of Medical


and J. STEENSGAARD University

of Aarhus,

8000 Aarhus

C, Denmark

Abstract-Precipitin reactions are traditionally explained as macromolecular interactions giving rise to very large molecular lattices or frameworks that due to their very large size are insoluble. We have questioned this concept experimentally by comparing the reactions of native and succiny~ated IgG (20 ‘I;, modified lysine residues). By use of a variety of different techniques we have found that (1 f succillylation of IgG does not change the principal ability of IgG to reitct with its antigen. (2) succinylation of IgG gives rise to altered prec&tin behaviour in the biologically important mild antigen excess region, (3) difference turbidity measurements of precipitin reactions in antibody excess show highly significant differences, (4) zonal centrifugations of selected immune complex mixtures show that succinylated complexes are more water soluble than complexes formed of native IgG and (5) theoretical calculations show that very large complexes (as typical lattice or framework aggregates) are thermodynamic unlikely. It is a unique feature of typical precipitin reactions that no free antigen is present before the equivalence zone. To explain this it is necessary to introduce two different molecular mechanisms or stages. in the first (and immune specific) stage of the reaction, a series of composition~~liy different complexes is formed. In a second stage some of these complexes create a hydrophobic phase by non-imn~une-specific inte~dctions. The remaining smaller complexes will be distributed between the two phases. According to this hypothesis the distinguishing absence of Free or complexed antigen in solution before the equivalence zone can be explained.


Precipitin reactions form the basis of numerous analytical laboratory methods. Yet, the molecular mechanisms leading to formation of immunological precipitates are not fully understood. Already, Marrack (1938) discussed whether or not the precipitin phenomenon could be explained solely by immune specific interactions. Theoretical considerations have led us to suggest that at least two different molecular mechanisms are involved in immunological precipitin reactions (Steensgaard & Frich, 1979). The present communication deals with an experimental study of this problem using human serum albumin (HSA) and rabbit anti-HSA IgG as antibody. To see whether or not the overall reaction can be experimentally divided in more stages, i.e. one stage involving only immune specific bonds, and a second stage involving non-specific reactions, we have conducted a series of comparisons between the reactions of isomolar concentrations of native and succinylated IgG. The precipitin reactions have been followed precipitated Steensgaard, supernatant,

by measurement of total precipitate, antigen, difference turbidity (Jacobsen & 1979), measurement of antigen in the and analysis of soluble complexes by

zonal centrifugation (Steensgaard & Funding, 1971). The results indicate that two different molecular mechanisms actually are involved in precipitin reactions, and that succinylation of IgG selectively decreases the velocity of the second stage of these reactions. 571

IgCi (rabbit anti-human serum albumin, DAKOPATTS, A/S. Copenhagen, code No. 10-001, lot No. 0086) (5.0 ml) with an initial protein concentration of approx C mg/ml was diluted with 100 ml I A4 NaHCO, buffer, pH 8.0 to give an IgG concentration of about 2.7 FM. Succinic anhydride (300 mg) was dissolved in 2.0 ml dry dioxane, and 1300 ~1 of this solution was gradually added to the continuously stirred IgG solution. The reaction was allowed to proceed for 1hr at 20’ C maint~~ining pH at 8.0 by adding 0.2 N NaOH. The final concentration of succinic anhydride was 18.6 miW. Finally, the whole portion was dialysed against 0.006 /M borate buffer, pH 8.4, 0.15 M in NaCI.

Native IgG was dialysed against the borate buffer and diluted with the same buffer to give an extinction of 0.866 at 280 nm as the succinylated IgG solution did have. Thus, the concentrations of the two solutions were identical as succinylation takes place only on amino p.-oups of lysine residues leaving tyrosine and tryptophane unaffected, and hence leaving extinctions at 280 nm unchanged (Jacobsen. 1972). The percentage of succinylated amino groups was subsequently determined fluorometrically (AmincoBowman Spectrophotofluorometer) using fluorescamine (Fluram ‘, Roche) and unsuccinylated IgG as standard (Bijhlen eI ol., 1973). With the concentrations used here 20”,, lysine residues were succinylated.

The concentration of specific antibody was determined from the decrease in E,,, in the supernatant at the point of maximum precipitation, where no free HSA exists (cf. Fig, 2). The maximum decrease in E,,, for both preparations was




0.183. Supposmg ~~‘:,;, -z_ I5 (Sober, 1970) and mol. wt 140,000, the isomolar concentration of specific native I@ as well as of spcc~lic succinylated lgcl \\as therefore taken as 0.9 bt,Ylm the final mixtures. The total IgG concentrations in the final mixtures were in all citscs ’_. 1 @f.

