UNIMOLECULAR FILMS OF NERVE PROTEINS' LYMAN FOURT AND FRANCIS 0. SCHMITT Department of Zoology, Washington University, St. Louis, Missouri Received June 11, 1936
Since the modern developments in the theory of protein structure little work has been done on the characterization of the individual proteins of nerve and on the significance of the colloidal properties of these proteins in determining nerve structure and function. The mere classification of the various nerve proteins is still in considerable doubt and, with the possible exception of the so-called neurokeratin, the localization of these proteins in the nerve axon is altogether unknown. Recent experiments on living axons by means of polarized light have shown that the proteins of the axon sheath are organized in a manner quite different from those in the axis cylinder, and there is some evidence that there may be considerable difference in the molecular drchitecture of these various proteins. Obviously before the ultimate question of the rBle of these protein structures in nerve function can be answered, these preliminary points must be settled. The present studies have been designed to aid in the characterization of certain of the nerve protein fractions now recognized. Comparison of the behavior of films of these proteins with films of various other proteins not only yields information of value for an interpretation of nerve structure, but furnishes additional facts for which the general theory of protein films must account. MATERIALS AND METHODS
The nerve proteins were prepared from the leg and claw nerves of lobsters, according to the method described by Schmitt and Bear (9, 10). The finely cut nerve bits were successively extracted in neutral saline, borate buffer of pH 9 and 11, and in N/100 sodium hydroxide. In the fraction previously called neurostromin we now recognize three subdivisions: one coming out in N/10 sodium hydroxide and exhausted with successive changes of alkali, another obtained by relatively brief extraction with N / 2 sodium hydroxide, and likewise diminishing in quantity in repeated extractions, and a third, removed by N / 2 alkali only after twelve 1 Presented a t the Thirteenth Colloid Symposium, held a t St. Louie, Missouri, June 11-13, 1936.
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to eighteen hours of reaction. I n N / 2 sodium hydroxide the nerve shreds eventually swell and soften, so that violent shaking suffices t o convert previously recognizable shreds into a strongly turbid solution. The main bulk of the nerve vanishes at this stage; strong centrifuging yields only a very small residue quite different from the original nerve fibers. The protein extracted in N/10 sodium hydroxide has a flocculation zone beginning at p H 5.5 with a maximum around pH 4.5, as determined turbidimetrically in acetate buffer. The first fraction coming out in hr/2 sodium hydroxide is also precipitated a t pH 4.5 by acetic acid. The fraction obtained after long standing in N / 2 sodium hydroxide is not precipitated under the same conditions even with prolonged centrifuging For the present film work only the fractions removed in N/lO sodium hydroxide and by brief extraction with N / 2 sodium hydroxide were used, because of the instability of the fractions obtained with weaker alkalinity. Both preparations were purified by thrice repeated precipitation with acetic acid at pH 4.5. The precipitates were fairly readily redissolved in S@rensen borate buffer of pH 9, yielding stable, slightly opalescent solutions, es. pecially if the precaution of washing out the acetate nith a little borete is observed. The final preparation was analyzed for nitrogen by microKjeldahl, the weight of protein being taken as 6.6 times the amount of nitrogen. The egg albumin used for comparison was recrystallized four times according t o the method of Sgrcnsen as described by Moriom (8), and dialyzed in the cold until barium chloride gave no test for sulfate. The protein concentration was determined by drying to constant weight Force-area measurements were made with the Adam modification of the Langmuir trough. Surface potentials were measured by a modification of the vibrating condenser method of Yarnins and Zisman (11). The film v-as applied by forcing the solution from a microburet, the capillary tip of which was held just inside the surface. By a rack and pinion stand the tip is pulled up slightly after tourhing the surface, to minimize the possibility of protein going into the bulk of the subsolution. GENFjRAL CONDITIONS O F FILM SPREADING
Three considerations arising from the method of preparation of the nerve proteins may be of significance with regard to their spreading as monomolecular lay The first is the possibility that nucleic acid from the nuclei abundant in nerve sheaths is dissolved In these extractions. Levene (7) obtained purine bases from the ammonium chloride extract of brain, as well as from a dilute alkali extract. However, Goiter, Ormondt, and Rleijer (4) have shown that the addition of nucleic acid to protein solutions has no effect, at least upon area per milligram extrapolated t o zero pressure.
