rule out the presence of multiple antigenic determinants sufficiently close to each other to make binding by more than one antibody fragment sterically impossible. It is, however, certainly con- sistent with a single determinant contributed to by amino acid residues from both heavy chains of yG or a pair of symmetrical, closely apposed determinants each arising from one of the heavy chains. Studies on the reactivity of 7MLay with various primate ~G’s have led us to similar conclusions (5).

The studies of the association constant of the Fab fragments provide the most direct test of the relative functional homogeneity of the Fab combining sites. In the equilibrium molecular sieving experiments a 5 to 60% site saturation was studied while in the ultracentrifuge 20 to 90% saturation could be examined. In neither case was there evidence for heterogeneity. On the other hand, the error in the experimental results is sufficiently large that K, at any single point can only be relied on to approximately

a factor of 2. Thus we cannot rule out that a variety of com- pensating errors have not obscured a moderate degree of hetero- geneity. It should be recalled, however, that induced anti- bodies usually show a considerably greater heterogeneity than could possibly pertain to yML,,. For example, in most hapten binding studies at least a lo-fold variation in Ka is found in a range containing 50 to 75% of the combining sites and much larger deviations are not unusual.

The discrepancy between the mean association constant cal- culated from the two methods used remains unexplained. The gel filtration studies represent true thermodynamic equilibrium and would be expected to yield more accurate absolute values. The centrifuge experiments may have been subject to a variety of nonequilibrium phenomena. For example, at the high pressures which exist at the speeds used, the relative molal volumes of reactants and complex, respectively, can profoundly influence the position of the equilibrium (E-20). We performed one centrifuge run at 40,000 rpm-a speed at which the centrifugal field is about half that at 56,000. Although no change in equilib- rium binding which exceeded our experimental error was found, such effects cannot be excluded entirely.

The important point is that with neither technique was there any trend toward lower association constants at progressively higher levels of saturation.

Valence of yMLay, Its Subunits, and Fragments-The valence of YML~~ has so far been t,ested only in a precipitating system (3). It was found that, above a yG:yM ratio at which all of the yM was precipitated, a maximum of 5 moles of yG per mole of YML~ could be found in the precipitate. The lack of an anti- genie fragment capable of reacting but not precipitating with ?MLay has prevented us from studying the binding properties of ?MLity directly.

The binding’properties of the subunit yM, appear to confirm the above finding in that the five subunits are each only able to bind 1 mole of Fey (Fig. 3). This finding was somewhat sur- prising since we had previously shown that, yM, had weak ag- glutinating activity (6) and that on a molar basis it appeared to be 2.5 times as active an inhibitor of rM,,,-rG precipitation as

was the Fab fragment (3). The explanation for these discrep- ancies can only be guessed at. While the agglutination experi- ments were conducted in such a way as to make yM contamina- tion an unlikely source of the activity, the loo-fold difference in titer for -yMLay compared to yM, makes such contaminating activity difficult to exclude entirely. Since yM, does in fact have two combining sites (below)-although it can only effectively bind 1 mole of antigen-the possibility exists that some distortion in a small percentage of the molecules makes the second site available. Alternatively, when one site in yM, is saturated, the effective binding constant for the second ma,y be considerably smaller than the intrinsic association constant. In either case binding experiments would only show a single binding site.

With respect to the inhibition data it can be shown that, at the low levels of inhibitor site saturation used in those experi- ments (1 to 2% (3)), a fragment with t)wo potential binding sites-all other factors being equivalent-would be twice as effective as an equimolar concentration of a univalent fragment.

This holds regardless of the fact that the divalent inhibitor can bind only 1 mole of ligand at a time.

The dimeric fragment F(ab’)z p had inconstant agglutinating activity but was as effective an inhibitor of yM precipitation as yM, (3, 6). Because its size is intermediate between the Fc

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Issue of November 25, 1968 M. J. Stone and H. Metxger 5983

fragment of rG and rG itself, binding studies will be difficult to perform and would better await the isolation of a low molecular weight antigenic fragment. We must assume at present that F(ab’)z p is functionally equivalent to y,M,.

Data on the activity of the Fab fragments have been presented, and are consistent with each Fab from TM,,, having an active site and with the functional equivalence of those sites.2

It cannot yet be estimated to what extent our conclusions on the valence of yMtiY can be generalized to include other yM immunoglobulins. The conflicting data in the literature may reflect differences among yM molecules or experimental pro- cedures (21-23). None of the results has involved specimens in which one can exclude intermolecular heterogeneity and more importantly, in none of the studies have the Fab fragments been investigated.3 Until such data are at hand it is difficult to ar- rive at any conclusions.

It is interesting to speculate about the structural implications of the fact that yM,, L8.y has two combining sites, only one of which can be saturated at a time. It suggests a model in which the combining sites are close to each other. It makes unlikely a model-such as that proposed for rG (24)-in which.the combin- ing sites are at opposite ends of a rigid cylindrical molecule. Recent electron micrographs are most compatible with a Y-like structure for rG in which the two arms may move with respect to each other (25,26). In other immunoglobulins the two arms may be more closely apposed. The recent finding that, in one of the subclasses of human yA as well as in six specimens of murine yA, the light chains are disulfide-linked to each other (27) is relevant. Although such a linkage has been excluded in those human yM’s so far examined the above data provide some support for a model in which the Fab fragments are closely aligned. The best micrographs of yM (28, 29) are compatible with such a model.

