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Effects of ionizing radiations upon the serological properties of proteins
164
International Symposium on the Effects of Ionizing Radiations on Immune Processes
protein become available for reaction. These include S H groups; - - S - - S groups and - - N H 2 groups, Conversely some groups that are accessible in the native protein become inaccessible in the irradiated protein. These effects have been observed in serum albumin, serum y-globulin, lysozyme and trypsin. With wool fibres the extensive rupture of hydrogen bonds has been demonstrated by mechanical tests. Radiation induced covalent changes lead to the introduction of new groups such as - - C O and unusual side chains due to decarboxylation of aspartic and glutamic acid residues. T h e chemical behaviour and general reactivity of the irradiated protein is therefore very different from that of the starting material. Electron spin resonance measurements have also shown that unusual radicals--which in our view are not related to sulphur--persist for a long time in the solid state. T h e changes produced when proteins are irradiated in solution (i.e. when the reaction is not directly due to an ionization but due to active entities produced in the surrounding water) are largely the result of covalent chemical alterations in the side chains. Most of the amino acid residues seem to be attacked if sparsely ionizing radiations are used and there is no great selectivity. New groups such as - - C O and peroxides are also introduced. An important reaction which is liable to affect the reactivity is the formation of stable covalent crosslinks between molecules. Denaturation can also be detected, for example, by revelation of disulphide bonds, but this in contrast to direct action, would appear to be a consequence of the rather extensive covalent chemical changes. If protein solutions are irradiated with a-rays, the changes produced are qualitatively different from those produced by sparsely ionizing X-rays. T h e attack is now largely confined to the tryptophane residues. It has been postulated that in the densely ionizing tract a reactive form (excited or isomeric) of hydrogen peroxide is formed which reacts differently from O H radicals.
E n e r g y R e q u i r e m e n t s for I n a c t i v a t i o n o f Prot e i n a n d V i r u s e s : * DWIGHT E. WILSON (Department of Biology, Rensselaer Polytechnic Institute, Troy, New York).
varies with temperature and oxygen tension during irradiation. Radiosensitivity of proteins is also influenced by the preparation techniques used before irradiation as in the case of desoxyribonuclease where the sensitivity varies depending upon the p H of the suspending m e d i u m prior to lyophilization and irradiation. Chemical compounds such as acetates and sucrose have been shown to increase radiosensitivity of enzymes. Sulfhydril compounds, on the other hand, have been found to decrease radiosensitivity. Energy transfer mechanisms may be invoked to explain the increase in radiosensitivity of hyaluronidase in combination with its substrate, and the decrease in radiosensitivity of the enzyme-substrate mixture for desozyribonuclease and ribonuclease. Little evidence is available to indicate the importance of the environment on the radiosensitivity ofvirns particles. Radiation studies indicate that virus particles contain a radiosensitive component which is generally m u c h less massive than the virus particle, and that one ionization in this component will cause inactivation. A linear relationship between nucleic acid content and radiosensitivity exists for plant and bacterial viruses. For the animal viruses containing R N A , the radiosensitive mass increases approximately linearly with particle mass and is equal to or greater than the nucleic acid mass. T h e animal viruses containing D N A , on the other hand, have radiosensitive masses which are smaller than their nucleic acid masses. Although both types of animal virnses contain radiosensitive nucleic acids, a simple relationship between nucleic acid content and radiosensitivity has not been found. It is concluded that R N A animal viruses also contain radiosensitive protein, and that the D N A animal viruses contain radiosensitive protein and radiosensitivc nucleic acid. T h e radiation insensitive nucleic acid of the D N A animal viruses may be nonspecific D N A carried over accidently from the host cell. Another possibility is that some D N A present in the virus is identical, or genetically homologous, to the D N A present in the host cell.
Effects o f I o n i z i n g R a d i a t i o n s u p o n the Serol o g i c a l P r o p e r t i e s o f Proteins:* CHARLES A. LEONE (Department of Zoology, the University of Kansas, Lawrence, Kansas).
Radiation inactivation studies on proteins in the dry state have shown that the release of energy equal to approximately one primary ionization in a molecule will cause inactivation. T h e radiosensitivity of proteins is, however, dependent to a certain extent on the physical conditions during irradiation and the chemical environment of the molecules. Radiosensitivity
Both in vivo and in vitro serological analyses of yirradiated proteins indicate that the principal effects of the radiation are to cause denaturation and a loss of normal biological activity in the affected molecules. In this report comparisons are presented of the anaphylactogenic, precipitin stimulating and serological
* This work was supported by grants from the National Institute of Allergy and Infectious Diseases.