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The second consideration is the unknown partition of the lipoids between the different fractions. Although lobster peripheral nerve contains relatively less lipoid than does vertebrate medullated nerve, Schmitt, Bear, and Clark (10) obtained x-ray diffraction patterns characteristic of lipoids even in nerve protein fibers spun into and extensively extracted with alcohol. Apparently the union is very tenacious; just how the properties characteristic of large arrays of short amino acid residues are modified by hydrocarbon or sterol groups, themselves capable of forming films, remains to be determined. The third consideration is the possibility of progressive hydrolysis and denaturation of the normal nerve proteins by the strong alkali solutions employed. It must be admitted that the extraction agents bring about permanent changes; for instance, the N/10 and first N/2 sodium hydroxide fractions after precipitation in acetic acid may be redissolved in weakly alkaline buffer solution of pH 9. However, the fact that at each of these stages the extractions go to completion rather than progressing steadily, seems to indicate that a definite portion of the large structure protein unit is being removed in each case, rather than that an indefinite and generalized attack on all the linkages is occurring. Of interest in this connection is the fact that upon N/300 acetate buffer of pH 4.7 the N/lO sodium hydroxide extract forms elastic patches, whereas the N/2 extract gives fluid films. This property is observed by the movement of talc particles upon the surface under the influence of gentle puffs of air from a medicine dropper. The validity of interpreting this as indicating a difference in the particle size of the two protein fractions is rendered doubtful by the fact that on dilute hydrochloric acid of pH 2.5 the N/10 sodium hydroxide extract forms fluid films which become elastic at a pressure less than 1 dyne per centimeter. A difference in the constitution of the two fractions is more probably responsible for this difference in adhesion. EXPERIMENTAL RESULTS
After the monomolecular layer of protein has been spread, either by its intrinsic spreading tendency or by the admixture of alcohol (3), it can be further studied by compression to smaller areas. Films not already elastic become so upon compression, and with increasing pressure become quite rigid. *Themanner of compression has a marked influence on the type of curve obtained. At each step of compression to smaller areas the pressure rises quickly to a maximum, then decreases to a steady value higher than the previous equilibrium value. The magnitude of this pressure readjustment is shown in figure 1, which presents an experiment in which special care was taken to obtain complete equilibi-ation. The small curves extending
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out to the side of each equilibration step having time as abscissae give the course of this adjustment. The irregularities imposed upon the equilibration curves a t high pressures are probably due t o the rigidity of the film and the difficulty of adjusting the torsion system to a n exact halance. This experiment shows several other features: (1) The approximately linear portion of the equilibrium force-area curves d i i c h , ext,rapolated to zero pressure, is used as a quantitative measure of spreading 4o
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FIQ.1. Egg albumin on N/300 acetate buffer, PIS 4.6. Unrestricted spreading a t 1.5 in.* per milligram. Ordinates, surface pressure; abscissae, for open circles, area per milligram of protein; for small points, time after ohtaining initial pressure values. The open circles give initial and equilibrium pressure values a t each area. The small points show the course in time of the corresponding equilibration. Thickness is calculated from tho specific volume (0.75 cc. per gram) for dissolved protein.
area, following the convention of Gorter. (2) At low areas t'he increase of pressure with decrease in area is less rapid than in the linear portion of the curve. Similar efTect,s have been observed also with casein and myosin. This region we shall refer t o as the plateau. (3) Both initial and equilibrium pressure values undergo a final and rapid rise at very small areas. This apparently corresponds t,o the final close-packed condensed state of molecules with long hydrocarbon chains, such as fatty acids. Other experiments show rapid increases in the equilibrium pressure at lower area's
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than those of the plateau. (4) An equilibration takes place in the opposite sense on reexpanding; this must be the reversal of the readjustments made in compressing the film. ( 5 ) The compression is reversible throughout the range of areas down to 0.25 m.z per milligram, if equilibration is allowed. Further compression brings about a complete buckling of the film, properly comparable to the collapse of a fatty acid film, and irreversible, as noted by Devaux (2). We find, as did he, a tendency for the collapsed film to form fibers. These fibers when transferred to a slide and examined with polarized light showed birefringence positive with respect to the fiber axis. While the stretching necessary in the manipulation may have been
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FIQ. 2. Egg albumin on N/300 acetate buffer, pH 4.6. Restricted spreading a t 0.63 m.2 per milligram. Coordinates as before. Initial and equilibrium pressure values shown a t each area. A to B, compression; B to C to D,expansion; D to E , adjust water level; E to F , recompression; F t o G to H , reexpansion.
responsible for a portion of the orientation, the fact is interesting as an indication that the protein in the film was in a state of partial degeneration or denaturation and capable, by interaction of side chains, of being integrated into a fibrous structure as postulated by Astbury, Dickinson, and Bailey (1). The initial points lie on a smooth curve only if the technique of compression, pressure adjustment, and further compression is maintained uniformly, whereas the equilibrium values are independent of further lapses of time, as figure 1 shows. In rapid compression an equilibration debt, as it were, is accumulated, which forces itself upon the attention of
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the observer only at relatively high pressures, if compression is rapid and uniform. The amount of equilibration in the first minute after obtaining the initial pressure is small at low pressure as the time curves show in figure 1. As the pressure increases, the fraction of the whole equilibration occurring within the first minute increases very greatly. This should not be confused with the more or less irreversible collapse of the protein film found after the condensed state is reached. That the compression is reversible above an area of 0.25 m S 2per milligram is further shown by the experiment presented in figure 2, in which a film of egg albumin is compressed, expanded, recompressed, and reexpanded. Only the initial and equilibrium points are shown a t each step.
