EFFECT O F ULTRASONIC WAVES ON PROTEINS
335
T H E EFFECT OF ULTRASONIC WAVES ON SERUM PROTEINS W. W. LEPESCHKINI Bethesda, Maryland Received M a y 6, 1948 INTRODUCTION
Since the first application of piezoelectric vibrations to the production of ultrasonic waves (Langevin, 1920-26) (8), the action of these waves on macromolecular substances has been investigated by several scientists. Szent-Gyorgi (19) showed that starch is dwomposed by ultrasonic waves to achroodextrin. A decomposition of the molecules of highly polymerized substances was observed by Szalay (lS), who claimed that the action of ultrasonic waves may be other than mechanical. Only in the case of very large molecules, such as those of hemocyanin, did a mechanical breakdown of molecules by ultrasonic vibrations seem to be possible. dccordirig to Brohult (1) the molecules of hemocyanin are decomposed by ultrasonic waves to molecules which have only 1/2, 1/4, 1/8, and 1/1G of their original size. Similar results were obtained in the esperiments of Sozaburo (17), Kausche, Pfartkuch, and Ruska (G), Sollner (E),and Schmid and coworkers (13). Ilausche, Pfankuch, and Ruska, using an electron microscope, observed a reversible decrease of the molecules of tobacco mosaic virus under the influence of ultrasonic waves. Schmid found that the molecular weight of polystyrenes dissolved in toluene decreases under the influence of ultrasonic waves. Schmid esplains the breakdown of the macromolecules produced by ultrasonic waves by the difference in the deformability between these molecules and the solvents. Sonic and ultrasonic waves disintegrate not only large molecules but also other particles and bring about a peptization of colloids (Sollner, 1938). Recently Lepeschlrin (10) concluded that salt-like inner bindings of protein molecules (-COONHs-) can be destroyed by a purely mechanical action of the displacement current in a high-frequency electromagnetic field (-COONH3= -COOH
+
KHL-),
In the case of high-frequency sonic vibrations, especially when cavities are formed in watery solutions (see Frenkel (3)), the bubbles produced may excite dissolved oxygen molecules and produce an oxidation which results in the formation of hydrogen peroside, nitrous acid, and nitric acid (Fox (2); Icling and Kling ( i ) ) .These substances can then affect other substances present in the solution and bring about, for instance, hydrolysis of polysaccharides. Even sucrose can be split if very powerful waves are used (Roikh (12); TVesenmeier (20)). On the other hand, ultrasonic waves can produce not only a disintegration but also an accumulation, aggregation, and coagulation of substances (11, 16). The experimental work upon which this paper is based was carried out by the author in the Institute Zeileis, Gdlspnch, Austria. Present address: 4513 Battery Lane, Bethesda, Maryland.
336
W. JV. LEPESCIIKIN
In the present paper, t’le action of ultrasonic waves upon serum proteins was investigated. The methods used have been previously applied by the author to the investigations of the action of heat and short electric waves on proteins (9, 10). MATERIALS 4 N D METHODS
-
Human serum was mixed with 2 per cent acetate buffer to maintain its pH constant. If not buffered, it loses carbon diovide and its pH gradually increases. It was poured into small Erlenmeyer flasks, 10 cc. into each flask. The flasks were put into a paraffin oil bath above the oscillating quartz crystals in a generator (300 watts) of ultrasonic waves of frequency 28: and 2871 kilocycles. The diameters of the openings in the brass frame holding the crystals were G 5 and 30 mm. The upper surface of the crystal was 40 mm. below the surface of the paraffin oil. The oil IVRS cooled by means of copper tubing immersed in it, through which tap water (13-15°C.) was continuously circulated. Kear the flask containing serum a stirrer \vas inserted into the oil bath. The temperature in the bath did not vary by more than 1°C. during the experiment. The change in the average molecular weight of the serum proteins was investigated by two methods. The first method was that of the longitudinal dispersion of light (9). A beam of dark red and infrared rays was passed through a plane-parallel cuvette (“glass cell”) filled with a protein solution and was intercepted by a horizontal brass strip cemented on the rear wall of the ruvctte a t its lower edge. The dispersed rays fell above the brass strip on a photographic plate (sensitive to infrared rays) where, after development, a dark circular segment appeared. The area of the semicircle corresponding to this segment, expressed in mme2,is a measure of the dispersion and can be computed according to the formula:
