MOLECULAR STRUCTURE AND BIOLOGICAL SPECIFICITY duly 17, 1647, 2:50 P.M. Main Theater, Royal Institution By Linus Pauling President of Section IX of lith International Congress of Pure and Applied Cheaistry The most striking and characteristic property of biological substances ia the specificity of activity which they show ~ the power to combine selective~ ly with or to influence the behavior of one substance, rejecting others with a precision and certainty seen in few physical and chemical phenomena, This specificity of activity fe shown by genes in their ability to reproduce them- Selvea, usually unchanged, and to produce the ensymes or other producta through which they detcrmine character} also by engsymes, which select from a mixture the molecules upon which they exert their catalytic action, by hormones, by therapeutic agents, and, in an especially striking way, by antibodies, A complete and reliable understanding of the phyeicochemical basis of biological Specificity would bring us auch nesrer to the solution of the great funda- mental problem of biology, that of the nature of life. My om interest in biological problems was formed first by a study of hemoglobin made a dozen years ago, which led to the discovery that hemo- globin itself (ferrohemoglobin) is paramagnetic, whereas oxyhemoglobin and sarbonmonoxyhemoglobin are diamagnetic. It was found that the magnetic prop- erties of hemoglobin provide a simple physicochemical methoi of neseuring ~Z— equilictria end rates of reaction involving hemoglobin and its derivatives, and the magnetic technique has since been found useful aleo in the atudy of cyto~ chrome ¢ and other fron-containing proteins, Drs Alfred Mirsky and I then de- veloped a theory of the denaturation of proteina,+ based upon the concepts that in a native protein the polypeptide chains are coiled together into a definite structure, with a configuration which determines the specific properties of the protein, and that heat, alkali, urea, send other densturing agents and conditions may cause the configuration of the polypeptide chain or chains to be altered, without necessarily breaking any peptide bonds or otherwise changing the covslent- bond structure of the molecules. Se pointed out that some protein molecules in the native estate might have thet configuration of coiled polypeptide chains which is the most stable of the configurations accessible to the chains, and thet these proteins might be capable of undergoing reversible denaturation, with the chains coliing back into the stable configuration characteristic of the native protein as the denaturing agent or condition 1s slowly removed. Other native proteins, however, might be built originally into a configuration which is not the acst steble of those accessible to the polypeptide chains; these protecing, when de- natured by uncoiling their chains, would not, on removal of the denaturing agent, settle into the original native configuration but instead into the postulated more stable configurations accordingly a protein of this sort would not undergo reversible denaturation, regenerating the original native protein, although it might be denatured and then renatured to give a well-defined and cr: stallizable protein, with properties different from those of the native orotein, PENT EETY A, E. Mirsky and Ly Pauling, Proce Nat. 4cad. Belo, 2a, 459 (1986). sw See When, in 1986, I became interested in the problem of the structure of antibodies, as the result of conversations with Dr. Karl Landsteiner, I found that the complex and at first confusing reported phenomena of immunology could be clarified and brought into order by a theory of the structure of antibodies based upon the idea of the folding of polyp:ptide chains into the most stable of the accessible configurations.* The theory of the structure and process of form ation of antibodies developed in thia way involved the acceotance of the sug- gestion that antibody and antigen have complementary structures, originally sade by Breinl and Haurowits, J. Alexander, and Stuart muda,® The picture of the serological precipitate as « tranewoik of sultivelent antibody and multivalent antigen, developed by Marrack and by Heidelberger, * also seemed to be so rea~ sonable and so in accord with rost of the observational data as to require its acceptance, “ The theory of the structure of antibodies and the process of antibody formation dependa upon the assumption that the antibody precursor is a polypep~ tide chain of such 1 nature as to be able to fold into a large number of alter- native configurations, which have nearly equal free energy, and hence nearly equal stabllity. Im the absence of an antigen in the region in which the anti- bedy precursor ie being formed the polypeptide chain will fold into one of the moat stable of the configurations acceptable to 1t, producing a nolecule of normal gamma globulin. However, if an antigen molecule is present, it met be considered as part of the environment acting upon the folding of the polypeptide chain, and the cost stable configur:tion of those accessible to the folding 1, Pauling, J+ Ams Ghem. Soce, 62, 2645 (1940). 