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Chromatography of influenza virus hemagglutination inhibitors
DISCUSSION
I44 Chromatography Hemagglutination
of Influenza
AND
PRELIMINARY
Virus
Inhibitors
The recovery of a- and y-hemagglutination inhibitors in different fractions of rabbit serum obt,ained by ammonium sulfate precipitation has recently been described (1). Inactivation of both these types of inhibitor by periodate suggests that they are mucopolysaccharides or mucoproteins, but their recovery in serum protein fractions does not in itself necessarily indicate that the active carbohydrate prosthetic groups are linked to protein, or if they are, how strong the linkages are. Some information concerning the relationship between the active inhibitory and protein components has now been obtained by chromatography of rabbit serum on calcium phosphate columns. Absorbed serum was eluted in a gradient of phosphate buffer of gradually increasing molarity at constant pH as described by Tiselius et al. (9). The column effluent was collected in IO-ml fractions which were analyzed for protein by measuring the ext,inction coefficient at 280 rnp, for phosphate by calorimetric estimation using the standard phosphomolybdate method, and for aand v-inhibitors by titrations with standard prototype viruses (1). These are the type A2 strain A/Singapore/i/57, which is sensitive in the unheated state to w and y-inhibitors, and the type B strain ROB or LEE, which in the heated state is sensit,ive only to a-inhibitor. Although some batches of calcium phosphate resolved albumin and globulin components into separate peaks eluting at different phosphate molarities (Fig. 2)) others failed to resolve them into more than one peak (Fig. 1). Nevertheless, the chromatographic patterns of inhibitory activity obtained were not widely different. A typical chromatogram of normal rabbit serum obtained at both pH 6.8 and 8.0 is illustrated in Fig. 1. The slight differences between the inhibitory patterns of the two test strains probably reflect, the composite nature of a-inhibitor previously described (1) Two broad peaks of inhibitory activity are distinguishable, one eluting between 0.01 and 0.02 M and the other between 0.06 and 0.1
REPORTS
M. The forward position of the initial peak close to the gradient origin indicates that some of the inhibitory activity is dissociable from the main protein components, and this was confirmed by analysis of t,he leading part of the chromatogram in a less steep gradient, which gives higher resolution. Chromat,ograms of untreated serum give no indication of the position of y-inhibitor relative to the or-inhibitory components. For its location, chromatography of serum treated with neuraminidase to remove the a-component is necessary. In most experiments where this has been done the neuraminidase-resistant y-inhibitor began to elute at 0.04 M and was closely associated with protein (Fig. 1). Inhibitor eluting below this molarity, separate from protein in the corresponding untreated serum, there-
70 Inhibition of WEE
60 .3-
FIG. 1. Gradient elution chromatograms at pH 6.8 of normal rabbit serum obtained before and after treatment with neuraminidase. (Top) Control without neuraminidase treatment (Bottom) After neuraminidase treatment. Both the control and treated sera were exposed to the same thermal conditions: 37°C for 5 hours followed by inactivation at 56°C for 30 minutes. Samples ahead of the gradient interface are plotted by tube number.
DISCUSSION
,4ND
PRELIMINARY
fore, represents all, or part of, the a-inhibitory components. These are found mainly in the pseudoglobulin and albumin fractions (1). Since the inhibitory component of the pseudoglobin fraction does not show any significant tendency to dissociate from protein on chromatography (Fig. 2), it may be concluded that the easily dissociable inhibitor revealed in chromatograms of whole serum is equivalent to that found in t,he albumin fraction. This conclusion is confirmed by the chromatogram of the albumin fraction illustrated in Fig. 2, which shows dissociation of inhibitor from protein. Also included in Fig. 2 are chromatograms of the euglobulin fractions. y-Inhibitor is located in the water-insoluble euglobulin (I), and it is of interest to note that most of the inhibitor in this fraction is closely associated with protein, although the y-component itself can be precisely defined only after neuraminidase treatment. Inhibitory act,ivity appears to be medi-
ated by serum mucoproteins; its partial separation from the protein moiety of t,hese compounds, by chromatography, suggests that with some inhibit.ors at least, the carbohydrate moiety alone may be capable of inhibitory activity. The closer association of r-inhibitory activity wit,11 protein xuggests that the distinctive characteristics of serum inhibitors may depend not only on the chemical structure of carbohydrate prosthetic groups, but also on the proteins to which they are attached or on the type and tenacity of the linkage between these two moieties. REFERENCES f. COHES, 4., and BELYAVIX, G. (1961), Virology 13,5%67. 2. TISELIUS, A., HJERT~, S., and LEVIA-, 6. (1956). Arch. Riochem. Riophys. 65, 132-155. G. BELYAVIX 11. COIIES
Department
of Bacteriology Gniversity College Hospital IYnizwsit y Street
Lontlon.