where B (mm.) is the width of the segment and a (mm.) is the distance between the light beam and the upper edge of the brass strip. The thickness of the layer of protein solution through which the light beam passes is 10 mm. Since serum shows Tyndall scattering of dark red rays, mainly produced by particles of lipids, its effect on the photographic plate must be considered. The magnitude of this effect was found by photographing, in the above manner, the dispersion of dark red and infrared rays produced by a colloidal suspension of cholesterol acetate which had a Tyndall effect of the same color, in white light, and of the same strength, in dark red rays, as the serum and which showed only negligible longitudinal dispersion. The area of the dispersion semicircle found was subtracted from that found for the serum. The absorption of dark red and infrared rays by serum expressed in per cent of incident light was determined by photographing on the same plate the rays which passed through the serum and those which did not pass through it and by comparing the blackening of the negative in both cases
EFFECT O F ULTRASONIC WBVES ON PROTEINS
337
in a photometer.2 The reflection of the rays from the cuvette walls was 4 per cent of the incident light, The area of the semicircle of dispersion must be increased by percentage of the ahsorption and reflection. As the longitudinal (lispersion is almost proportional t o the cube root of the molecular ]\.eight of proteins, the average molecular weight, relative to its original value, \vas computed according to the formula 11 = (P/Po)3,where P is the longitudinal dispersion aft'er the action of ultrasonic naves and Po is the original dispersion. The second method was that of the precipitation of serum by acetone. This met,liod, proposed by Schulz (14) and Jirgensons (5), consists in titration of protein solutions from a micropipet by acetone. The amount of acetone necessary to produce t8hefirst cloudiness (or its first increase) in serum is the greatel,-, the smaller is the molecular \\,eight of the proteins. The change in the hydrabion of the protein molecules was investigated by the titration of sei'um with a saturated solution of ammonium sulfate (micropipet,). By determining the relative viscosity of serum by means of an 0stn.ald viscosimeter, :It constant temperature (ISOC.), not only the change in the hydration but also tlie change in t'he molecular shape of the proteins was tested. RESULTS
1. Changes i n the average molecular weight of serum proteins
I n the following tables B is the width of the dispersion segment (mm.); F is the area of tmhecorresponding semicircle (mm.2);Fo is this area after considei*ing the nlnsorpt'ion and reflection; T is the area produced by the Tyndall effect in a fresh suspension of cholesterol acetate; P is the longitudinal dispersion ( F 2'); ;lf is the average relative molecular weight of the proteins; t is the temperature (in "C.) of the solution after the action of ultrasonic waves; a is the dista.nce bet,\veen the light beam and the upper edge of the brass strip. From table 1 it may be seen that the Tyndall effect was not perceptibly affected by ultrasonic waves, while the average molecular weight of serum proteins increased during the first minute of the action of ultrasonic waves and then decreased and became smaller t'han the original molecular weight.3 h similar result was obtained in other experiments independently of the temperature of the serum after the action of ultrasonic waves, as may be seen from table 2. The first increase in the molecular weight of the serum proteins observed in slightliy alkaline solutions was, however, not observed if the reaction was acid, as may be seen from table 3. It should be emphasized that buffered serum does not change its pH during the action of ultrasonic waves. The experiment's on the action of ultrasonic Jvaves of frequency 2871 kc. gave similar results. If the reaction of the serum was slightly alltaline, they produced first a polymerization while their longer action always led to the destruction of L' The nlisorpt~ionincludes the scattering of t h e rays by Tyndnll effec,t t o the sides :md hack. a As \v:is stated in previous papers (J. Phys. Colloid Cliein. 51, 875 (1947)), tlie Tyndnll effect of proteins in dark red rays is negligible in coinparison with their lo~igit,udinaldispersion.
338
W. W. LEPESCHKIN
protein molecules. The energy of ultrasonic waves of frequency 2871 kc. was found to be greater than that of ultrasonic waves of frequency 285 lrc. in the apparatus used: The polymerization occurred, in the first case, during the first second and probably in less than half a second after the beginning of the action of ultrasonic waves (2871 kc.), but the arrangement used could only detect this TABLE 1 Experiment 1: Effcct of ultrasonic waves on huinan serum p H = 7.8; frequency of ultrasonic n'avcs = 287 kc.; exposure in photographing the dispersions = 120 sec.; a = 3.5 n m . ; stirrer n-as removed t o allow thc temperature t o rise TIME OF EXPOSURE TO ULTRASONIC NAVES
min.