38. Breinl and F, Haurowits, Zeit. physio}. Chem. » 192, 48 (1980); J. Alex- ander, J. Protoplages, 14, 296 (1 Judd, J. Immupole, 28, 428 (1982). 43, R. Marrack, "The Chemistry of Antigens and Antibodies," Report 280 of the Medical Research Gouncil, H.M. Stationery Office, London, 1954; 2nd ed., 1938; M. Heidelberger et al., J. Exp. Meds, G1, 565 (1988); Shem. Revs, 24, B28 (1959). ~4- chain now become different from those in the absence of the antigen; they are now configurations which take the greatest advantage of the opportunity of inter~ action with the antigem, of such a nuture aa to atabllise the system - that ia, of the opportunity of assuming such 4 structure as to lead to attraction between the forming antibody and the antigen, and hence to the form:tion of an antigen- antibody bond, A structure of this sort would be one in which the surface atoms | of the antibody molecule are able to come into the closest possible proximity to the surface atoms of the antigen molecule. This could be achieved in case that the folding antibody molecule were to mold itself over a portion of the surface of the antigen molecule, reproducing the configuration of the antigen in the game way as 8 coin docs its die. The principal forces of attraction which are operative are the general van der Waals forces (electronic dispersion forces), the forces described. as hydrogen-bond forces, and the electroststic forces be- tween positively charged and negatively charged ionised groups. A very high degree of specificity can be obtained if the surface area over which the com plenentariness in structure ip exercised is great enough to inelude a good nua- ber of Interacting structural unites, The assumption that antibodies are bivalent, or have still grester valence ~- that is, that each antibody molecule has two or sore surface regions eapable of combining specifically with the homologous entigem ~ is necessary in case, the framework theory of the serological precipitate is socepted. The gen- eral evidence, of varied nature, for the framework theory, as suamarized by Mar- rack and Heidelberger, is strong bit not complete, Further evidence was ob- tained by studies aacs ty my collaboretors(Professor Dan H. Ccmpbell, Dr. Devid Preseman, Dr. Carol Ikeda, Dr. M. Ikava, Mr. David R. Brown, Mr. de Le Grose~ berg, Dr. Stanley M, Swingle, Dr, Jobn T, Maynard) and myself by the study of ~S~ the precipitation of antibodies with simple cheaical substances of known struc- ture, It was discovered by Landsteiner and van der Scheer® that a precipitate is formed when a dye nade by coupling two or sore haptenie groups with resorcinol or tyrosine is added to an antiserum obtained by injecting an animal with an asoprotein containing the same haptenic group, We investigated the interaction of many substances containing the para-asobensenearsonic acid group and anti~para- asobensenearsonic acid serum, and found thet all dyes containing two or nore of these hapteniec groups were able to form a precipitate with the serum, vhereas these containing only one haptenic group were not. It wae alao found that under certain conditions the ratio of the number of molecules of dihaptenie precipitat- ing antigen to the number of molecules of antibody in the orecivitate was equal to unity, and if the assumption is sade that each of the two haptenie grouns is operative in bond formation the bivalence of the antitody is proved, However, it was aleao found that the same molecular ratio of unity held for the crecipitate formed by trihaptentc and tetrahaptenic dyes. This confusing result can be ex- plained by the reasonable assumption that the steric interaction of the large antibody molecules about 5s anall dye molecnle is so great aa to prevent sore tha two antibodies usually from combining with the haptenic groups of the same dye molecule, the steric repulsion thus effectively limiting the valence cf the noly~ haptenic substance to two, and the data then indicate bivalence of the antibody, A deterainative experiment has also been carried out, involving the aimultensous precipitation of antibodies from two different antisere by a single substence, which is incapable of precipitating either of the antisera alone. The substance is a dye containing one haptenic group of each of tro different kinds, and the two Bx Landsteiner and J. van jer Scheer, Proc. eee « Meds, » 747 (1982)5 J. Exptl. Meds, 56, 399 (1982); brs 63 658 (1958), 67, a eet See ~~ specific antisera which when mixed are simultaneously precipitated by the dye are those made by injecting separate rabbits with asoprotcins containing, re- spectively, the two haptenic groups, This experiment provides very strong evi- dence for the framework theory of serological precipitation.