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Interaction of protein and lipoprotein monolayers with nitrogen dioxide-trans-2-butene gaseous mixtures
COMMUNICATIONS
962 of the lytic factor. Increasing concentration releases the proceeds.
the external osmotic tension, and lysis
REFERENCES GASCON, S., OCHOA, A. G., NOVAES, M., AND VILLANUEVA, J. R., Arch. f. Mikrobiol. 61, 156167 (1965). DUELL, E. A., INOUE, S., AND UTTER, M. F., J. Bmteriol. 88, 1762-1773 (1964). JAIME MONREAL ELWYN T. REESE] Inst. Biol. Celular, Consejo Superior de Investigaciones Cientijicas Madrid 6, Spain Received June 21,1968 1 Pioneering Research Laboratory, Natick Laboratories, Natick, Mass. Interaction layers
of Protein
and
with Nitrogen tene Gaseous
U. S. Army
Lipoprotein
Mono-
Dioxide-tram-2-BuMixtures*
Insoluble monomolecular films of lipids have been used extensively to study the interaction of numerous compounds with components of biomembranes. A number of studies have related these interactions to in vivo properties of the reacting compounds, and thus established the general usefulness of this model as a means of studying membrane properties. We recently reported on the interactions of nitrogen dioxide-olefin gas atmospheres with saturated and unsaturated lecithin monomolecular films using surface pressure measurements. Whereas films of dipalmitoyl lecithin, a saturated phospholipid, showed no interaction with any of the test atmospheres used, films of egg lecithin, an unsaturated phospholipid, showed significant changes in the surface pressure-surface area (z-.4) curves in the presence of all atmospheres containing nitrogen dioxide (1). Since proteins also constitute an important part of the cell membrane of living organisms, it was of interest to study the interactions of pollutant atmospheres with oriented protein and lipoprotein films. The gas-train assembly, Teflon-coated trough, and Wilhelmy plate method of surface pressure measurement used in this study were described previously (1). * This work was supported by Grant APO0487 from Research Grants Branch, National Center for Air Pollution Control, Bureau of Disease Prevention and Environmental Control.
Crystalline bovine albumin, 100% pure as determined by electrophoresis (Nutritional Biochemical Corp.), was spread from aqueous solution by the method of Trurnit (2). Various lipid-protein mixed films were formed by spreading the protein from aqueous solution onto monomolecular fdms of phospholipids (dipalmitoyl lecithin or natural bovine cephalin) according to the method of Vilallonga et al. (3). All films were spread on a phosphate buffered subphase (pH = 7.2) and equilibrated for 15 minutes at a surface pressure of l-2 dyn/cm prior to exposing them to the gases. Further equilibration produced no changes in surface pressure values over a period of two hours. The films were then exposed to a standard atmosphere (i.e., air flowing at the rate of 300 ml/ min) or to the following test atmospheres, all flowing at this same rate of 300 ml/min: (a) 0.33% nitrogen dioxide in air; (b) 0.08% trans-2-butene in air; and (c) 0.33yo nitrogen dioxide and 0.08yo trans-2-butene in air. In all cases, the films were exposed to the flowing gases for one hour before manual compression of the film was initiated. Surface pressure readings were then obtained at various film areas. Significant changes in the Z--A curves for the
16
5
I
I
6
7 METER?/MG.
8
9
X IO
FIG. 1. IDA curves of protein films exposed to standard atmosphere (air) and test atmosphere b (0) and to test atmospheres a and c (0 ).