Primary Versus Secondary TM,,,-yG Interactions-The results presented here lead us to the following conclusions with respect to the interaction of 7MLay with rG. We assume for this dis- cussion that the equilibrium constant determined for the Fab fragments is the same as that for the sites as they exist in TM. The primary union between the combining site and the antigenic determinant is energetically similar to that found with other

2 The tryptic digestion of unreduced YML*~ (and other human +yM proteins that we have studied) is incomplete under the condi- tions used here. The Fab fragments are, therefore, not quantita- tively released from all of the molecules in the digestion mixture. While all of the Fab fragments which are released are active the possibility (admittedly unlikely) existed that only active frag- ments were being released. The digestion of yM, can, however, be readily driven to completion and the resulting Fab (reduced) fragments were all active. In the actual experiment some 24yo of the protein precipitated during the digestion for unknown reasons. Sephadex G-200 chromatography of the soluble products resulted in a full yield of Fab fragments, 90% of which were bound in an ultracentrifuge experiment.

3 In a previous communication we briefly described studies on a rheumatoid factor (human anti-human rG antibody), ~MB... This preparation was known to consist of a heterogeneous popula- tion of rM antibodies and was of interest for comparative purposes with respect to ~ML~~. Preliminary results suggested that only one-half of the Fab fragments of YMB*~ were active. A low bind- ing constant and considerable intermolecular heterogeneity were, however, considered to be possible causes (6). While we have never been able to get more than about 50yo of the Fab to bind to rG, we have found that those Fab which do bind have a binding constant less than 104. We are unable, therefore, to come to any conclusions regarding the valence of YMB%~.

protein antiprotein systems (Table II) (16, 17). It shows only a moderate dependence on temperature and is quite insensitive to pH variations near neutrality and to differences in ionic strength. When rG is added to yM the sites become progressively satu- rated and higher order complexes are built up. Because the antigen is effectively univalent (Fig. 2), rM(rG)5 is the largest complex that can form. With ordinary heterogeneous, multi- valent antibodies reacting with a multivalent antigen, compli- cated molecular matrices are formed (30-32), but this is clearly not possible with the system under consideration here. At the concentrations at which the precipitation studies were conducted (4 X lo+ M yM, 2 X lo+ M effective combining sites) we expect only 1 y0 of the yM sites to be saturated when 2 moles of yG are added per mole of yM. Consequently, a maximum of 5% of TM should precipitate. That instead 75 to 80% precipitated under these conditions (upper curve, Fig. 7) shows that the in- soluble complexes are largely taken out of the equilibrium, thereby shifting the reaction toward completion. It seemed to us clear, therefore, that the sensitivity of the precipitation re- action to environmental influences largely reflected the solubility properties of the complexes. We predicted on this basis that at higher pH and ionic strength complexes larger than 1 :l, yG:yM, would be required to obtain precipitation. The data shown in Table III confirmed this prediction.

The marked influence of ionic strength and pH on precipita- bility is thus explicable by the limited size of the complexes which can form and by their solubility characteristics. It clearly does not reflect the properties of the primary antibody site-antigen interaction. A similar explanation may be valid for other cryoprecipitating systems, particularly those in which yM and rG are present together (so-called “mixed” cryopro- teins) and where the yM component is the reactive species (33, 34). Until the Fab portions of the yM have been directly im- plicated in this reactivity and an appropriate stoichiometry shown, one cannot be sure that this is true antibody activity, but it seems likely. By and large these reactive yM’s are hetero- geneous; they contain both K and X light chains. It is striking, however, that many of them show very similar precipitating properties to those of yMLay; i.e. precipitation is extremely sensitive to pH and ionic strength as well as to temperature. It seems possible, therefore, that in these instances too, the rG “antigen” has only a single effective determinant, thereby limit- ing the size of the yM-yG complexes which can form. Under physiological conditions these small complexes will remain solu- ble in the serum. Upon exposure to cold in peripheral parts of the body, precipitation of these complexes could produce the clinical signs and symptoms noted in some patients with such yM proteins.

REFERENCES

1. EISEN, H.N., LITTLE, J.R., OSTERLAND,~. K., AND SIMMS, E. S., Cold Spring Harbor Symp. Quant. Biol., 32,75 (1967).

2. BEaUMONT, J.-L., Compt. Reid. He&L Seances,.264, 185 (1967). 3. METZGER. H., Proc. Nat. Acad. Sci. U. S. A.. 67. 1490 (1967). 4. Bull. Wo;ld Health Organ., 30, 447 (1964); imrkunoch‘emistry,

1, 145 (1964). 5. STONE, M. J., AND METZGER, H., J. Immunol., in press. 6. STONE, M. J., AND METZGER, H., Cold Spring Harbor Symp.