® The precipitation of antibody and antigen is closely similar to that of, say, silver ion by cyanide fon, end the similarity extends also to the re-solutiog of the precipitate in an excess of one of the reactants (cyanide ion or antigen), The cyanide precipitate dissolves in an excess of cyanide lon because of the formation of a silver cyanide complex Ag(C)p , and similarly the antibody-antigen precivitate redissolves in an excess of antigen because the antigen molecules combine with both (or all) of the combining groups of the antibody, saturating thea and forming a soluble complex. It would be of inter- eat to physical chemists to investigate this reaction quantitatively, and to find whether the same simple lawe of cheaical equilibrium apply as to the silver cy- anide precipitation and re-solution. It is found that these Simple laws do not apply, but that inatesd the behavior of antisera and antigens is that which would be expected if the antiserum contained antibody molecules of many diff:rent kinds, with thelr combining groups differing by several kilocalorics per wole in free energy of combination with haptens, correaponding to a several hundred-fold or thousand-fold range in equilibrium constants for the combination reaction. The data indicate clearly that natural antibodies are very heterogeneous. This is, of course, to be expected from the theory of antibody production deseribed above, The nature of the specific forces operative between antigen and antibody has been investigated especially by the quantitative study of the phenomenon of hapten inhibition, A monohaptenic substance ia able to combine with antibody, but not te form a precipitate, Through combination with the antibody, however, the formation of a precipitate by a polyhaptenic substance cen be inhibited, This SL. Pauling, D. Pressuan, and D.H. Campeell,, J. Ame Qhem. Soce, 68, 330 (1944), wo Poe phenomenon of hapten inhibition was discovored by Landsteiner. Quantitative stud- ies of the inhibiting power of different haptena of known structure have been made, and subjected to physicochemical interpretation by the use of a theory of heterogeneous antibody, This theory 1s based upon the assuaption th:t the dis- tribution function for the heterogeneous antilody is an error function in the free energy of interaction of antibody and haptenic group. The sassuaption of en error function in the free energy (that is, in the logarithm of the equilibrium com stant) is seen to be a reasonable one by the argument that the total free energy of combination of the combining group of an antilody with the hapten aay depend upon several structural features, which may be present or «bsent independently of one another; if the number of structural featares were large, there would re~ sult an error function distribution in the free energy of Interaction with ant4i- body, to which they sake their independent additive eontributions.” It has been found that the hapten inhibition constants of different haptens depend very strongly upon the degree of conformity in shape of the haptens “@ the immunizing haptenic group. The requirement for Similarity in shape is eush that the conclusion can be drawn that the antibody reflects or reproduces, in a negative way, the shape of the haptenic group of the immunizing antigen to withis about 1 &, 8 Moreover, it has been found for a series of related haptensa contain- ing substituent groups in the position para to the charged group (the arsonie acid group) that the average equilibrium constants for combination with antibody denend significantly upon the optical polariaabllity of the para group, in the way indicated by the London thoory of electronic dispersion forces, the magnitude of the effect being such as to indicate approximation of the antibody to within 1 & of the haptenic group. A third test has been made, that of the contribution "he Pauling, D. Presaman, end A.L. Grossberg, J. dm. Chem. Socs, 56, 784 (1944), and later papera, a. Pauling and D. Preasman, Je da. Chen. des., &7, 1008 (1945). Gee of an electrical charge to the antibody~antigea forces.’ This test involves com parison of a hapten containing the trisethylammonium ion group and ome contain- ing the uncharged tertiary butyl group, and the determination of their hapten inhibition constants. The difference in free energy of combination indicated by these hapten constants can be expressed in terme of s distence between the positiv charge of the charged haptenic group and a complementary negetive charge of the antibody, with the use of effective dielectric constants as indicated by the in- vestigations of Schwaraenbach,# 2° The distance ao found is 7,0 i. Since the radius of the phenyltrimethylammonium ion isa 5,5 he, and the sinimum distance to which a negative charge could aporoach the surface of an antibody is le4 he (the radius of an oxygen atom), the value 7,0 i. shows that the complementary negative cherge of the antibody is within 2.1 i, of the minimus possible distence froa the positive charge of the immunizing heptenie group. This evidence also accordingly supports the thesis that the forces of specific attraction between antibody and antigen depend upon the very close approximation of the antigen and sntibedy mole- cules. - Por a long time there remained unrecognised a striking analogy between the highly specific phenomenon of serological interaction end another highly specific phenomenon of the chemistry of siapler substances; namely, the phen- omenon of crystallization. The process of the crystallization of a substance from a complex solution ia in general highly specific - often a very pure aub- Stance can be grown as crystals from a complex aixture, as is shown by the ex ample of the formation of pure crystals of cream of tartar from grape jelly. It ia clear that the sa@peificity of erystallisation is the result of the same in~ teratomic and intermolecular forces and the same striving toward complementaria- esa thst are resonsible for the spaificity of antibodies, A molecular crystal ‘ %p, Pressman, A.L Grossberg, LsH. Pence, and Le Paubing, J. Ame Chea. Soo, 68, 250 (1946). 