963
COMMUNI [CATIONS
lecithin does not interact with NO2 (l), it interacts strongly with bovine albumin (as determined by previous VA experiments). The protective action of dipalmitoyl lecithin for the protein film was observed at all phospholipid:protein ratios tested (71X36to 40:60, w/w). Moreover, the expansion of the bovine cephalin-protein mixed films on exposure to the NO, containing atmospheres corresponded to that observed for monolayers of unsaturated phospholipids alone when exposed to the NOz containing atmospheres (1). These data, although limited to only two phospholipids and one protein, suggest that the effect of NOz on the lipoprotein films studied appears to be a function only of the phospholipid component of the film. In general, membrane lipoproteins contain a large proportion of unsaturated phospholipids attached to structural and functional protein. In vivo interaction of the supporting phospholipid with nitrogen dioxide, or other reactive pollutants, could result in an expansion of the exposed cell membrane. This expansion would then lead to a change in the conformation of the attached protein. In the case of a functional protein, changes in conformation would be accompanied by changes in enzyme activity. Nelson (4) has suggested that the key to mem1 I I brane function resides in the protein-lipid 100 SO 35 60 and that lipid-lipid or lipid-eninteractions, PERCENT OF TROUGH AREA vironmental interactions have less physiological FIG. 2. 7r-il curves of protein-cephalin films ex- significance. Our data further suggest that proposed to standard atmosphere (air) and test attein-environmental interactions also have less mosphere b (0) and to test atmosphere a and c biological significance than lipoprotein-environ(0). mental interactions. pure protein films were observed in the presence of all atmospheres containing nitrogen dioxide, while the trans-2-butene did not interact with the film, nor did it appear to inlluence the nitrogen dioxide-film interaction. However, whereas exposure of unsaturated phospholipid films to nitrogen dioxide containing atmospheres resulted in a large expansion of the film (l), exposure of bovine albumin films to these same test atmospheres resulted in a significant contraction of the films (Fig. 1). When the dipalmitoyl lecithin-protein mixed films were exposed to the various pollutant atmospheres, no film-pollutant interaction was observed. However, exposure of the natural bovine cephalin-protein mixed films to the nitrogen dioxide containing atmospheres resulted in a large expansion of the films (Fig. 2). On the basis of the p.4 data obtained, the presence of dipalmitoyl lecithin appears to prevent the bovine albumin-NOz interaction. It should be pointed out that, whereas dipalmitoyl
REFERENCES 1. FELMEISTER,
A., M.
Environmental 2. TRURNIT, H. J. 3. VILALLONGA, FERNANDEZ,
AMANAT, N. WEINER, Sci. Technol. 2, 40 (1968).
J. Colloid Sci. 16, 1 (1969). F., R. ALTSCHUL, M. S. Biochim. Biophys. Acta 136,
406 (1967). 4. NELSON,
G. J., Biochim.
Biophys.
Acta
144,
221 (1967). ALVIN FELMEISTER~ MOHAMMAD AMANAT NORMAN D. WEINER
College of Pharmaceutical Sciences Columbia University New York, New York 10025 Received March 22, 1968; accepted July
2, 1968
1 Present address: Rutgers-The State versity, College of Pharmacy, Newark, Jersey.
UniNew
962 of the lytic factor. Increasing concentration releases the proceeds.
the external osmotic tension, and lysis
REFERENCES GASCON, S., OCHOA, A. G., NOVAES, M., AND VILLANUEVA, J. R., Arch. f. Mikrobiol. 61, 156167 (1965). DUELL, E. A., INOUE, S., AND UTTER, M. F., J. Bmteriol. 88, 1762-1773 (1964). JAIME MONREAL ELWYN T. REESE] Inst. Biol. Celular, Consejo Superior de Investigaciones Cientijicas Madrid 6, Spain Received June 21,1968 1 Pioneering Research Laboratory, Natick Laboratories, Natick, Mass. Interaction layers
of Protein
and
with Nitrogen tene Gaseous
U. S. Army
Lipoprotein
Mono-
Dioxide-tram-2-BuMixtures*
Insoluble monomolecular films of lipids have been used extensively to study the interaction of numerous compounds with components of biomembranes. A number of studies have related these interactions to in vivo properties of the reacting compounds, and thus established the general usefulness of this model as a means of studying membrane properties. We recently reported on the interactions of nitrogen dioxide-olefin gas atmospheres with saturated and unsaturated lecithin monomolecular films using surface pressure measurements. Whereas films of dipalmitoyl lecithin, a saturated phospholipid, showed no interaction with any of the test atmospheres used, films of egg lecithin, an unsaturated phospholipid, showed significant changes in the surface pressure-surface area (z-.4) curves in the presence of all atmospheres containing nitrogen dioxide (1). Since proteins also constitute an important part of the cell membrane of living organisms, it was of interest to study the interactions of pollutant atmospheres with oriented protein and lipoprotein films. The gas-train assembly, Teflon-coated trough, and Wilhelmy plate method of surface pressure measurement used in this study were described previously (1). * This work was supported by Grant APO0487 from Research Grants Branch, National Center for Air Pollution Control, Bureau of Disease Prevention and Environmental Control.