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FIQ.3. Nerve protein extracted in N/10 sodium hydroxide. Spread on N/300 acetate buffer, pH 4.6, with aid of 4.26 per cent alcohol. Coordinates as before. Equilibrium points only, of four separate films, except open circles, which show both initial and equilibrium points. Comparison of figures 1and 2 shows that the areas at zero pressure extrapolated from the approximately linear portion of the equilibrium pressure curves are different in the two cases, although the buffer subsolutions were practically the same. This variation has been studied in other experiments and seems to be due to incomplete spreading of the protein when the surface available for its spreading is limited. It is not clear whether this is due to an incomplete uncoiling or degeneration, to use Astbury's term, of the protein from the dissolved (globular) state to its extended condition on the surface, or is caused by a partial passage of protein into the body of the subsolution in the trough. I n such restricted spreading the pressure
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rises rapidly a t first, then gradually approaches an equilibrium value. This spontaneous pressure rise is quite sensitive to the character of the subsolution and to the precise manner of application from the tip of the microburet. Nerve proteins resemble egg albumin in showing equilibration, but the plateau effect between the linear portion and the final condensed packing is absent. In figure 3 are plotted the equilibrium points of four successive films of nerve protein extracted in N/10 sodium hydroxide, spread on N/300 acetate of pH 4.6 with the aid of 4.25 per cent alcohol. One set of initial points is included as well to show the similarity to other proteins. However, instead of showing a plateau, the curves at the lowest area seem to be passing over into the condensed packing. This is not so far-reaching a distinctioh between the proteins as might be thought, however, since casein, which shows the plateau markedly on some subsolutions, s h o w hardly any upon others. Moreover, definite indications of this plateau have been obtained for nerve proteins of the neurostromin group on other buffers. Fibrinogen films, although showing the plateau upon water, show little or none upon McIlvaine’s phosphate-citrate buffer. The general relations of the phase boundary potentials observed with these films are quite similar to those reported by Hughes and Rideal (6) and by ter Horst (5), using the polonium electrode method. At great areas the potentials fluctuate. With decrease of area, the time average of these potentials tends to rise and the range of fluctuation to decrease, until the potential becomes steady a t the area a t which the whole available surface is covered with film. I n the case of elastic patch films this can be made plainly visible by dusting talc upon the surface previous to spreading. Depending on the resistance of the elastic patches, more or less pressure is required to deform them and thus to fill the available space. The steady potentials increase with decreasing area without showing any equilibration to correspond to the force changes within the sensitivity of our measurements (1or 2 millivolts). At lower areas the potential levels out, becoming constant and independent both of area and pressure. This constant potential region extends into the final upturn of the force-area curves for condensed packing. Through the region of largest equilibration the potentials remain constant. I n spreading at restricted areas the potential changes cease long before the increase in pressure reaches equilibrium. This has been observed with fibrinogen on phosphate-citrate buffer of pH 7.4. Egg albumin on N/300 acetate, pH 4.65, shows a somewhat greater tendency for the potentials and pressure to change together, but even here the potential curve is changing less with time than the pressure curve. This probably indicates that in spreading at restricted areas there is an initial period in which the actual surface concentration of protein is changing, owing to tranclatjon
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of protein chains along the surface, but that the later and longer period of the changes is due t o readjustments similar to the equilibration at each step in compression. The experiments here presented extend t o low pressures the range of the metastable state given by Hughes and Rideal (6) for gliadin films. The importance for the theory of film structure of the independence of potential and pressure in the plateau region has been emphasized by ter Horst (5). The facts shown in the present experiments, that potential depends only partially upon the area of the film (or surface concentration) and not at all upon equilibration of pressure, must be taken into consideration in any general theory of film structure. SUMMARY
The method of preparation of certain nerve protein fractions and the general features of the unimolecular films formed by them on various subsolutions are described. A phenomenon of pressure equilibration following change of area of these film has been observed. This equilibration is not associated with changes in phase boundary potentials and is distinct from irreversible collapse. The relation of this equilibration to film spreading at restricted areas is discussed. REFERENCES (1) ASTBURY, If'. T., DICKINSON, S., AND BAILEY,K.: Biochem. J. 29, 2351 (1935). (2) DEVAUX, H.: Compt. rend. soc. biol. 119, 1124 (1935). L., AND PERLEY, A. M.: Proc. SOC.Exptl. Biol. Med. 33, 201 (1935). (3) FOURT, (4) G O R T E RE., ~ ORMONDT, H. V., AND MEIJER,T. &I.: Biochem. J. 29, 38 (1935). M. G.: Rec. trav. chim. 66, 33 (1936). (5) TER HORST, (6) HCGHES, A . H., AND RIDEAL,E. K.: Proc. Roy. Soc. London 137A, 62 (1932). (7) LEVESC,P. A , : Arch. Neurol. Psychopathology 2 , 1 (1899). (8) Momow, C. 8.:Biochemical Laboratory Methods. John TWey and Sons, New York (1927). (9) SCHMITT, F. O., A N D BEAR, R. S.: (a) Proc. Soc. Exptl. Biol. Med. 32, 943 (1935); (b) Am. J. Physiol. 113, 116 (1935). (le) SCHMITT, F. O., BEAR,R. S., A N D CLARK,G. L.: Radiology 26, 131 (1935). (11) YAMINS,H.G., AKD ZISMAX,. W. A , : J. Chem. Physics 1, 656 (1933).
