Chemistry and Biology of Proteins - F. Haurowitz 1953

The Role of Proteins in Immune Reactions
Antigens

The Emergence of Immunity was first observed in bacterial infections. An individual who had recovered from an infectious disease became refractory to reinfection by the same microorganism. Upon investigating the causes of this refractoriness, it was found to be due to the ability of Blood serum to specifically agglutinate or lyse the Bacteria causing the infection, as well as to increase the susceptibility of these bacteria to phagocytosis. In attempting to explain this phenomenon, Ehrlich concluded that Antibodies must be present in immune serum, and designated as antigens those substances that stimulate The production of these antibodies.

In 1897, Kraus [1] demonstrated that not only bacteria or Cells, but also certain soluble substances can act as antigens. Antigens are precipitated by their corresponding antibodies. This phenomenon, known as the precipitation reaction, serves as the simplest (and formerly the only) method for studying the species Specificity of Proteins. The immense importance of this method to Protein Chemistry is entirely self-evident.

Originally, it was believed that protein antigens differed fundamentally from bacterial and cellular antigens. Subsequently, however, it was shown that bacteria or cells cannot be regarded as individual antigens, as they consist of a multitude of various substances—a sort of "mosaic" of substances—some of which act as antigens, whereas others possess no antigenic activity. Antigenic properties are characteristic of the majority of proteins found in the bodies of animals, plants, and bacteria. Unlike proteins, CARBOHYDRATES, Lipids, and other substances generally do not function as antigens, i.e., they lack the capacity to elicit antibody formation.

It has long been known that certain low-molecular-weight compounds, such as iodine, picryl chloride (1-chloro-2,4,6-trinitrobenzene), and other nitro compounds, are capable of inducing an allergic state when administered parenterally. It is hypothesized that in such cases, the allergy develops As a result of antibody formation directed not against the injected substances themselves, but against their derivatives. This assumption is based on the fact that all of the aforementioned substances readily combine with proteins in vitro. It is conceivable that upon Introduction into the Organism, these substances combine with proteins at the site of injection, and the resulting protein complex constitutes the actual antigen [2]. Similar results have been obtained with lipids, which likewise acquire antigenic properties after being mixed with protein solutions. Low-molecular-weight substances that serve as precursors of antigens are termed proantigens [3]. Most researchers believe that Proteins are essential for The conversion of proantigens into antigens.

Since most proteins are digested by Pepsin or Trypsin, it is readily understood that proteins lose their antigenic properties when passing through the digestive tract. Therefore, to elicit antibody formation, proteins must be administered parenterally.

Any natural protein possesses antigenic specificity. Antibodies arising in response to the administration of a specific protein form precipitates exclusively with that protein and not with other proteins. Cross-reactivity is observed only when the test antigen is very closely related to the antigen used for immunization. For instance, antibodies formed upon the injection of horse Serum proteins also precipitate donkey serum proteins; duck egg albumin is precipitated by antibodies elicited by immunization with chicken egg albumin [4]. On the other hand, the serological properties of Myoglobin differ sharply from those of Hemoglobin, even though both substances contain the exact same hemin [5]. Obviously, in this case, specificity is determined by the protein moiety rather than the hemin. Human hemoglobin is serologically distinct from bovine hemoglobin. Nevertheless, using the Complement fixation inhibition test, a certain degree of affinity between these two substances can be established [6].

Proteins exhibit not only species specificity but also organ specificity. For example, serum proteins differ serologically from hemoglobin or Muscle Proteins of the same animal. However, certain globulin fractions isolated by fractional precipitation with ammonium sulfate are very similar to each other and can be distinguished by the precipitation reaction only with great difficulty [7]. Ferritin (see Chapter XI) is species-specific, but lacks organ specificity [8].

Landsteiner [2] discovered that the specificity of proteins is not governed by the protein molecule as a whole, but rather by specific chemical groups within that molecule. The validity of this proposition was proven by complexing proteins with various chemical groups. The primary method utilized by Landsteiner for this purpose involves the diazotization of proteins with various diazo compounds. This method offers several advantages: 1) virtually all substances possess The ability to form diazo derivatives, and consequently, any desired groups can be attached to the protein molecule in this manner; and 2) the complexation of diazo compounds with proteins occurs at 0°C in a slightly alkaline solution (pH 9), i.e., under conditions where most proteins do not undergo Denaturation. Using this approach, highly interesting data have been obtained, some of which will be discussed below.

To determine whether antibodies exhibit specificity toward optical isomers, Landsteiner coupled proteins with d- and l-tartaric acids. To prepare the diazo derivatives of these acids, they were subjected to Condensation with nitroaniline, after which the nitro tartaranilic acid was reduced to the corresponding amino compound and the latter was diazotized:

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The resulting diazo compounds readily combine with the Tyrosine and Histidine residues present in the protein, forming colored azoproteins:

As a result of these experiments, Landsteiner found that azoproteins containing d-tartaric acid and analogous azoproteins containing l-tartaric acid behave as distinct antigens. Antibodies generated in response to the administration of a protein coupled with one isomer do not react with the protein coupled with the other isomer [9]. Similarly, leucylglycine and glycyl-leucine differ from one another in their antigenic properties [10]. In another series of experiments, it was demonstrated that diazo derivatives of o-, m-, and p-aminobenzenesulfonic acids yield entirely different antigens when coupled with proteins [11]. From these and many other findings, which cannot be elaborated upon here, it follows that the specificity of proteins treated in this manner depends primarily on the chemical groups introduced into their molecule. For instance, antibodies against arsanilazodervatives of horse serum proteins can precipitate arsanilazoovalbumin and other arsanil proteins. Chemical Treatment may cause proteins to lose their species specificity. If, for example, A large number of azo groups are introduced into the horse serum globulin molecule, the resulting compounds are not precipitated by antibodies directed against this globulin. Likewise, iodination or Acetylation of serum globulin leads to the loss of its species specificity if the number of introduced iodine atoms or acetyl groups exceeds a certain minimum [12].