1r.c.1.
Specific
Phosphate Holmty
Medical
School
lc,lgla?ld
Neutralization
FIG. 2. Gradient elution chromatograms at pH 8.0 of rabbit serum fractions obtained by ammonium sulfate precipitation. F/lA, water-soluble euglobulin; F/lB, water-insoluble euglobulin; F/ II, pseudoglobulin ; F/III, albumin.
145
REPORTS
of Interferon
by
Antibody’
Earlier attempts to immunize rabbit,s and chickens against interferon derived from the chick allantois, with or without the addition of adjuvants, did not meet with success (1, Z?,3). This report describes the preparation of antisera in guinea pigs which specifically neutralized the activity of interferon. Interferon was obtained from L cells exposed to ultraviolet (UV) -irradiated Newcastle disease virus (NDV,,,) as described previously (4). The materials after tenfold concentration contained between 300 and 1000 units per 0.5 ml, as determined by the plaque reduction test with vesicular stomatitis virus (VSV) (4). Guinea pigs received over a period of 5 months, at roughly evenspaced intervals, a total of 13 intraperi1 The work described has been supported b> Grant E-2405 and Training Grant 2E-101 from the National Institutes of Health, United States Prtblic Health Service.
I44 Chromatography Hemagglutination
of Influenza
AND
PRELIMINARY
Virus
Inhibitors
The recovery of a- and y-hemagglutination inhibitors in different fractions of rabbit serum obt,ained by ammonium sulfate precipitation has recently been described (1). Inactivation of both these types of inhibitor by periodate suggests that they are mucopolysaccharides or mucoproteins, but their recovery in serum protein fractions does not in itself necessarily indicate that the active carbohydrate prosthetic groups are linked to protein, or if they are, how strong the linkages are. Some information concerning the relationship between the active inhibitory and protein components has now been obtained by chromatography of rabbit serum on calcium phosphate columns. Absorbed serum was eluted in a gradient of phosphate buffer of gradually increasing molarity at constant pH as described by Tiselius et al. (9). The column effluent was collected in IO-ml fractions which were analyzed for protein by measuring the ext,inction coefficient at 280 rnp, for phosphate by calorimetric estimation using the standard phosphomolybdate method, and for aand v-inhibitors by titrations with standard prototype viruses (1). These are the type A2 strain A/Singapore/i/57, which is sensitive in the unheated state to w and y-inhibitors, and the type B strain ROB or LEE, which in the heated state is sensit,ive only to a-inhibitor. Although some batches of calcium phosphate resolved albumin and globulin components into separate peaks eluting at different phosphate molarities (Fig. 2)) others failed to resolve them into more than one peak (Fig. 1). Nevertheless, the chromatographic patterns of inhibitory activity obtained were not widely different. A typical chromatogram of normal rabbit serum obtained at both pH 6.8 and 8.0 is illustrated in Fig. 1. The slight differences between the inhibitory patterns of the two test strains probably reflect, the composite nature of a-inhibitor previously described (1) Two broad peaks of inhibitory activity are distinguishable, one eluting between 0.01 and 0.02 M and the other between 0.06 and 0.1
REPORTS
M. The forward position of the initial peak close to the gradient origin indicates that some of the inhibitory activity is dissociable from the main protein components, and this was confirmed by analysis of t,he leading part of the chromatogram in a less steep gradient, which gives higher resolution. Chromat,ograms of untreated serum give no indication of the position of y-inhibitor relative to the or-inhibitory components. For its location, chromatography of serum treated with neuraminidase to remove the a-component is necessary. In most experiments where this has been done the neuraminidase-resistant y-inhibitor began to elute at 0.04 M and was closely associated with protein (Fig. 1). Inhibitor eluting below this molarity, separate from protein in the corresponding untreated serum, there-
70 Inhibition of WEE
60 .3-
FIG. 1. Gradient elution chromatograms at pH 6.8 of normal rabbit serum obtained before and after treatment with neuraminidase. (Top) Control without neuraminidase treatment (Bottom) After neuraminidase treatment. Both the control and treated sera were exposed to the same thermal conditions: 37°C for 5 hours followed by inactivation at 56°C for 30 minutes. Samples ahead of the gradient interface are plotted by tube number.
