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Electrophoresis as an aid to preparative ultracentrifugation
NOTES FROM THE BIOCHEMICAL RESEARCH FOUNDATION. Electrophoresis as an Aid to Preparative Ultracentrifugation.--THOMAS J. DIETZ. (Physico-Chemical Department of
the Biochemical Research Foundation of the Franklin Institute, Philadelphia, Pa.) The field of protein chemistry has been extended materially by the development of the analytical ultracentrifuge. This apparatus makes possible the subjection of protein solutions to centrifugal force sufficiently intense to cause the protein in solution to sediment, and permits the observation and recording of this sedimentation under controlled conditions by means of a suitable optical system. In consequence of ultracentrifuge studies, many proteins are now regarded as possessing characteristic sedimentation constants (for details see " T h e Ultra-Centrifuge and the Study of High Molecular Weight Compounds," by Prof. The Svedberg, Nature, 139: lO51, 1937). Since a differential sedimentation can result in the separation of proteins of unlike sedimentation constants, ultracentrifuges of the air-driven type have been devised for this purpose (a review of air-driven ultracentrifuges may be obtained from " H i g h Speed Centrifuging," by Dr. J. W. Beams, Review of Modern Physics, 1o: 245, 1938). From these ultracentrifuges, which are designed to handle relatively large volumes of material, the supernatant and sediment fractions of the centrifuged specimens can be recovered for chemical or biological assay. The preparative type of ultracentrifuge may be either: (I) a quantity ultracentrifuge which is capable of handling volumes up to 200 cc. at one time, or (2) a continuous separation ultracentrifuge through which fluids to be fractionated may be passed continuously. Ultracentrifuges of the latter type offer opportunities for application in commercial preparative procedures. Together with the ultracentrifuge analytical method for determining molecular homogeneity and sedimentation constant of a protein, have come electrophoretic methods for 396
Sept., 1939.] BIOCHEMICAL RESEARCH FOUNDATION.
397
determining electrochemical homogeneity and electrophoretic mobility. Probably the most suitable method for measuring electrophoretic mobility is that devised by Tiselius, the design of which was the result of considerable research (for more complete information, see " A New Apparatus for Electrophoretic Analysis of Colloidal Mixtures," by Arne Tiselius, Trans. Farad. Soc., 33: 524, I937). The electrophoresis apparatus of Tiselius enables the obtaining of data on the electrochemical state of any one of several proteins in solution. Electrophoretic analysis of a complex mixture of proteins in solution defines the number and r e l a t i v e concentrations of all electrochemically distinct components present if they are not .in too small concentration to be beyond the sensitivity of the optical system of the apparatus. The electrochemical homogeneity and the electrophoretic mobility of each component are also included in the definition, and if mobility measurements are made at different hydrogen ion concentrations, the iso-electric point of each component is determinable by interpolation. Therefore, it is possible to correlate alterations in electrochemical homogeneity with the deleterious effects of chemical or physical treatment, to correlate changes in electrophoretic mobility with chemical combination, and to define the specificity and efficiency of ultracentrifuge fractionation procedures from measurement of relative concentrations of components found in the supernatant and sediment fractions. The usefulness of such data in preparative ultracentrifugation is unquestionable because the results of ultracentrifuge fractionation are often indefinite concerning the identity of proteins in a fraction. Examples are presented in the following paragraphs to show how etectrophoretic analysis can be utilized in ultracentrifuge fractionation procedures. Applications of the preparative ultracentrifuge are limited when fractionation is dependent alone upon native molecular weight differences. The possible ultracentrifuge purification of virus proteins for use as immunizing agents is evident, but at present such procedure is impracticable for technical reasons related to pathogenicity. The use of electrophoresis in conjunction with ultracentrifuge purification of virus proteins has not been investigated extensively. Conse-
398
BIOCHEMICAL RESEARCH FOUNDATION.
[J. F. I.