Not all groups that can be introduced into a protein molecule possess the ability to alter its serological properties and induce The formation of specific antibodies. The serological specificity of antigens is determined exclusively by polar groups—primarily acidic groups, such as —COOH, —SO₃H, —AsО₃H₂ [2], or basic groups, such as the quaternary ammonium group [13]. Polar groups presumably also determine the antigenic specificity of natural proteins. At present, we are unable to specify precisely which chemical groups dictate the antigenic specificity of these proteins. It is highly probable, however, that this specificity is not associated with any single defined type of group, but rather depends on a specific spatial arrangement of various polar groups On the surface of the protein molecule [12].

The antigenic Properties of Proteins are abolished following their Cleavage by Proteolytic Enzymes. Denaturation may result in either an alteration or a partial loss of the native protein's original antigenic properties. For example, antibodies against native egg albumin react very weakly with denatured egg albumin [14]. Conversely, immunization with denatured egg albumin leads to the production of antibodies that react specifically only with the denatured protein [15]. It should be noted, however, that no marked differences in the antigenic activity of native and denatured horse serum globulin could be detected [16]. The quantitative Study of the antigenic Properties of Denatured Proteins is considerably hindered by their tendency to form aggregates, since aggregation entails a nonspecific increase in the mass of the precipitate. The reduction in antigenic activity observed in denatured proteins is most likely due to the fact that they are hydrolyzed by proteolytic enzymes significantly faster than native proteins (see p. 149). Consequently, denatured proteins may be degraded within the organism before they have time to trigger antibody production.

Gelatin, obtained by heating Collagen, lacks antigenic properties. This fact was initially attributed to the absence of tyrosine in this protein. However, gelatin does not acquire antigenic properties even after The addition of tyrosine [17], diazo compounds [18], or iodine [19]. Currently, the lack of antigenicity in gelatin is ascribed to several factors: 1) gelatin is a protein denatured by heat and consequently lacks a defined internal Structure [20]; 2) upon introduction into the organism, it does not localize at the sites of antibody formation, but is rapidly eliminated from the body [18, 19]; and 3) gelatin contains a high proportion of Glycine. Because glycine lacks a side chain at the a-position, peptide chains containing glycine can rotate freely about their long axis, leading to a disruption of their spatial conformation [21]. In this regard, the peptide chains of gelatin lack the rigid structure that is a prerequisite for the immunological specificity of proteins.

The necessity of a defined, stable structure for the preservation of antigenic properties is corroborated by the fact that these properties are lost upon exposure of proteins to high pressure. For instance, serum proteins lose their antigenic properties after being subjected to a pressure of 6,000 atm [28].

The immunological specificity of proteins is a highly characteristic property that remains unaltered under normal conditions. However, by attaching foreign compounds to proteins or by creating complexes of two or more proteins, their antigenic properties can be modified. For example, if serum globulin is gently heated in the presence of serum albumin, a complex endowed with novel serological properties is formed [23]. Similarly, the iodination of proteins results in an alteration of their serological specificity [24, 25]. The specificity of iodinated proteins is conferred by the presence of diiodotyrosine residues. Nevertheless, antibodies directed against these proteins do not react with thyroglobulin—an iodine-containing protein of The Thyroid Gland [26]. This indicates that the diiodo groups within thyroglobulin are buried inside the large thyroglobulin hormone molecule and are therefore inaccessible to antibodies against diiodotyrosine. It is entirely evident that only reactive groups exposed on The surface of the molecule can determine the Specificity of the antigen molecule.

Summarizing the data presented, it can be stated that the presence of antigenic properties in the molecules of a given compound depends on the following conditions: 1) the molecule must contain a protein acting as a carrier for the determining groups; 2) the protein moiety must possess certain minimum dimensions; 3) its internal structure must be stable and not subject to rapid alteration; and 4) the protein must bear a specific number of polar groups on its surface.

The absence of antigenic properties (or more precisely, the presence of only negligible antigenicity) in Insulin and many Pituitary Hormones is most likely attributable to their low molecular weight. In some cases, the reason why a particular compound is devoid of antigenic properties remains unknown. For example, it is unclear why yellow enzymes (Flavoproteins) are not antigenic [27], whereas Other Enzymes, notably urease, are highly active in this respect.

The Combination of an antigen with an antibody does not obligatorily require the participation of the antigen's protein component; for instance, antibodies against arsanilazoproteins also bind to arsanilic acid or arsanilazotyrosine [28, 29]. It has likewise been demonstrated that the reaction between sulfonylazoprotein and its corresponding antibody is inhibited by low-molecular-weight compounds containing the same phenylsulfonic acid group [2]. Such low-molecular-weight non-protein compounds are termed haptens due to their ability to bind to antibodies (derived from the Greek word haptein, to bind). Typically, The process of antibody-hapten binding is not accompanied by precipitation, although precipitation does occur in certain instances. Haptens differ from true antigens in that they are incapable of eliciting the formation of corresponding antibodies upon parenteral injection.