* Supported in part by U S P H S Grant E-1809 and A E C Contract AT(11-1)-83, Project No. 4.
Abstracts of Papers properties of native and irradiated ovalbumins. Some serological comparisons were also made between native and irradiated samples of human gamma globulin and arthropod hemocyanins. Lyophilized ovalbumin received doses between 32 and 300 x 10s rep; one per cent solutions received doses between 105 and 8.5 x 10e rep. Below6 x 105 rep the capacity of irradiated ovalbumin to be precipitated by antisera against native protein is essentially unchanged from the native protein. At higher doses there is an exponential loss of serological activity without a loss in protein that is soluble at pH 7. Irradiated lyophilized ovalbumin exponentially lost its capacity to induce anaphylaxis in guinea pigs previously sensitized against native protein. Zn irradiated solutions the loss of anaphylactogenic activity, in relation to dose, is sigmoidal. Some irradiated solutions receiving as much as 6 x 10s rep had anaphylactogenic capacities which were 30 to 100 x greater than that of native protein. Solutions of irradiated ovalbumins were fractionated by isoelectric precipitation of the radiationdenatured protein. Thermolabile components were also derived from the isoelectrically soluble proteins in the supernatants. The relative serological activity of the various fractions were compared to that of native protein. The relationship A(I) = [F] .A(F) + (1 -- [F]) .A(S) appears valid, where A(I) is the serological activity of the solution of irradiated protein, A(S) is the activity of the supernatant after the removal of a known amount of an isoelectrically insoluble fraction IF] and A(F) is the activity of that fraction. Radiation-denatured, isoelectrically insoluble protein is a much poorer antigen than native protein. All radiation-denatured proteins were serologicaUy equivalent, regardless of the radiation exposure, when tested with antisera produced against radiation-denatured protein. Proteins denatured by heat and chemicals gave strong cross reactions with these antisera. Reciprocal tests with antlsera produced against heat-and urea-denatured proteins confirmed the high amount of serological correspondence between the variously denatured proteins and radiation-denatured protein. • Effects of Radiation on Natural I m m u n i t y and W a y s o f I t s S t i m u l a t i o n VICTORL. TROITSK!
(Gamaleya's Institute of Epidemiology and Microbiology, Academy of Sciences of the USSR Moscow, USSR). Experiments carried out in our laboratory during the last few years have shown that damage of natural immunity plays a dominant role not only in the decrease of resistance of irradiated animals to ex- and 4
165
endogenous infections but also in the suppression of acquired immunity and primarily in the suppression of artificial, immunity. Bearing in mind the variety of protective physiological mechanisms of organisms that are destroyed by radiation, one can suppose that there are different ways of restoring the damaged natural immunity. It is of especial interest, however, to stimulate hemopoiesis because its decline seems to be the essence of suppression of both natural immunity and immunogenesis in irradiated animals. Destruction or inhibition of a natural immunity mechanism, such as hemopoiesis, provides opportunities to use transplantations of bone marrow to stimulate the depressed natural immunity. The majority of our experiments employing bone marrow treatment of radiation disease were made on mice. With rats the experimenter encounters a situation in which the difference between the radiation dose that suppresses transplantation immunity and the dose that induces radiation-syndrome in the intestine is too small. Therefore, after rats are irradiated with doses designed to produce only bone marrow syndrome, the immunological systems of the recipients are often insufficiently suppressed. One kind of treatment elaborated in our laboratory consists of multiple injections of bone marrow. Repeated daily injections of homologous bone marrow for 10 to 25 days after irradiation proved to be effective in irradiated rats. Irradiated rats that received bone marrow repeatedly under certain conditions survived, during the first 30 days, in higher percentages than rats that were given a single injection. Late mortality--between 30 and 60 days--was not observed among the rats receiving repeated injections of bone marrow. A considerable amount of the protection obtained within 2 to 3 weeks after irradiation may be explained by the development of anaphylactic antibodies against introduced homologous bone marrow cells. The presence of such antibodies was proved in our studies experimentally. The multiple injections of bone marrow ceils may be regarded, in such cases, as desensitization. The absence of late mortality indicates that the introduced bone marrow cells failed to get implanted. Experiments made in our laboratory on large numbers of rats have shown that the bone marrow, by restoring hemopoielis in irradiated anita,/Is, sharply raises natural immunity to infection and brings it to the level of the resistance of animals not subjected to irradiation. However, treatment with the bone marrow had no effect on immunogenesis in irradiated rats actively immunized against typhoid bacteria. Another possible way of overcoming secondary reaction is based on induction of the development of hemopoietic tissue from elements of an irradiated