Crystalline bovine albumin, 100% pure as determined by electrophoresis (Nutritional Biochemical Corp.), was spread from aqueous solution by the method of Trurnit (2). Various lipid-protein mixed films were formed by spreading the protein from aqueous solution onto monomolecular fdms of phospholipids (dipalmitoyl lecithin or natural bovine cephalin) according to the method of Vilallonga et al. (3). All films were spread on a phosphate buffered subphase (pH = 7.2) and equilibrated for 15 minutes at a surface pressure of l-2 dyn/cm prior to exposing them to the gases. Further equilibration produced no changes in surface pressure values over a period of two hours. The films were then exposed to a standard atmosphere (i.e., air flowing at the rate of 300 ml/ min) or to the following test atmospheres, all flowing at this same rate of 300 ml/min: (a) 0.33% nitrogen dioxide in air; (b) 0.08% trans-2-butene in air; and (c) 0.33yo nitrogen dioxide and 0.08yo trans-2-butene in air. In all cases, the films were exposed to the flowing gases for one hour before manual compression of the film was initiated. Surface pressure readings were then obtained at various film areas. Significant changes in the Z--A curves for the
16
5
I
I
6
7 METER?/MG.
8
9
X IO
FIG. 1. IDA curves of protein films exposed to standard atmosphere (air) and test atmosphere b (0) and to test atmospheres a and c (0 ).
963
COMMUNI [CATIONS
lecithin does not interact with NO2 (l), it interacts strongly with bovine albumin (as determined by previous VA experiments). The protective action of dipalmitoyl lecithin for the protein film was observed at all phospholipid:protein ratios tested (71X36to 40:60, w/w). Moreover, the expansion of the bovine cephalin-protein mixed films on exposure to the NO, containing atmospheres corresponded to that observed for monolayers of unsaturated phospholipids alone when exposed to the NOz containing atmospheres (1). These data, although limited to only two phospholipids and one protein, suggest that the effect of NOz on the lipoprotein films studied appears to be a function only of the phospholipid component of the film. In general, membrane lipoproteins contain a large proportion of unsaturated phospholipids attached to structural and functional protein. In vivo interaction of the supporting phospholipid with nitrogen dioxide, or other reactive pollutants, could result in an expansion of the exposed cell membrane. This expansion would then lead to a change in the conformation of the attached protein. In the case of a functional protein, changes in conformation would be accompanied by changes in enzyme activity. Nelson (4) has suggested that the key to mem1 I I brane function resides in the protein-lipid 100 SO 35 60 and that lipid-lipid or lipid-eninteractions, PERCENT OF TROUGH AREA vironmental interactions have less physiological FIG. 2. 7r-il curves of protein-cephalin films ex- significance. Our data further suggest that proposed to standard atmosphere (air) and test attein-environmental interactions also have less mosphere b (0) and to test atmosphere a and c biological significance than lipoprotein-environ(0). mental interactions. pure protein films were observed in the presence of all atmospheres containing nitrogen dioxide, while the trans-2-butene did not interact with the film, nor did it appear to inlluence the nitrogen dioxide-film interaction. However, whereas exposure of unsaturated phospholipid films to nitrogen dioxide containing atmospheres resulted in a large expansion of the film (l), exposure of bovine albumin films to these same test atmospheres resulted in a significant contraction of the films (Fig. 1). When the dipalmitoyl lecithin-protein mixed films were exposed to the various pollutant atmospheres, no film-pollutant interaction was observed. However, exposure of the natural bovine cephalin-protein mixed films to the nitrogen dioxide containing atmospheres resulted in a large expansion of the films (Fig. 2). On the basis of the p.4 data obtained, the presence of dipalmitoyl lecithin appears to prevent the bovine albumin-NOz interaction. It should be pointed out that, whereas dipalmitoyl
REFERENCES 1. FELMEISTER,
A., M.
Environmental 2. TRURNIT, H. J. 3. VILALLONGA, FERNANDEZ,
AMANAT, N. WEINER, Sci. Technol. 2, 40 (1968).
J. Colloid Sci. 16, 1 (1969). F., R. ALTSCHUL, M. S. Biochim. Biophys. Acta 136,
406 (1967). 4. NELSON,
G. J., Biochim.
Biophys.
Acta
144,
221 (1967). ALVIN FELMEISTER~ MOHAMMAD AMANAT NORMAN D. WEINER
College of Pharmaceutical Sciences Columbia University New York, New York 10025 Received March 22, 1968; accepted July
2, 1968
1 Present address: Rutgers-The State versity, College of Pharmacy, Newark, Jersey.
UniNew