1’wu/‘1llll (‘111’1 C’, l’recipttin CLI~-LC\were produced as described prc\lously (Stccn\~aard r’r (ii.. 1975). Aliquots of300 /,I of HSA and IgG wtrc u\ed an alI cxpcriment\. The HSA concentrations are glccn in the text ;~nd legends to ligures whenever needed. lodinatcd ( ’ “I)-HSA (Amcrsham. code IBt 7P) was alk>i;tted and monomerllcd as described prebiously (Hail ct Stecnsga;lrd. 1971 1. In diil’crence turbidimetric moasurments :I corresponding cold iodinated HSA preparation was used tJacoh\en & Steensgaard. 1979). Radioactivity was measured in ;I P;lckard-g;lmma-spectl-onl~lcr. All radioactive data was recalculated to the data of purchae.

The method used has been described in details previously. An ,4minco I3W-2 sp~ctrophotometcr in split beam mode, and ri set of matched tandem quart7 cuvettes (Hellma 23X) lvere u\cd (Jacobsen tl: Steensg~lard. 1979).

All tonal centrlfugation c%pcriments were performed with a B-XIV ronal rotor of Andersons destgn (Anderson. 1966, 1968). obtained from M.S.E. it‘rawlcq. U. K.). An isokinetic sucr*se gradient (3-21”,, u ‘+) was calculated as described previously (Stccnsgaard, 1970). The overlay volume was IO0 ml, the sample volume 7.0 ml. and the rotor temperature 8 C. Centrit’ugation w;ts iontinucd at 46.000 re\:min until ;tj,~~~G dt-value ofO.ZX x IO” set was reached. Th~acorrcspnnd~ to ;I run time at m~~\m~um apecd of 3 hr. Fraction of9 ml were collected on ;t lunc ha\is. Samples were incubated l’ol- 30 min at 37 C‘ bcforc ccntrifupation.

Equl\alent sedimentation coefficients were calculated from the formula of Martin and Ames (1961) by numerical integration as described hy Stecnsg:t:rrd. Mecller and Funding (1978). The presentation of results in Fig. 4 was drawn directly by the computer. In these figures mathematical corrections arc made for sectorial dilution in the B-XlV zonal ro1or. Hence. the areas below these cur+es are directly integratable.

To make equations (I) and (5) equal in form LI must be ?./3, whereas h is defined b) the following expression (after insertion of all constants) and cletining ,\ in Svcdherg unit\ IO’ = 1.1962 IO


It follows I’rorn equation (6) thus h ib ;I rather complicated function of I; and (//,h,). Bccausc interpretations concerning complex sizes depend very much on this rehttionuhip we have calculated the h-isotherms shown in Fig. I. For h-values inside the area of interest this tigure give5 i- and (//frl)+aluca that arc inherent to a givol h-values. It is of special interehl that because (f,f,,) is likcl~- to increuscs l\ith incrcnsing complex SIZL’, it follows that r must decrease to malntaln II tixed h-value and hence the density of the comple\ mu&t Incre3se3ccordingl~. II should finnlly be noted that the hea\> antigen excessexperiments shown later (Fig. 4) indicate th,tt ;I h-value of -2.5 &Ives the best numerical tit of the experimental results. and this value )h thcreforc u\ed to estimate complex sizes. The shaded a~e;t of Fig. I includes ~hc arca in which it IS most likely that the true h-value ih to be found.


curves of’ native and in isomolar concentrations were made, and are compared in Fig. 2(a). It appears from this part of the figure that the ascending parts of the curves are identical. Moreover, the two precipitin curves reach rna~inlunl of the same height and at the same antigen concentration. We take this appearance of the two precipitin curves as evidence of that mild succiny~~~tion does not harm the principal capability of Conventional




b - ksotherms




IO& s = a log (M) + b


involves the problem of esCm;ltlng u and h. The Svedberp equation (Svedberg Pr Pedersen. 1940) say5 .\ = M(

1- t’p,/ f .f)





where p is the partial specific volume. /J the density of the media, 1. Avogadros number. and f the frictional coefficient. Inserting (Tanrord. 1961)


0.68 1.0

where,! is themscosity andR IS Stoke’sradius,and defined by the expression

IettmgR be









Fig. I. h-Isotherms for equivalent sedimentation coefficients calculuted from equaGon t I ). The constant (0) is physrcally fixed as 0.67: h is a complicated function of i: and //ii, (equation (6)). The b-isotherms give those paired i and f/r,,values that are inherent to II given h-value. The shaded area deptclts the area of special immunolo~ic~ll interest.