104, schwarsenbach, Z phyaik. Chem», AL76, 155 (1986). =Qe is stable because all of the molecules pile themselves into a configuration such that each molecule is surrounded as closely as oossible by other molecules, in euch a way as to make the forces of sttraction of the molecules within the crystal ae grest se possible. This result is achieved if the cavity im the orya- tal into which each molecule fita conforas as closely as possible to the shape of the molecule, and if also there is a complementariness in structure, with respeot to hydrogen bond formation and fonic interactions, between the molecule and the surrounding molecules, Other molecules, with different shape and struc- ture, would not fit into this cavity nearly so well, and in consequence other molecules would not in general be incorporated in the growing crystal. Only if the other molecules were very similar to the molecules of the crystal would deviation from specificity occur, leading to the formation of solid solutions. It is well known, for example, that organic compounds containing sethyl groups tend to form solid solutions with those containing chlorine atoas substituted in the corresponding positions. The replacement of nethyl groups by chlorine atoms similarly leads to blological cross-reactivity ~ a hapten comtaining a methyl group interacts nearly ss strongly with the serum homologous to a hapten con- taining a chlorine group in this position ss does the schlorine-substituted hapten itaelf, The value of a physicochemical attack on the bidlogical problem of the nature of the structure and properties of antibodies has been indicated. The usefulness of this attack is limited by the complies tion introduced by the het- erogenelty of antibody. Other natural proteins with apecifie properties cay be far sore homogeneous, and aight be still more profitebly subjected to physico- chemical atudy. For examnle, the ensymes, for which the value of the physico~ chemical attack has been shown by the early work of Michaelis, are in general ~1Os homogeneous proteing, and a quantitative study of auch phenomena as sub- strate inhibition and competition aight well provide very valuable informetian about the configuration of the active region of the ensyme noleculea and the nature of the phenomenon of enzymatic catelysia, The theory of biological specificity described above, which is strongly Bupported by the results of lumunochemical experiments, requires that the mole-~ Gules which attract one another specifically de #0 only when they are in imaed- iate juxtaposition, that is, when their surfaces are within a few iingstroms of one another. These specific forces, depending on the satomio-scale comple- mentariness of structure of the two interacting molecules, decrease in magni- tude very rapidly sith incressing intermolecular distance, and are negligible at distances greater than about 10 g. between the surfaces of the moleaules, Long-range non-specific forces between molecules exist, of course, but it ia very difficult to develop any theory of long-range specific forces on the basis of our present knowledge of molecular structure. + The discovery of long-range intermolecular forcea of such great specificity as ia shown in bi- ological phenomena would be of the greatest int«rest. Ingasamch as the exiatence 2f short-range specific forces, derendent upon the contact between the molecules, must be accented, in view of the strength of the evidence that haa been accumulated for it, the discovery thet long-range a@pecific forcea betwem antibody molecules end antigen molecules also exist would be very surpriging. The experiments reported during the last two years by Rothen and interpreted ag indicatin: the existence of these long-range specifie forces are accordingly of great interest, !” Rother has shown that a Ly, Pauling and M. Delbriek, Science, 92, 77 (1940). 124, Rothen, J» Blol. Chem, 183, 348 (1946); 167, 299 (1947); 168, 78 (1947). w~lle layer of protein antigen, such as bovine albugin, deposited on a metal plete 1s able to combine with its homolovous antibody, or to be hydrolyzed through the enaymitie action of trypsin, even efter some othor Materiel, such ca Porge var (a volyvinyl-formal resin), hae aleo been deposited on the plate. On the as- sumption that the Formvar or other screening saterialis iy apread uniforaly over the surface of the plate, and thus covers the antigen, these experimental results have been interpreted ag showing that the forces of specifie attraction between antigen and antibody or the forces of catalytic action of the engyme on the pro- tein are eble to operate over distances as great et 100 or 200 i, the average thickness of the screening layers, I think it is highly probable that it will be found that the specific interaction of antigen and antibody and of enzyme and aubstr