0 1/60 1 3 15
[
27.5 30
20
I
314 370
25 30
45
25
262
i
1 1
34i 414 428 364 293
199 199
199 199 199
1 3 3.7 1.4 0.25
14s 215 229 165 94
TABLE 2 Average relatiue ~ n o l e c d a rweight of proteins in lizciiian s e r u m following e x p o s u m to ziltmsonic waucs pH = 7.3-7.7; frequency of ultrasonic waves = 285 kc.; exposure and a are the same as in table 1; t h e stirrer was removed only in experiments 2 and 3; t h e Tyndall effect was not changed by ultrasonic waves EXPERIMENT NO.
. . .. . . . . . .
~
TIME OF EXPOSURE TO ULTRASONIC W A V E S
4
5
6
7
nr
M
M
M
M
1
1
1
1 2.s
1 1.6
2.3 2.7
2
3.5
2.3 0.7 21
3
_______
nr
~
i ~
8
M
nrin.
0 1/120 1/60 1/2 1
1 ~
n
J
5 20 t , "C
2
30
1.4
I
1
j
3 0.1
1
42
'
,
2.5
1
0.i 0.3
0.3
0.2
25
22
22
1 2 2.8 2
~
2 0.5 21
change after half a second of their action. Destruction and no polymerization was observed if the reaction of the serum was acid. Similar results were obtained in experiments in which the method of titration Theoretically the energy of ultrasonic waves is proportional t o the square of their frequency (1).
339
EFFECT O F ULTRASOSIC lV.4VES ON PROTEIXS
of serum with acetone was employed, as may be seen from table 4; the amount of acel one decreased in the first minute of the action of ultrasonic waves (285 kc.) TABLE 3 Azwagc i d n l i c c m o l c c i t l u ~ireighl of s c r u v ~protcius following e.rposcirc to dtrnsonic u w c s Frcquciicy, csposure, and n nrc the same as in tables 1 and 2; the stirrer n'ns removed in espcriinent 11 EXPER1UE:NT SO... , .
PH OF
TEE S E R C U . . ACIlOS
.. . ... . . . ... ... ...
.. . .
,
. . . . ..
. .. . . . ... .. . , .., , . , ., . ... , , . ,.
OF ULTRASONIC \VA\'ES LASTED
11
12
9
10
6.1
4.9
4.8
1 0.3
1 0.5
1 0.7
0.3
0.4
0.4
-
2.4
df
1 0.7 0.6 0.5 0.4 0.3
1
2 6 10 t , " C. . . . . . . . . ,
...... ..........., ...
22
0.5 0.1
20
30
0.7 0.4 20
EjJcct of irllrasonic iuaues on serum proteins Ainount of acetone in cc. necessary to produce an increase of cloudiness in 2 cc. of human seruiu, a t 20°C.; every number is an average of four numbers det'erinined i titration . , ., .. , . .....
13 ___-7.3
7.4
7.4
5,26
5.26
FREQUENCY OF ULTRASONIC WAVE
258 kc.
285 kc.
2871 kc.
265 kc.
2871 kc.
EXPERIYEST NO.
pH
, ,
OF Tmc SERUU
16
ACTION OF ULTRASONIC \ Y A W S LASTED
17
____
wiu.
0 1/60 1/.1 1/a 3/4 1 2
0.56 0.52 0.45
10
0.40 0.53
0.40 0.61
0.55
0.6s
0.57
0.62
0.53
0.70
0.62
0.69
0.5s
0.70
0.62
0.70
0.68
0.72
0.67
0.70
0.52
n
20
0.65 0.68
0.41
d
5
0.65 0.54
0.62 0.63 0.G2
arid then increased again if the reaction was alkaline; it increased, however, without any preliminary decrease if t,he reaction was acid.
340
IY.
\V.