0 20

c $ N


0 16

0 I2



gives rise to a more efficient precipitation of HSA than does succinylated IgG. This difference continues in the region of antigen excess. The appearance of HSA (in free and/or in complexed form) in the supernatant was measured too. The results are also given in Fig. 2(b) and show an agreement with the radioactive precipitin curve.

s 9 5


9 004




/// 15





0 02






0 06

, mg/ml

Fig. 2. Precipitin curves of native and succinylated IgG obtained by four different techniques. The symbols (o), ( n) denote native IgG and (o), (0) denote succinylated IgG. In all experiments the total IgG concentration was 2. I /1.44in the final mixtures; (a) curves obtained by measuring absorbance at 280 nm of precipitates redissolved at pH 2.2; (b) (O), (0) curves from measuring radioactivity ofthe iodinated antigen in the precipitate, and ( n), (CJ)in the supernatant; (c)curves obtained by measuring difference absorbance at 260 nm after turbidity has been allowed to develop IO min.

IgG to react with its corresponding

antigen. However, in moderate antigen excess (on the descending part of the precipitin curve) a marked difference is apparent. Native IgG produces more precipitate than does succinylated IgG. The use of radioactively-labelled and purified human serum albumin gives the possibility of following the fate of the antigen in these precipitin reactions. The results of these measurements are shown in Fig. 2(b). It appears that the shape of the precipitin curve in antibody excess is the same for both kinds of IgG. However, an interesting difference occurs in the zone of equivalence where native IgG

Difference turbidity precipitin curves were measured, and the results are shown in Fig. 2(c). Final turbidity recordings were taken after 600 set of reaction and at 260 nm. It appears from this tigure that in succinylated isomolar native and IgG concentrations give rise to two markedly different difference turbidity precipitin curves. The difference turbidity of succinylated IgG is at a whole half the size of the difference turbidity of native IgG, although they have maximum at the same antigen concentration. It is of particular interest that the difference turbidity precipitin curves reach their maximum at antigen concentrations where the amount of precipitate [cf. Fig. 2(a)], and the amount of precipitated antigen [cf. Fig. 2(b)] do not show any differences. This indicates in itself that native and succinylated IgG with identical capability of precipitating their antigen exhibit differences in mechanism in at least one stage of the overall mechanism. The development of difference turbidity with time for identical mixtures of HSA and either native or succinylated IgG also show marked differences. When measured in the minute range [Fig. 3(a)] the reaction velocity is clearly decreased by succinylation. When measured over 3 hr [Fig. 3(b)] the difference is also clear. The maximum difference turbidity of succinylated IgG corresponds to about 213 of the maximum of native IgG. However, both preparations show a development ofdifference turbidity that follow similar patterns. Inside less than half an hour they reach their maximum difference turbidity followed by a period of stable difference turbidity, and after 3 hr they both show a disappearence of the difference turbidity signal nearly synchronously. The examples shown in Figs. 3(a) and (b) correspond to the point of maximum precipitation in Fig. 2(c). Zonal centrijtigation A series of zonal centrifugations were carried out to compare the formation of soluble antigen-antibody complexes for both preparations. The sample compositions are marked with arrows in Fig. 2. The computer processed results are shown in Fig. 4. Equivalent sedimentation coefficients were calculated as described under Materials and Methods. Inside each part of the figure the highest point have been set equal to loo’&, and all other points are given in relation hereto. It appears from Fig. 2 that the total amount of sample material was largest in the upper example (a), and the total amount of complexes decl.eased drastically as equivalence was approached. The following parameters were used for Table I: a = 2/3 and h = -2.5. The mol. wt of human serum albumin was taken as 66,500 (Meloun c’tal., 1975), and that of rabbit IgG as 140,000 (Mamet-Bradley, 1970). The following features should be noted in Fig. 4: (i) The amount ofcomplexes formed of succinylated





















I-ig. 3. The daelopmcnt ofturh~d~ty In :I HSA-IgG mixture in dependence on tlmc. The lower curves in (a) as well II> in (b) arc for \uccinylated IgG. The concentrations of HSA were 0.015 mgml and oflgG. natlbe or 5uccinylated. 2. I u.M.