quently, the question of the possibility of electrophoretic control of methods for inhibiting pathogenicity preparatory to ultracentrifugation cannot be answered at present. Since, however, native viruses are infective in minute concentrations, i.e. beyond the sensitivity of the optical system of t h e electrophoresis apparatus, the only apparent possibility for use lies in the control of degradation of the purified product. The conditions for ultracentrifuge purification of virus protein are ideal in principle in t h a t the readily separable c o m p o n e n t is the virus protein itself; more general applications of ultracentrifuge purification present difficulties because the protein to be purified is contaminated with undesirable proteins whose sedimentation constants are often of the same order. Therapeutic antisera are examples of this type of system, where the protective antibody is the desired protein c o n t a m i n a t e d with an excessive a m o u n t of inert protein. The molecular weight of the antibody from antipneumococcus horse serum is represented in the literature to be roughly six times t h a t of any of the inert protein components. These molecular weight determinations were taken from ultracentrifuge data, the analyses being made on antiserum obtained from freshly immunized horses and diluted three or more times. In undiluted serum the antibody protein tends to dissociate into components of lower molecular weights. It has also been found t h a t the antibody fraction from the serum of repeatedly immunized horses is only partially of the homogeneous high molecular weight species. A practical ultracentrifuge m e t h o d for antibody purification is not set up to meet limitations such as are mentioned above. The only apparent alternative is to change the molecular weight of the antibody c o m p o n e n t so as to insure efficient fractionation. Experiments conducted in this laboratory have indicated t h a t it is possible to heat-treat antipneumococcus horse serum so as to cause aggregation of some of the serum proteins. Ultracentrifuge tests proved that, under controlled conditions, the aggregation is semi-specifically confined to the antibody fraction of the serum. The possibilities for practical ultracentrifuge fractionation based upon such procedure have not been fully determined, b u t it appears t h a t the idea of aggregating a specific c o m p o n e n t for purification purposes is not
Sept., I939. ]
B I O C H E M I C A L RESEARCH
FOUNDATION.
399
untenable. In this connection, it was evident from electrophoretic control experiments t h a t the technique of electrophoretic analysis can be used to guide ultracentrifuge fractionation. Electrophoretic control of chemical or physical t r e a t m e n t of therapeutic antisera materially aids in defining the condition of the system. For instance, when antipneumococcus horse serum is subjected to mild heating at temperatures ranging from 56 ° C. to 65 ° C., certain progressive changes take place in the appearance of the electrophoretic diagram. The protein c o m p o n e n t with which the antibody activity is associated appears to undergo some minor electrochemical change evidenced by a slight broadening of the boundary; 1 this change accompanies the semi-specific aggregation of the antibody component. Another component, usually referred to as the fi component, exhibits a moderate increase in concentration. With increased heat t r e a t m e n t the electrochemical homogeneity of the antibody component is further reduced, as is also its concentration. The fi component at the same time exhibits an additional increase in concentration. Heat t r e a t m e n t has not been carried beyond this point because further heating results in gelation of the antiserum. Similar alterations can be produced by allowing the antiserum, to which a small a m o u n t of formalin has been added, to stand over night at room temperature. On the following day the antiserum has an appearance of opalescence similar to that resulting from heat treatment. Electrophoresis of the formolized antiserum indicates changes qualitatively similar to those accompanying heat treatment, but with the formolized antiserum, the alteration can be carried further without causing gelation. A slight increase in the concentration of formalin added causes an aggregation which apparently includes all globulin components because electrophoretic analysis reveals only one globulin boundary in the formolized antiserum, i.e., the aggregated component. This component 1 The term " b o u n d a r y " refers to the interface or the refraction gradient which is created between the protein in solution and its solvent at the beginning of the experiment. The behavior of the boundary in an electric field is correlated with electrophoretic characteristics of the protein which constitutes the boundary. VOL. 228, NO. I365--28
400
BIOCHEMICAL RESEARCH FOUNDATION.
[J. F. I.
is homogeneous with respect to charge, and has the approximate mobility of the original ¢~component. If electrophoretic analyses are made on the supernatant and the sediment fractions of ultracentrifuged antipneumococcus horse serum (undiluted), the electrophoretic diagrams of the two fractions appear qualitatively alike. All serum protein components are present in the sediment fraction in slightly greater concentration than in the supernatant fraction. In some specimens of antiserum, the globulin component associated with antibody activity may be relatively increased in the sediment fraction. In general, the similarity of the electrophoretic diagrams of the supernatant and sediment fractions of the ultracentrifuged antiserum indicates that there can be no specific or semi-specific sedimentation of any protein component in the unaltered antiserum. In the case of heat-treated antipneumococcus serum, the situation is somewhat changed. When a certain protein component is aggregated as a result of heating, the sedimentation constant of this component will differ markedly from those of unaggregated components. Electrophoretic analysis of the supernatant and sediment fractions of the ultracentrifuged heated antipneumococcus horse serum reveals: first, that the component presumably associated with antibody activity is almost entirely absent from the supernatant fraction; second, that a highly aggregated, electrochemically monodisperse component is present in the sediment fraction, its mobility being approximately that of normal B globulin. About seventy-five per cent. of this component is specifically precipitable with homologous polysaccharide; specific precipitability is normally an attribute of the antibody component. Considering the cases mentioned above, it seems evident that electrophoretic analysis of a protein system, such as antipneumococcus serum, yields data useful to the quantitative control of preparative ultracentrifugation. Such analytical data are particularly helpful when physical or chemical treatment is required for specific fractionation. A Titrimetric Modification of the Glyoxalase Method for the Estimation of Reduced Glutathione.--E. F. SCHROEDER AND GLADYS E. WOODWARD. (Journalof Biological Chemistry,
