Chemistry and Biology of Proteins - F. Haurowitz 1953

The role of proteins in immune reactions
Antibodies

The presence of Antibodies is detected through their specific reaction with Antigens. Antibodies formed in response to the Introduction of Cells or Bacteria are capable of agglutinating or lysing the corresponding cells and bacteria. Antibodies against soluble Proteins form insoluble complexes with these proteins. Toxins lose their toxicity upon binding with antibodies. All these phenomena are essentially manifestations of a single reaction—the Combination of an antibody with an antigen. If whole cells or bacteria are used for immunization, A large number of antibodies are produced against various antigenic substances present in the injected antigen complex. The situation is considerably simplified when soluble substances, such as pure proteins, are used as antigens. In such cases, Antibodies Are Formed against only a single antigen.

It has long been known that antibodies reside in the globulin fraction of immune serum. This observation served as the basis for the concept that antibodies are more or less loosely bound to serum globulins. When immune serum is fractionated using ammonium sulfate, antibodies are found mainly in the y-globulin fraction [30]. Fractionation of the globulin fraction with ethyl alcohol showed that antibodies are present in fractions II-1, II-2, and II-3, as well as in fraction III-1 [31]. Some antibodies precipitate together with euglobulins upon dialysis against distilled Water, whereas other antibodies are found in the pseudoglobulin fraction [32]. As a rule, immune serum contains more y-globulins than normal serum. In addition to the a-, β-, and y-globulins also present in normal serum, immune serum sometimes contains a new fraction (T) which, upon Electrophoresis, migrates between the β- and y-fractions. The T-fraction also contains antibodies [33, 34]. Recent studies have shown that the serum of many newborn animals, such as foals [35] or rabbits [36], is poor in globulins. This is in good agreement with the fact that antibodies are absent from the serum of newborn animals [37].

The PHYSICOCHEMICAL PROPERTIES OF antibodies are very close to those of normal serum y-globulins. In most cases, their isoelectric point lies near pH 6 [38]. The Molecular Weight of antibodies from rabbit and monkey Blood is 157,000, whereas that of horse, sheep, and bovine blood antibodies is 920,000 [39]. Hydrolysis of antibodies yields the same Amino Acids that are detected in the hydrolysate of normal y-globulins [40]. In normal rabbit serum globulins and in rabbit blood antibodies, the Amino acids are arranged in the same sequence. Both proteins contain aspartic acid, valine, and leucine, and Alanine with a free amino group is located at the ends of the peptide chains [41].

To resolve the question of whether antibodies are serum globulins or merely associated with this fraction, Structure/129.html">Specific Methods for antibody purification can be employed. Typically, these methods include two main stages: 1) Formation of the antigen-antibody precipitate, and 2) dissociation of the precipitate and Isolation of the pure antibody. The first successful experiments of this kind were undertaken by Felton [42], who precipitated pneumococcal antigenic Polysaccharides with the corresponding immune serum and then decomposed the precipitate by treating it with barium hydroxide. Following this Treatment, the antibodies pass into solution, while the insoluble barium salts of the polysaccharides remain in the precipitate. Heidelberger and his coworkers successfully cleaved such precipitates by treating them with concentrated sodium chloride solutions [43, 44]. In the author's laboratory, to obtain pure antibodies, azoprotein precipitates were treated with dilute acids in the presence of neutral salts. In this process, the majority of the antibodies dissociated and passed into solution, whereas the antigen and the undissociated portion of the antibodies remained in the precipitate [45]. Antibody solutions obtained by these methods contain globulins that do not differ in properties from the normal globulins described above. Further studies showed that more than 90% of the globulins isolated in this manner are precipitated by the corresponding antigen. This strongly Supports the Conclusion that these globulins are indeed identical to true antibodies.

Antibodies differ from normal serum globulins, as well as from one another, in their Specificity. Each antibody combines only with the antigen used for immunization and does not combine with any other antigen. To explain this specific ability of antibodies to combine with the corresponding antigen, Haurowitz and the author of the present book put forward a theory according to which antibodies are globulins whose molecules possess a configuration that is geometrically complementary to the configuration of the determining groups of the antigen molecule [46]. The authors of this theory suggest that the polar groups of the antigen influence The process of globulin formation from amino acids, thereby altering the normal course of this process and resulting in The formation of globulin molecules with a different spatial configuration [46–48].

The complementary surface shape of the antibody is determined, on the one hand, by the manner in which the peptide chain is folded [50] and, on the other hand, by the presence of ionic groups of opposite sign in that region of the antibody molecule which directly binds to the antigen (Fig. 46). The site of normal serum globulin formation has not yet been definitively clarified; however, most researchers believe that antibodies are formed in the reticuloendothelial cells or macrophages of the Liver, Bone Marrow, and Spleen [51]. It was found that upon injection of such a potent antigen as arsanilazoglobulin into a rabbit, the highest amount of antigen accumulates in The Liver and bone marrow of the experimental animals [29]. Upon administration of phosphovetellin labeled with P³², radiophosphorus is rapidly taken up by the liver and lung Tissues [52]. This observation indicates that these Organs play an essential role in the process of antibody formation.