IgG is in all cases larger than the amount ofcomplexes formed of native IgG. This agrees with the finding that succinylated IgG forms less precipitate than does native IgG. This supports the idea that succinylated IgG have retained its ability to form complexes with IgG, but the complexes are seemingly more soluble in an aqueous phase than corresponding complexes of native IgG. (ii) In case of native IgG the I5 Speak is consistently lower than the 5 S peak. In case ofsuccinylated IgG a contrasting growth of the size of the I5 S peak in relation to the 5 S peak can be seen when approaching equivalence. (iii) The first peak in all cases has ;I sedimentation coefficient of 5 S and represent free and unreacted HSA. There is in all cases also ;I clearly dominating peak with a sedimentation coefficient of about 1.5 S. Comparison with Table I lead to the suggestion that the 15 S peak consists of complexes containing 1and 2 antibody molecules. Thcsc rather small complexes

seem to be a consistent part of the outcome of reactions between HSA and rabbit-anti HSA IgG. (iv) Larger complexes are formed also in both preparations. but there is a clear indication of that the amounts of complexes formed in these reactions decrease with increasing complex size. This finding is in accordance with the thermodynamic predictions shown in Table 1, being calculated as described by Steensgaard c/t nl. (1977).. (v) In example (a) the major part of the complexes have sedimentation coefficients below 40 S. Most complexes are actually smaller and contain only ;I few antibody molecules. Large complexes, as for instance those containing 8 antibody molecules are formed only in minute amounts. In the two other examples a relative shift in the direction of somewhat larger complexes can be seen. Yet. the larger complexes are still formed in lesser amounts than small complexes. (vi) The kind of complexes formed of both preparations appear similar, and the differences

Table I. Calculated equivalent scdimentatlon coefficients of HSA-anti-HSA IgCi complexes grouped according to the number antibodymolecule\ in the complexes. The antigenic valence was taken as 8. The table also gives the thermodynamic most likely antigen content at Ag-Ab ratios of I:3 and I:l. The numbers are calculated as precentages making them comparable to the experimental results in Fig. 3


No. of antibody m the complexes

Smallest II I6 20 23 26 30 33 35 38 41 43 46

Equi\alent \-\:ilues (Sbedberg units) Largest I3 IS 25 29 34 3x 42 45 49 53 56 59

Amount Average 12 IX 23 26 30 34 38 40 44 41 48 53

(in ” ,,) of antigen In complexes

Antigen-antibody= 6.86 8.07 X.38 X.25 7.92 1.53 7.13 6.73 3.92 3.70 3.49 3.29


Antigen-antlbody x.49 IO.81 I I.53 11.14 10.09 x.75 7.35 6.03 4.84 3.84 3.01 2 33












15 Sedimentation





30 coefficient,







I 60


Fig. 4. Computerized analyses of the size distributions of HSA-rabbit-anti HSA complexes obtained by zonal centrifugation in a B-XIV rotor. The abscissa gives the equivalent sedimentation coefficients in Svedberg units. The ordinate gives the radioactivitv (from the ‘311-labelled HSA) as percentage of the maximum ialue in each part figure. The upper curve in each part figure qhows complexes of succinylated IgG. The HSA concentrdtlons were (a) 0.06 mg/ml, (b) 0.04 mgjml and (c) 0.035 mg/ml in the final samples. The samples correspond to the points denoted by arrows in Fig. 2. between zonal profiles of succinylated and native IgG complexes are likely to reflect differences in the amounts formed rather than a change in types of complexes. It should be noted here that Table 1 includes all the complexes that can be formed in this system. DISCUSSION