International Symposium on the Effects of Ionizing Radiations on Immune Processes
protein become available for reaction. These include S H groups; - - S - - S groups and - - N H 2 groups, Conversely some groups that are accessible in the native protein become inaccessible in the irradiated protein. These effects have been observed in serum albumin, serum y-globulin, lysozyme and trypsin. With wool fibres the extensive rupture of hydrogen bonds has been demonstrated by mechanical tests. Radiation induced covalent changes lead to the introduction of new groups such as - - C O and unusual side chains due to decarboxylation of aspartic and glutamic acid residues. T h e chemical behaviour and general reactivity of the irradiated protein is therefore very different from that of the starting material. Electron spin resonance measurements have also shown that unusual radicals--which in our view are not related to sulphur--persist for a long time in the solid state. T h e changes produced when proteins are irradiated in solution (i.e. when the reaction is not directly due to an ionization but due to active entities produced in the surrounding water) are largely the result of covalent chemical alterations in the side chains. Most of the amino acid residues seem to be attacked if sparsely ionizing radiations are used and there is no great selectivity. New groups such as - - C O and peroxides are also introduced. An important reaction which is liable to affect the reactivity is the formation of stable covalent crosslinks between molecules. Denaturation can also be detected, for example, by revelation of disulphide bonds, but this in contrast to direct action, would appear to be a consequence of the rather extensive covalent chemical changes. If protein solutions are irradiated with a-rays, the changes produced are qualitatively different from those produced by sparsely ionizing X-rays. T h e attack is now largely confined to the tryptophane residues. It has been postulated that in the densely ionizing tract a reactive form (excited or isomeric) of hydrogen peroxide is formed which reacts differently from O H radicals.
E n e r g y R e q u i r e m e n t s for I n a c t i v a t i o n o f Prot e i n a n d V i r u s e s : * DWIGHT E. WILSON (Department of Biology, Rensselaer Polytechnic Institute, Troy, New York).
varies with temperature and oxygen tension during irradiation. Radiosensitivity of proteins is also influenced by the preparation techniques used before irradiation as in the case of desoxyribonuclease where the sensitivity varies depending upon the p H of the suspending m e d i u m prior to lyophilization and irradiation. Chemical compounds such as acetates and sucrose have been shown to increase radiosensitivity of enzymes. Sulfhydril compounds, on the other hand, have been found to decrease radiosensitivity. Energy transfer mechanisms may be invoked to explain the increase in radiosensitivity of hyaluronidase in combination with its substrate, and the decrease in radiosensitivity of the enzyme-substrate mixture for desozyribonuclease and ribonuclease. Little evidence is available to indicate the importance of the environment on the radiosensitivity ofvirns particles. Radiation studies indicate that virus particles contain a radiosensitive component which is generally m u c h less massive than the virus particle, and that one ionization in this component will cause inactivation. A linear relationship between nucleic acid content and radiosensitivity exists for plant and bacterial viruses. For the animal viruses containing R N A , the radiosensitive mass increases approximately linearly with particle mass and is equal to or greater than the nucleic acid mass. T h e animal viruses containing D N A , on the other hand, have radiosensitive masses which are smaller than their nucleic acid masses. Although both types of animal virnses contain radiosensitive nucleic acids, a simple relationship between nucleic acid content and radiosensitivity has not been found. It is concluded that R N A animal viruses also contain radiosensitive protein, and that the D N A animal viruses contain radiosensitive protein and radiosensitivc nucleic acid. T h e radiation insensitive nucleic acid of the D N A animal viruses may be nonspecific D N A carried over accidently from the host cell. Another possibility is that some D N A present in the virus is identical, or genetically homologous, to the D N A present in the host cell.
Effects o f I o n i z i n g R a d i a t i o n s u p o n the Serol o g i c a l P r o p e r t i e s o f Proteins:* CHARLES A. LEONE (Department of Zoology, the University of Kansas, Lawrence, Kansas).
Radiation inactivation studies on proteins in the dry state have shown that the release of energy equal to approximately one primary ionization in a molecule will cause inactivation. T h e radiosensitivity of proteins is, however, dependent to a certain extent on the physical conditions during irradiation and the chemical environment of the molecules. Radiosensitivity
Both in vivo and in vitro serological analyses of yirradiated proteins indicate that the principal effects of the radiation are to cause denaturation and a loss of normal biological activity in the affected molecules. In this report comparisons are presented of the anaphylactogenic, precipitin stimulating and serological
* This work was supported by grants from the National Institute of Allergy and Infectious Diseases.