While the Blood Plasma of rabbits and horses contains large amounts of soluble antibodies, in the plasma of other animals, such as guinea pigs, soluble antibodies are present only in small quantities (even when these animals are sensitized with soluble antigens). The allergic state in these animals is manifested by severe anaphylactic Shock occurring upon repeated injection of the same antigen. The antibodies in these animals apparently cannot penetrate from the tissues where they are formed into the blood plasma because they represent insoluble proteins. Evidently, here too, There is a correspondence between the spatial configuration of antibodies and antigens that determines the high selectivity of the reaction between them [53]. Some researchers have succeeded in extracting antibodies from lymphoid cells. In this regard, it has been suggested that some antibodies are formed in lymphoid cells [54, 55, 56] or in plasmacytes [57]. In the opinion of the author of this book, antibodies can be formed in all cells where proteins are synthesized, provided these cells are capable of binding the molecules of the injected antigen [58].

Based on experiments with amino acids labeled with N¹⁵, it can be concluded that the formation of antibodies in the Organism proceeds at approximately the same rate as the formation of normal serum globulins. The half-life of antibodies is about two weeks [59]. Of great interest are experiments in which non-immunized rabbits were injected with rabbit antibodies against bacterial polysaccharides taken from another immunized animal, along with simultaneously administered N¹⁵-labeled amino acids. It turned out that N¹⁵ is not incorporated into preformed antibodies [59]. However, other researchers, using a similar methodology, demonstrated that passive immunization results in the incorporation of C¹⁴-labeled leucine into antibodies [60]. Due to contradictory literature data, it is still not clear whether antibody molecules introduced into the organism of another animal can undergo any restructuring within it.

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Fig. 46. Combination of the m-azophenylsulfonyl derivative of a protein (left) with an antibody (right) [49].

Antibody formation, much like the Synthesis of Other proteins, depends on Nutrition. Animals fed a complete diet produce significantly more antibodies than animals maintained on an inadequate diet [61]. In frogs, the antibody titer depends on body Temperature: at 20° it is significantly higher than at 8°. A decrease in temperature causes the adsorption of antibodies by liver cells with a corresponding drop in the antiserum titer [62].

Some authors attempted to explain antibody specificity by assuming that the determining group of the antigen becomes incorporated into the antibody molecule during its formation. Therefore, they suggested viewing antibodies as natural globulins that have incorporated this group into their molecular structure [63]. This hypothesis is refuted by the fact that antibodies against iodinated and brominated proteins, arsanilazoproteins, and Phosphoproteins contain neither iodine, bromine, arsenic, nor phosphorus [64]. It is also known that a single antigen molecule is capable of inducing the formation of a large number of antibody molecules. For instance, upon immunization with bacteria, The amount of antibodies produced is approximately 600 times greater than the amount required for the agglutination of the injected bacteria [65].

Initially, it was assumed that each antigen elicits the appearance of only a single type of antibody, adapted to the determining group of the antigen molecule. If each molecule of the injected antigen contains several different determining groups, several different antibodies are formed. Immunization with a globulin into which iodine and azobenzenearsonic acid have been introduced leads to the appearance of Two Types of antibodies: antibodies against diiodotyrosine and antibodies against azobenzenearsonic acid [66]. These two types of antibodies can be separated from each other by precipitating one with iodovalbumin and the second with arsanilazoovalbumin. If iodarsanilazoovalbumin is added after the precipitation of these antibodies, no additional precipitate is formed.

A completely different picture is observed upon immunization with sheep serum arsanil-globulin. In this case, at least Three types of antibodies are formed: 1) antiarsanil, 2) antisheep, and 3) antiarsanilsheep [67]. The latter antibodies are precipitated by sheep serum arsanilglobulin and are not precipitated by either arsanilazoovalbumin or sheep serum globulin. It follows from these experiments that the specificity of antibody molecules depends on the arrangement of the determining groups within the antigen molecule. If an antigen (A+B) is introduced whose determining groups A and B are located at different sites on the antigen molecule's surface, two types of antibodies will be formed: anti-A and anti-B, whereas antibodies of the anti-AB type will not be produced. However, if groups A and B are located close to each other, then in addition to anti-A and anti-B antibodies,

anti-AB antibodies will also be formed [68] (see Figs. 47 and 48).

Fig. 47. Combination of an antigen with an antibody.

A and B are two different determining groups of the antigen (A+B).

Fig. 48. Combination of an antigen with an antibody.