The precipitin reactions of HSA and rabbit-antiHSA IgG exhibit two distinguishing features. The first



is a very clearly expressed zoning phenomenon, and the second is a complete precipitation of HSA in antibody excess and until the point of maximum precipitation. Both features are clearly expressed in Fig. 2. Reactions of this kind are traditionally explained as reactions leading to the formation of very large molecular lattices or frameworks that due to their size are insoluble in an aqeous phase (Marrack, 1938; Pauling, 1940; Day, 1972). These very large molecular aggregates are supposedly formed exclusively by bonds between antigenic determinants and corresponding antibody binding sites. We have questioned the validity of this hypothesis in different ways. Previously, thermodynamic considerations have shown that very large complexes are unlikely as the sole product of antigen-antibody interactions (Steensgdard rt a/., 197.5, 1977; Steensgaard & Frich, 1979). If larger complexes are formed, smaller ones should exist in detectable amounts also, as seen in Table 1. The absence of free or complexed HSA in the supernatant on the antibody excess side of the equivalence zone is therefore difficult to explain. Moreover, studies of the development of difference turbidity with time have indicated that the total set of interactions comprises some fast reactions (second range) and some substantially slower (minute range) reactions (Jacobsen & Steensgdard, 1979). The present experimental study was undertaken to investigate whether or not other interactions than the purely immune-specific interactions are involved in precipitin reactions. Succinylation of IgG was chosen because it changes positive surface charges into negative ones. Thus, if succinylated IgG could retain its ability to form complexes with HSA, but differences in its precipitating behaviour could be observed, the involvement of non-immune-specific interactions would be strongly indicated. Conventional precipitin curves show that native and succinylated IgG produce the same amount of precipitate in antibody excess. The precipitin curves reached maximum of the same height and at the same antigen concentration. Both preparations evoked a complete precipitation of HSA in antibody excess. In slight antigen excess less precipitate is formed of succinylated IgG, but correspondingly, larger amounts of soluble complexes are formed, as judged from zonal centrifugation experiments. As a whole we take these results as evidence of that succinylation as described here is only little if at all harmful to the reactivity of IgG. Difference turbidity measurements revealed considerable differences between the precipitating behaviour of the two IgG preparations. These differences are especially interesting because they appear in the region of antibody excess where the other precipitin measurements gave identical results for both preparations. Differences in precipitating behaviour of succinylated and native IgG were observed also in the antigen excess region. Succinylated IgG gave rise to less precipitate and precipitated less antigen than native IgG. The differences between reactions of succinylated IgG and native IgG in antigen excess may be a clue in the identification of those complexes that are precursors of the precipitate. The zonal centrifugation




experiments show that the same size range 01 complexes are formed of both preparations. but substantially more of the soluble complexes are formed of succinylated IgG. Precursor complexes are believed to be those that precipitate when native IgG is used but arc soluble when succinylated IgG is used. It appears from Fig. 4 that these are surprisingly small complexes ranging from an antibody content of one molecule to a maximum of IO, but decreasing in amount with increasing complex size. Such complexes can speculatively turn insoluble in two different ways. One possibility is that they grow further by mutual specific interactions (involving determin~lnts and binding sites). and the other is that they crate a hydrophobic phase (the precipitate) by 21confluence process involving parts of the antibody molecule outside the binding sites. We think that the first possibility is less likely, because immune specific growth of complexes would imply that larger complexes should be formed in increasing amounts. oppositely of what is found experimentally. Moreover, the absence of free antigen and small complexes at equivalence can only be explained if these participate in another type of reaction, like a phase separation process. Finally, succinylation will take place randomly, and only few groups are likely to hit the binding sites. The changed precipitating behaviour of succinylated IgG is therefore believed to be due to changes in non-imnlune specitic interactions. The findings presented here are in accordance with this concept. The results presented here together with the previously mentioned thermodynamic collsider~tions lead us to propose that two different mechanisms in two different stages are required to explain the features of precipitin reactions. In the first stage. a series of compositionally different complexes will be formed. In the second stage a hydrophobic phase eventually appearing 3s the precipitate, will be formed by nonimmune specific confluence processes of complexes. The zoning phenomenon arises because some complexes (having inherent excess antibody hydrophobic properties) are required to initiate the confluence processes in the second stage.

A&riou tr%$?~lc~rr,.uThi\ by The Danish Medical Foundation.

pork ha\ hccn hupporled rn part\ Rc~earch C~wncrf ami the D&l The skilful irchnic;ll ~~~~isi;~nce 01‘ M\. A. M. I. Kjrrrgaard and ILI. Hansen is very much

Bundsgaard, appreciuled. Mr. ivmputattonal

J. K. I rich work.

hi15 ~C’II

Evidence of a two stage nature of precipitin reactions.

EVIDENCE OF A TWO STAGE NATURE OF PRECIPITIN REACTIONS C. JACOBSEN Institute of Medical Biochemistry, and J. STEENSGAARD University of Aarhus, 80...
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