LEPESCHKIN
2. Injzietice of heat ~ i p o the t ~ actioti of ultrasonic waves
The temperature increase of serum subjected to the action of ultrasonic waves was, in the experiments described, not sufficient to produce a denaturation of proteins (maximum 45°C.). Moreover, no great difference was observed between the action of ultrasonic waves on serum proteins whether the stirrer had been removed or not and whether the final temperature was 21°C. or 45°C. Some experiments were made in which the serum \vas either cooled with ice before the action of ultrasonic waves or not cooled. The effect of the ultrasonic waves lasted for 15 min. and no distinct difference was observed betveen the changes in molecular weight in either case, as may be seen from table 5 . We may therefore conclude that a moderate temperature rise in the serum does not influence the action of ultrasonic waves upon its proteins. 3 . Di%ferencein the change produced by ultrasonic waves a i d by tetiipemturc
It should be pointed out that changes in the molecular weight of serum proteins produced by ultrasonic waves gradually disappear under the influence of molecular movement. Restoration of the original molecular weight was observed in TABLE 5 E.fect of heat upon the action of ultrusonic u w e s cipon s e r u m pi,olei/is Avernge relative niolecular weight of serum proteins (computed from lorigitutlinal dispersion) after t h e action of ultrasonic waves during 15 min. E X P E R M C N T NO... , .
. . . , . . . , . .. . .
22
-~
___
4-5 days a t 3°C. and in several hours a t 18°C. In a recently published paper (lo), the author showed that the average molecular weight of serum proteins decreases with increasing temperature but the restoration of the original molecular weight, after heating human serum to 25°C. or to 34"C., required only 5 min. after the temperature had been lowered to 14°C. Only heating to 47°C. led to a more constant decrease in the molecular weight, so that in 7 min., after lowering the temperature to 14"C., the molecular weight was one-fifth of the original weight. Moreover, the difference in the action of the temperatllre increase and that of ultrasonic waves became marked if the reaction of the serum was slightly alkaline; an increase in the molecular weight was never observed after a temperature rise. At the same time, in experiment 1, this increase was observed in spite of an increase in the temperature of the serum to 45°C.
4. Titration with ainmonium sulfate arid viscosity of scrim The results of the titration of human serum with a saturated solution of ammonium sulfate before and after the action of ultrasonic waves are given in table G.
EFFECT O F ULTR.\SOSIC JV.L\'ES
34 1
ON PROTEIXS
The volume of the ammonium sulfate solution remained constant after the action of ultrasonic waves for 10 min. That is, the number of hydrophilic groups in the molecules of serum proteins did not change during 10 m h . of this action, while :after 15 min. a small increase in hydrophily \vas observed. That not only the number of hydrophilic groups but also the shape of the molecules did not change during 10 min. of the action of ultrasonic waves is seen from the experiments in which the viscosity of human serum was determined before and after the action of ultrasonic waves, as demonstrated by table 7. TABLE 6 Eject o j itlirnsoriic icat~eson l ~ i i i i i u seriiiri i~ Volumes (cc.) of saturated solution of nniiiioniuiii sulfate added t o 2 cc. of human serum t o produce the increase in i t s cloudiness
I EXPERIUENT NO.
ACTION O F ULTRASOKIC W A V E S LASTED .-
SONICWAVES
___15 miii.
kc.
0.76 0.78
23 24 25 26
TABLE 7 Ejccl o j iillimonic wai'cs 011 Itiirrinn s e r t i i t i Time (seconds) (luring ivliicli serum flowed through t h e cnpillarv t u b i n g of the viscosimeter at 18°C. I
1
1 min.
~-
' 1/60min.
'
3 min.
~
10 min.
____. -pI-i
"C .