DISCUSSION
,4ND
PRELIMINARY
fore, represents all, or part of, the a-inhibitory components. These are found mainly in the pseudoglobulin and albumin fractions (1). Since the inhibitory component of the pseudoglobin fraction does not show any significant tendency to dissociate from protein on chromatography (Fig. 2), it may be concluded that the easily dissociable inhibitor revealed in chromatograms of whole serum is equivalent to that found in t,he albumin fraction. This conclusion is confirmed by the chromatogram of the albumin fraction illustrated in Fig. 2, which shows dissociation of inhibitor from protein. Also included in Fig. 2 are chromatograms of the euglobulin fractions. y-Inhibitor is located in the water-insoluble euglobulin (I), and it is of interest to note that most of the inhibitor in this fraction is closely associated with protein, although the y-component itself can be precisely defined only after neuraminidase treatment. Inhibitory act,ivity appears to be medi-
ated by serum mucoproteins; its partial separation from the protein moiety of t,hese compounds, by chromatography, suggests that with some inhibit.ors at least, the carbohydrate moiety alone may be capable of inhibitory activity. The closer association of r-inhibitory activity wit,11 protein xuggests that the distinctive characteristics of serum inhibitors may depend not only on the chemical structure of carbohydrate prosthetic groups, but also on the proteins to which they are attached or on the type and tenacity of the linkage between these two moieties. REFERENCES f. COHES, 4., and BELYAVIX, G. (1961), Virology 13,5%67. 2. TISELIUS, A., HJERT~, S., and LEVIA-, 6. (1956). Arch. Riochem. Riophys. 65, 132-155. G. BELYAVIX 11. COIIES
Department
of Bacteriology Gniversity College Hospital IYnizwsit y Street
Lontlon.
1r.c.1.
Specific
Phosphate Holmty
Medical
School
lc,lgla?ld
Neutralization
FIG. 2. Gradient elution chromatograms at pH 8.0 of rabbit serum fractions obtained by ammonium sulfate precipitation. F/lA, water-soluble euglobulin; F/lB, water-insoluble euglobulin; F/ II, pseudoglobulin ; F/III, albumin.
145
REPORTS
of Interferon
by
Antibody’
Earlier attempts to immunize rabbit,s and chickens against interferon derived from the chick allantois, with or without the addition of adjuvants, did not meet with success (1, Z?,3). This report describes the preparation of antisera in guinea pigs which specifically neutralized the activity of interferon. Interferon was obtained from L cells exposed to ultraviolet (UV) -irradiated Newcastle disease virus (NDV,,,) as described previously (4). The materials after tenfold concentration contained between 300 and 1000 units per 0.5 ml, as determined by the plaque reduction test with vesicular stomatitis virus (VSV) (4). Guinea pigs received over a period of 5 months, at roughly evenspaced intervals, a total of 13 intraperi1 The work described has been supported b> Grant E-2405 and Training Grant 2E-101 from the National Institutes of Health, United States Prtblic Health Service.