the Biochemical Research Foundation of the Franklin Institute, Philadelphia, Pa.) The field of protein chemistry has been extended materially by the development of the analytical ultracentrifuge. This apparatus makes possible the subjection of protein solutions to centrifugal force sufficiently intense to cause the protein in solution to sediment, and permits the observation and recording of this sedimentation under controlled conditions by means of a suitable optical system. In consequence of ultracentrifuge studies, many proteins are now regarded as possessing characteristic sedimentation constants (for details see " T h e Ultra-Centrifuge and the Study of High Molecular Weight Compounds," by Prof. The Svedberg, Nature, 139: lO51, 1937). Since a differential sedimentation can result in the separation of proteins of unlike sedimentation constants, ultracentrifuges of the air-driven type have been devised for this purpose (a review of air-driven ultracentrifuges may be obtained from " H i g h Speed Centrifuging," by Dr. J. W. Beams, Review of Modern Physics, 1o: 245, 1938). From these ultracentrifuges, which are designed to handle relatively large volumes of material, the supernatant and sediment fractions of the centrifuged specimens can be recovered for chemical or biological assay. The preparative type of ultracentrifuge may be either: (I) a quantity ultracentrifuge which is capable of handling volumes up to 200 cc. at one time, or (2) a continuous separation ultracentrifuge through which fluids to be fractionated may be passed continuously. Ultracentrifuges of the latter type offer opportunities for application in commercial preparative procedures. Together with the ultracentrifuge analytical method for determining molecular homogeneity and sedimentation constant of a protein, have come electrophoretic methods for 396
Sept., 1939.] BIOCHEMICAL RESEARCH FOUNDATION.
397
determining electrochemical homogeneity and electrophoretic mobility. Probably the most suitable method for measuring electrophoretic mobility is that devised by Tiselius, the design of which was the result of considerable research (for more complete information, see " A New Apparatus for Electrophoretic Analysis of Colloidal Mixtures," by Arne Tiselius, Trans. Farad. Soc., 33: 524, I937). The electrophoresis apparatus of Tiselius enables the obtaining of data on the electrochemical state of any one of several proteins in solution. Electrophoretic analysis of a complex mixture of proteins in solution defines the number and r e l a t i v e concentrations of all electrochemically distinct components present if they are not .in too small concentration to be beyond the sensitivity of the optical system of the apparatus. The electrochemical homogeneity and the electrophoretic mobility of each component are also included in the definition, and if mobility measurements are made at different hydrogen ion concentrations, the iso-electric point of each component is determinable by interpolation. Therefore, it is possible to correlate alterations in electrochemical homogeneity with the deleterious effects of chemical or physical treatment, to correlate changes in electrophoretic mobility with chemical combination, and to define the specificity and efficiency of ultracentrifuge fractionation procedures from measurement of relative concentrations of components found in the supernatant and sediment fractions. The usefulness of such data in preparative ultracentrifugation is unquestionable because the results of ultracentrifuge fractionation are often indefinite concerning the identity of proteins in a fraction. Examples are presented in the following paragraphs to show how etectrophoretic analysis can be utilized in ultracentrifuge fractionation procedures. Applications of the preparative ultracentrifuge are limited when fractionation is dependent alone upon native molecular weight differences. The possible ultracentrifuge purification of virus proteins for use as immunizing agents is evident, but at present such procedure is impracticable for technical reasons related to pathogenicity. The use of electrophoresis in conjunction with ultracentrifuge purification of virus proteins has not been investigated extensively. Conse-
398
BIOCHEMICAL RESEARCH FOUNDATION.
[J. F. I.
quently, the question of the possibility of electrophoretic control of methods for inhibiting pathogenicity preparatory to ultracentrifugation cannot be answered at present. Since, however, native viruses are infective in minute concentrations, i.e. beyond the sensitivity of the optical system of t h e electrophoresis apparatus, the only apparent possibility for use lies in the control of degradation of the purified product. The conditions for ultracentrifuge purification of virus protein are ideal in principle in t h a t the readily separable c o m p o n e n t is the virus protein itself; more general applications of ultracentrifuge purification present difficulties because the protein to be purified is contaminated with undesirable proteins whose sedimentation constants are often of the same order. Therapeutic antisera are examples of this type of system, where the protective antibody is the desired protein c o n t a m i n a t e d with an excessive a m o u n t of inert protein. The molecular weight of the antibody from antipneumococcus horse serum is represented in the literature to be roughly six times t h a t of any of the inert protein components. These molecular weight determinations were taken from ultracentrifuge data, the analyses being made on antiserum obtained from freshly immunized horses and diluted three or more times. In undiluted serum the antibody protein tends to dissociate into components of lower molecular weights. It has also been found t h a t the antibody fraction from the serum of repeatedly immunized horses is only partially of the homogeneous high molecular weight species. A practical ultracentrifuge m e t h o d for antibody purification is not set up to meet limitations such as are mentioned above. The only apparent alternative is to change the molecular weight of the antibody c o m p o n e n t so as to insure efficient fractionation. Experiments conducted in this laboratory have indicated t h a t it is possible to heat-treat antipneumococcus horse serum so as to cause aggregation of some of the serum proteins. Ultracentrifuge tests proved that, under controlled conditions, the aggregation is semi-specifically confined to the antibody fraction of the serum. The possibilities for practical ultracentrifuge fractionation based upon such procedure have not been fully determined, b u t it appears t h a t the idea of aggregating a specific c o m p o n e n t for purification purposes is not
Sept., I939. ]
B I O C H E M I C A L RESEARCH
FOUNDATION.