20 20
ACTION O F U L T R A S O N I C W A V E S L A S T E D
, ~
61 61
1 ~
GO 61
~
el 62
~
60 GO
DISCUSSION
I n the esperiments described in the present paper only relatively short exposures to ultrasonic ivaves were used. The effect of a long esposure is certainly more complicated. As nearly all the available osygen in blood is present in the form of a chemical compound ivith hemoglobin, serum contains little dissolved oxygen; but even this small quantity present in serum can be excited by cavities and eventually oxidize the substances in it. Indeed, if the action of ultrasonic waves was prolonged to 1 hr. or more, the color of the serum chnngecl from yellow t o green. However, osidntion of proteins could not lie observed. Such oxidation cannot be espected either if the ultrasonic waves act only for 10 min. Also, the formation of acids in serum \Vliicli could produce hydrolysis
342
W. W. LEPESCHKIN
of the proteins is impossible in such a short time and is proved by the fact that the pH of the buffered serum is not altered by ultrasonic waves. As we have seen, a small increase in temperature does not influence the action of ultrasonic waves distinctly. The change in the average molecular weight of the serum proteins was observed if the temperature was almost constant and did not exceed 20°C. One may think that the temperature of protein molecules could be higher than the temperature of the solution, because ultrasonic waves produce heat passing from one substance to another. However, the relative surface of protein molecules is so great that the heat produced would be at once absorbed by the solution. Krasny-Ergen (7a) calculated the possible increase in the temperature of particles with diameter of 0.01 mm. and found it to be only 0.00001"C. above the temperature of the solution; still smaller must be the difference in the case of protein particles which are only 0.00001 mm. in diameter. Therefore, the changes in the molecular weight of serum proteins observed in the experiments described when the temperature of the serum did not exceed 45°C.can be ascribed only to the direct action of ultrasonic waves. This action can be only a mechanical one, as was explained by Sclimid for polystyrenes (see introduction). The difference in the action of ultrasonic waves upon serum when its reaction was alkaline or acid can be explained by the fact that the molecular size of proteins diminishes as the pH departs from the isoelectric point which lies on the acid side of neutrality (pH = 4.8 for serum albumin and pH = 5.2 for serum globulins). But the larger the molecules, the more easily they are destroyed hy mechanical action. As to the synthetic action of ultrasonic 'ivaves when serum has an alltaline reaction, it can be explained by the impact of protein molecules shaken by ultrasonic waves, similar to that observed in the case of water vapor which condenses to droplets under the influence of ultrasonic waves. Brought in contact with each other the protein molecules may react chemically, forming salt-like bindings (-COOH XH2- = -COONH,-). Such bindings seem to be present also in intact molecules of proteins and can probably be torn asunder by the mechanical action of ultrasonic waves so that proteins with a smaller molecular weight are produced, but the normal molecules can be restored again in time by the action of molecular movement. That the bindings mentioned are responsible for the change in molecular weight under the influence of ultrasonic waves is confirmed by the fact that this change is not accompanied by a change in the number of hydrophilic groups in the molecule.
+
SUMMARY
Ultrasonic waves of frequency 285 kc. increase the average molecular weight of serum proteins if the reaction of the serum is alkaline and if the exposure is short (about 1 min.). A longer exposure decreases this weight, however. If the reaction of the serum is acid, no increase but only a decrease in the nioleculnr weight is observed. Ultrasonic waves of a higher frequency (2871 kc.) bring about an increase in the molecular weight if they act only for 1 sec.; their further action decreases tliis weight. As no changes in liydrophily or viscosity are observed during the action of ultrasonic waves, it \\.as concluded
EFFECT O F ULTRBSONIC WAVES ON PROTEINS
343
that the supposed salt-like bindings COOT\"^-) of the protein molecules are destroyed ]\.hen the molecular weight decreases, or they are formed if it increases. Acid reaction increases the size of protein molecules and as a consequence makes them more unstable toward the mechanical action of ultrasonic Ivaves, so that even a very short action of ultrasonic waves results in a decrease in tile average molecular weight of serum proteins. REFERENCES (1) BROHVLT, S r . : Nature 140, 805 (1037). (2) Fox, F.: J . Wash. Xcad. Sci. 30, 402 (1940). (3) FRENKEL, Y A : Acta Physicocliini. U.S.S.R. 12, 317 (1940); Chein. Abstracts 34, 7682 (1940) ; 35, 3132 (1041). , E. : Gmudlagcn ztnd E r g c h i s s o der Ult,ascliallfoor,schuny, p. 173. W. de Gruyter mid Compnny, Bedin (1039). S , J . prakt'. Chem. 159, 303 (1042); 160, 21, 65 (1942); 161, 30 (1942); (5) J I I Z G E S S ~ NB.: Biochem. Z.310, 325 (1942); 311, 332 (1042); Kolloid-Z. 98, 72 (1942). ( 6 ) I ~ A L W HG.EA., , PFANKLCH, E., A N D RUSICA, H.: Katurwissenscliaften 29, 573 (1941). (7) I