AB is an antigen containing two determining groups, A and B, located adjacent to each other.

It follows from these observations that immune serum must inevitably contain diverse antibodies even when only a single simple antigen is used for immunization. The production of multiple antibodies in response to the Introduction of a single antigen is due, on the one hand, to the fact that the antigen may contain several different determining groups that elicit the appearance of various antibodies adapted to them, and, on the other hand, to the imperfection of adaptation of the resulting antibodies. Antibody molecules are formed from amino acids, and since The structure of the peptide chain can vary only up to a certain limit, their configuration can never completely match that of the antigen molecule. Consequently, immune serum must contain both well-adapted and poorly adapted antibodies (Fig. 49) [13, 69–72]. Therefore, even crystalline protein antigens, such as egg albumin, induce the formation of several different antibodies [72].

In immune serum, there are likely all intermediate forms between normal serum globulins and well-defined antibodies [73]. Some authors even suggest that all globulins in immune serum are antibodies, i.e., they are characterized by structural adaptation to antigen molecules [74, 75]. This view is supported by the fact that antibody specificity decreases upon prolonged immunization; antibodies formed during the initial days of immunization are capable of precipitating only the antigen used for immunization, whereas antibodies formed As a result of immunization lasting several weeks or months can also effectively precipitate other similar antigens [76–78].

Fig. 49. Determining groups of the antigen (phenylarsonic acid) and various types of antibodies.

A — antigen-determining group; B — ideal antibody; C — well-adapted antibody; D — poorly adapted antibody.

The diagrams shown in Figs. 47 and 48 demonstrate that each antibody molecule, even when a multivalent antigen is used for immunization, possesses only a single specific group capable of binding to the antigen. The concept that antibodies are univalent [67, 79] is supported by the following observation. Diphtheria antitoxin, which has a molecular weight of 184,000, is cleaved by Pepsin into two fragments. One of these fragments, with a molecular weight of 98,000, retains all The activity of the uncleaved antitoxin, whereas the other is completely devoid of antitoxic activity. Obviously, the specific reactive group is present in only one part of the original antitoxin molecule [80]. However, the notion of antibody univalency is far from universally accepted among immunochemists. Many investigators [50, 81, 82] maintain that each antibody molecule possesses two or more specific groups capable of binding antigens. They explain the low reactivity of antibodies formed during the early days of immunization by their univalency and the inability of univalent antibodies to form precipitates [83].

Since antibodies are proteins, it is hardly surprising that their activity is affected by any agents that induce Protein Denaturation. The efficacy of antibodies is diminished or entirely destroyed by heating, high pressures [84], as well as by treatment with iodine, formaldehyde, or diazo compounds [85].

At the same time, it cannot be assumed that the specific active group of antibodies is highly labile [16]; indeed, it is often more stable than the antigen-determining group. This applies primarily to antitoxins. Treating a toxin–antitoxin precipitate with Denaturing Agents destroys only the toxin, yielding a non-toxic antitoxin preparation. The compositional differences between antibodies and normal serum globulins are not substantial enough to cause a marked difference in their antigenic properties. If horse serum antibodies are injected into a rabbit, precipitins are formed in its body that react not only against the horse antibodies but also against normal horse serum globulin [86].

Antibody production in animal organisms is very often viewed as a process entirely unrelated to other continuous physiological processes in a normal body. However, antibody formation involves the synthesis of new protein molecules, and there is no reason to believe that this process differs significantly from standard Protein Synthesis [58, 87]. The Mechanism of protein formation in living cells will be discussed below (see Chapter XVII). At this point, it is only necessary to note that the process apparently proceeds in two stages [58]: 1) the formation of a copy of an extended protein template, and 2) The conversion of this two-dimensional copy into a three-dimensional globular protein molecule through folding. The Influence of the antigen comes into play primarily during the Second Stage of protein synthesis. It is likely that the polar groups of the antigen disrupt the normal folding of the peptide chain in such a way that the resulting globular particles acquire a configuration that is, in effect, a geometric Complement to the configuration of the polar-determining groups of the antigen [58]. The electrostatic forces of the polar-determining groups of the antigen apparently establish a specific force field, the dimensions of which vary from one antigen to another. Since glycylleucine is immunologically distinct from leucylglycine (see the beginning of this chapter), it must be concluded that antibody specificity is determined not by a single isolated antigen group, but by the entire region of adjacent polar groups. Based on this concept, it becomes clear why antibodies directed against Peptides are also capable of binding with peptide analogues — peptamines [89]:

Several researchers have attempted to synthesize antibodies in vitro by treating normal serum γ-globulins with mild denaturing agents in the presence of an antigen [90]. However, no definitive Conclusions can yet be drawn from these experiments, as denatured γ-globulins tend to form non-specific precipitates with the antigens used [91, 92].