399
untenable. In this connection, it was evident from electrophoretic control experiments t h a t the technique of electrophoretic analysis can be used to guide ultracentrifuge fractionation. Electrophoretic control of chemical or physical t r e a t m e n t of therapeutic antisera materially aids in defining the condition of the system. For instance, when antipneumococcus horse serum is subjected to mild heating at temperatures ranging from 56 ° C. to 65 ° C., certain progressive changes take place in the appearance of the electrophoretic diagram. The protein c o m p o n e n t with which the antibody activity is associated appears to undergo some minor electrochemical change evidenced by a slight broadening of the boundary; 1 this change accompanies the semi-specific aggregation of the antibody component. Another component, usually referred to as the fi component, exhibits a moderate increase in concentration. With increased heat t r e a t m e n t the electrochemical homogeneity of the antibody component is further reduced, as is also its concentration. The fi component at the same time exhibits an additional increase in concentration. Heat t r e a t m e n t has not been carried beyond this point because further heating results in gelation of the antiserum. Similar alterations can be produced by allowing the antiserum, to which a small a m o u n t of formalin has been added, to stand over night at room temperature. On the following day the antiserum has an appearance of opalescence similar to that resulting from heat treatment. Electrophoresis of the formolized antiserum indicates changes qualitatively similar to those accompanying heat treatment, but with the formolized antiserum, the alteration can be carried further without causing gelation. A slight increase in the concentration of formalin added causes an aggregation which apparently includes all globulin components because electrophoretic analysis reveals only one globulin boundary in the formolized antiserum, i.e., the aggregated component. This component 1 The term " b o u n d a r y " refers to the interface or the refraction gradient which is created between the protein in solution and its solvent at the beginning of the experiment. The behavior of the boundary in an electric field is correlated with electrophoretic characteristics of the protein which constitutes the boundary. VOL. 228, NO. I365--28
400
BIOCHEMICAL RESEARCH FOUNDATION.
[J. F. I.
is homogeneous with respect to charge, and has the approximate mobility of the original ¢~component. If electrophoretic analyses are made on the supernatant and the sediment fractions of ultracentrifuged antipneumococcus horse serum (undiluted), the electrophoretic diagrams of the two fractions appear qualitatively alike. All serum protein components are present in the sediment fraction in slightly greater concentration than in the supernatant fraction. In some specimens of antiserum, the globulin component associated with antibody activity may be relatively increased in the sediment fraction. In general, the similarity of the electrophoretic diagrams of the supernatant and sediment fractions of the ultracentrifuged antiserum indicates that there can be no specific or semi-specific sedimentation of any protein component in the unaltered antiserum. In the case of heat-treated antipneumococcus serum, the situation is somewhat changed. When a certain protein component is aggregated as a result of heating, the sedimentation constant of this component will differ markedly from those of unaggregated components. Electrophoretic analysis of the supernatant and sediment fractions of the ultracentrifuged heated antipneumococcus horse serum reveals: first, that the component presumably associated with antibody activity is almost entirely absent from the supernatant fraction; second, that a highly aggregated, electrochemically monodisperse component is present in the sediment fraction, its mobility being approximately that of normal B globulin. About seventy-five per cent. of this component is specifically precipitable with homologous polysaccharide; specific precipitability is normally an attribute of the antibody component. Considering the cases mentioned above, it seems evident that electrophoretic analysis of a protein system, such as antipneumococcus serum, yields data useful to the quantitative control of preparative ultracentrifugation. Such analytical data are particularly helpful when physical or chemical treatment is required for specific fractionation. A Titrimetric Modification of the Glyoxalase Method for the Estimation of Reduced Glutathione.--E. F. SCHROEDER AND GLADYS E. WOODWARD. (Journalof Biological Chemistry,