Quant. Biol., 32, 83 (1967). 7. MILLER, F., AND METZGER, H., J. Biol. Chem., 240,3325 (1965). 8. FAHEY, J. L., AND HORBETT, A. P., J. Biol. Chem., 234, 2645

(1959). 9. MILLER, F., AND METZGER, H., J. Biol. Chem., 240,474O (1965).

10. MILLER, F., AND METZGER, H., J. Biol. Chem., 241,1732 (1966).

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Dow

nloaded from

5984 Walden&&n Macroglobulin Antibody Vol. 243, No. 22

11. HELMKAMP, R. W., GOODLAND, R. L., BALE, W. F., SPAR, I. L., AND MUTSCH~ER, L. E., Cancer ties., 20; 1495 (i960).

12. DEUTSCH, H. F.. THORPE. N. 0.. AND FUDENBERG. H. H.. lmmun&ogy, 6; 539 (1963). ’

13. FAHEY, J. L., AND MCKELVEY, E. M., J. Iwrmunol., 94, 84 (1965).

14. NUENKE, R. H., AND CUNNINGHAM, L. W., J. Biol. Chem., 236, 2452 (1961).

15. STONE, M. J.. AND METZGER. H.. J. Biol. Chewz.. 243, 5049

17. SINGER; S. j., AND CAMPBELL, D. H., J. Amer. Chem. Sot., 77,

(1968). ’ ,

16. SINGER, S. J., AND CAMPBELL, D. H., J. Amer. Chem. Sot., 77,

4851 (1955).

3499 (1955).

18. JOSEPHS, R., AND HARRINGTON, W. F., Biochemistry. 6. 3474 (1966):

“,

19. KEGELES, G., RHODES, L., AND BETHUNE, J. L., Proc. Nat. Acad. Sci. U. S. A., 68, 45 (1967).

20. TENEYCK, L. F., AND’ KAUL&NN,‘~., Proc. Nat. Acad. Sci. U. S. A., 68, 888 (1967).

21. ONOUE, K., YAGI, Y., GROSSBERG, A. L., AND PRESSMAN, D., Immunochemistry, 2, 401 (1965).

22. CLEM, L. W., AND SMALL, P. A., JR., J. Exp. Med., 126, 893 (1967).

23. MERLER, E., KARLIN, L., AND MATSUMOTO, S., J. Biol. Chem., 243, 386 (1968).

24. EDELMAN, G. M., AND GALLY, J. A., Proc. Nat. Acad. Sci. U. 6’. A., 61, 846 (1964).

25. FEINSTEIN, A., AND ROWE, A. J., Nature, 206, 147 (1965). 26. VALENTINE, R. C., AND GREEN, N. M., J. Mol. Biol., 27, 615

(1967) . 27. GREY, H., ABEL, C., AND KUNKEL, H., Fed. Proc., 27, 617

(1968).

29. CHESEBRO, B., BLOTH, B., AND SVEHAG, S.-E., J. Exp. Med., 127, 399 (1968).

28. SVEHAQ, S. E., CHESEBRO, B., AND BLOTH, B., Science, 168,933

(1967).

30. MARRACK, J. R., The chemistry of antigens and antibodies, H. M. Stationary Office, London, 1934.

31. HEIDELBERGER, M., AND KENDALL, F. E., J. Exp. Med., 62, 697 (1935).

32. SINGER, S. J., J. Cell. Comp. Physiol., 60, 51 (1967).

33. KRITZMAN, J., KUNKEL, H. G., MCCARTHY, J., AND MELLORS, R. C., J. Lab. Clin. Med., 67,905 (1961).

34. MELTZER, M., AND FRANKLIN, E. C., Amer. J. Med., 40, 828 (1966).

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Marvin J. Stone and Henry MetzgerBinding Properties of a Waldenström Macroglobulin Antibody

1968, 243:5977-5984.J. Biol. Chem. 

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CORRECTIONS

In the paper by S. S. Subramanian and M. R. Raghavendra Rao (Vol. 243, No. 9, Issue of May

10, 1968, page 2367), the authors have submitted the following acknowledgments.

Acknowledgments-We are thankful to the Society of the Sigma-Xi and to Dr. I. C. Gunsalus for the gift of numerous biochemicals.

In the paper by Marvin J. Stone and Henry Metzger (Vol. 243, No. 22, Issue of November 25,

1968, page 5977), the values of AH0 and AS0 for the binding of Fab ray to yG were incorrectly calcu-

lated to be 2 kcal mole+ and 26 e.u., respectively (Table II, page 5981). The correct values are

AH0 = -7 f 1 kcal mole-r and ASo = -5 f 1 cal deg-1 mole-r. While the over-all conclusions

do not thereby require revision, it is clear that these values are more similar to those seen in some anti-hapten systems than those obtained with the anti-protein systems referred to in the article.

In the paper by E. E. Brumbaugh and G. K. Ackers (Vol. 243, No. 24, Issue of December 25, 1968, page 6315), on page 6323 the fraction line was inadvertently dropped from Equation 8 so

that the equation should read as follows.

v, - vo K, = - Vt - vo (8)

780