* Supported in part by U S P H S Grant E-1809 and A E C Contract AT(11-1)-83, Project No. 4.
Abstracts of Papers properties of native and irradiated ovalbumins. Some serological comparisons were also made between native and irradiated samples of human gamma globulin and arthropod hemocyanins. Lyophilized ovalbumin received doses between 32 and 300 x 10s rep; one per cent solutions received doses between 105 and 8.5 x 10e rep. Below6 x 105 rep the capacity of irradiated ovalbumin to be precipitated by antisera against native protein is essentially unchanged from the native protein. At higher doses there is an exponential loss of serological activity without a loss in protein that is soluble at pH 7. Irradiated lyophilized ovalbumin exponentially lost its capacity to induce anaphylaxis in guinea pigs previously sensitized against native protein. Zn irradiated solutions the loss of anaphylactogenic activity, in relation to dose, is sigmoidal. Some irradiated solutions receiving as much as 6 x 10s rep had anaphylactogenic capacities which were 30 to 100 x greater than that of native protein. Solutions of irradiated ovalbumins were fractionated by isoelectric precipitation of the radiationdenatured protein. Thermolabile components were also derived from the isoelectrically soluble proteins in the supernatants. The relative serological activity of the various fractions were compared to that of native protein. The relationship A(I) = [F] .A(F) + (1 -- [F]) .A(S) appears valid, where A(I) is the serological activity of the solution of irradiated protein, A(S) is the activity of the supernatant after the removal of a known amount of an isoelectrically insoluble fraction IF] and A(F) is the activity of that fraction. Radiation-denatured, isoelectrically insoluble protein is a much poorer antigen than native protein. All radiation-denatured proteins were serologicaUy equivalent, regardless of the radiation exposure, when tested with antisera produced against radiation-denatured protein. Proteins denatured by heat and chemicals gave strong cross reactions with these antisera. Reciprocal tests with antlsera produced against heat-and urea-denatured proteins confirmed the high amount of serological correspondence between the variously denatured proteins and radiation-denatured protein. • Effects of Radiation on Natural I m m u n i t y and W a y s o f I t s S t i m u l a t i o n VICTORL. TROITSK!
(Gamaleya's Institute of Epidemiology and Microbiology, Academy of Sciences of the USSR Moscow, USSR). Experiments carried out in our laboratory during the last few years have shown that damage of natural immunity plays a dominant role not only in the decrease of resistance of irradiated animals to ex- and 4
165
endogenous infections but also in the suppression of acquired immunity and primarily in the suppression of artificial, immunity. Bearing in mind the variety of protective physiological mechanisms of organisms that are destroyed by radiation, one can suppose that there are different ways of restoring the damaged natural immunity. It is of especial interest, however, to stimulate hemopoiesis because its decline seems to be the essence of suppression of both natural immunity and immunogenesis in irradiated animals. Destruction or inhibition of a natural immunity mechanism, such as hemopoiesis, provides opportunities to use transplantations of bone marrow to stimulate the depressed natural immunity. The majority of our experiments employing bone marrow treatment of radiation disease were made on mice. With rats the experimenter encounters a situation in which the difference between the radiation dose that suppresses transplantation immunity and the dose that induces radiation-syndrome in the intestine is too small. Therefore, after rats are irradiated with doses designed to produce only bone marrow syndrome, the immunological systems of the recipients are often insufficiently suppressed. One kind of treatment elaborated in our laboratory consists of multiple injections of bone marrow. Repeated daily injections of homologous bone marrow for 10 to 25 days after irradiation proved to be effective in irradiated rats. Irradiated rats that received bone marrow repeatedly under certain conditions survived, during the first 30 days, in higher percentages than rats that were given a single injection. Late mortality--between 30 and 60 days--was not observed among the rats receiving repeated injections of bone marrow. A considerable amount of the protection obtained within 2 to 3 weeks after irradiation may be explained by the development of anaphylactic antibodies against introduced homologous bone marrow cells. The presence of such antibodies was proved in our studies experimentally. The multiple injections of bone marrow ceils may be regarded, in such cases, as desensitization. The absence of late mortality indicates that the introduced bone marrow cells failed to get implanted. Experiments made in our laboratory on large numbers of rats have shown that the bone marrow, by restoring hemopoielis in irradiated anita,/Is, sharply raises natural immunity to infection and brings it to the level of the resistance of animals not subjected to irradiation. However, treatment with the bone marrow had no effect on immunogenesis in irradiated rats actively immunized against typhoid bacteria. Another possible way of overcoming secondary reaction is based on induction of the development of hemopoietic tissue from elements of an irradiated