Effects of ultrasound on catalase and malate

dehydrogenasea)
H. Ali Kashkooli, James A. Rooney, and Robert Roxby
Departmentsof Physicsand Biochemistry,Universityof Maine, Orono,Maine 04473
(Received6 July 1979;acceptedfor publication25 January 1980) ,

Catalaseand malate dehydrogenase

(MDH) were subjectedto the soundfield producedby a transversely
o,scillatingwire drivenat 20 kHz. Catalasewasnot inactivated
underany conditions
of sunication
whereasMDH inactivationincreasedexponentiallywith the duration of sunicationand dependedupon
the initial enzymeconcentration.The inactivationwas not the result of collapsecavitationor thermal
inactivationand was probablyrelated to the presenceof acousticmicrostreaming.

PACS numbers: 43.80. Gx

INTRODUCTION activity/mgprotein (1 unit is the amountof enzymecat-

The effects of ultrasound on enzyme systems has been
studiedby many investigators and reviewed byEl'p e r. x
Research into the mechanisms involved in enzyme
inactivation has shown that effects associated with
lapse cavitation can'be important. Investigators
as DeGrois and Baldo2 have emphasized mechanical
damage caused by the cavitation as the mechanism for
suchinactivationWhileCoakleyet al. s believe that in-
teractions with free radicals produced by cavitation is
the important mechanism. There have also been re-
ports of inactivation of enzymes caused by exposure to
noncavitating ultrasound including studies on the en-
zymes trypsin and a-amylase by Stefanovic et al. 4'5
However, Macleod and Dunn6 showed that the enzyme
inactivation observed by Stefanovic et al. 4' probably
resulted from chemicals sonically released from a rub-
ber membrane that was part of the sample holder for
the enzyme solutions. In addition Dunn and Macleod?
have shown, for five selected enzymes, that collapse
cavitation was a necessary condition for enzyme inac-
tivation. The question remains whether noncavitating
ultrasound can cause inactivation of enzyme systems
and what mechanisms may be involved. In order to
address that question the enzymes catalase and malate
dehydrogenase were selected to be exposed to a con-
trolled noncavitating ultrasonic field.
I. MATERIALS AND M fiTHODS
;Beef liver catalase (Worthington ;Biochemical Compa-
ny type CTR) was obtained as a crystalline suspension
in saturated ammonium sulfate solution. Solutions
prepared by dialysis of the sample into 0.05M sodium
phosphate buffer and dilution to concentrations appro-
priate for sonication and enzyme assay. ;Enzyme activ-
ity was measured by the assay of Beers and Sizer 8 in
which the decrease in absorbance of H.O.at 240 nm is
followed spectrophotometrically. As supplied, the
sample has a specific activity of 20 000 units of enzyme
,,
a) Extractedfrom the thesisof H. A. Kashkooliin partial
fullfilment of the requirements for the M. S. degree in Phy-
sics, University of Maine, Orono, ME, 1977. Parts of
this work were reported at the 95th meeting of the Acoustical
Society of America.
alyzing conversionof I /M of substrate/min)anda
protein concentrationof 6.7 mg/ml. The material was
substantially pure by the criterion of polyacrylamide
gel electrophoresis in SDS. Integration of spectropho-
col- tometric scans of gels stained for protein indicated that
such 95% of the material present was in a single zone mi-
grating at a rate consistent with the molecular weight
of the catalase subunit. Solutions for sonication were
preparedby diluting the dialyzed sample 1/1000 with
dialysate to a final concentrationof 6.7 /g/ml. The
sonicated solution was diluted further by a factor of 30
just prior to assay. Control experiments showed that
the specific activity of the enzyme was unaffected by
dilution.
Malate dehydrogenase(MDH) partially purified from
pig heart mitochondria (Worthington type MADHC) was
obtained in ammonium sulfate suspension and dialyzed
into 0.1M phosphate buffer/H 7.4. As supplied, the
samplecontained3.7 mg protein/ml and hada specific
activity of 1200units/mg protein. The samplewas not
pure by gel electrophoresis. In this case the MDH band,
which migrates at a rate indicating a molecular weight
of 35 000 in an SDS polyacrylamide gel electrophoresis
experiment, represented only about 40%-50% of the
staining material in the gel. This result was confirmed
by electrophoresis under conditions in which the enzyme
remained active. In these experiments the MDH band
was identified by the local precipitation of reduced ni-
fro blue tetrazolium dye in the presence of malate and
NAD. MDH assays in the sonication studies were car-
ried out in the reverse direction, monitoring oxidation
were of NADH by oxalacetate by absorbance at 340 nm. So-
lutions of MDH for sonication were prepared by dilut-
ing the dialyzed sample 1:20 and 1:1000 with dialyzate.
The final total protein concentrations were then 190
g/ml while the actual MDH concentrations
were ap-
proximately half these values. The more concentrated
solution was further diluted by a factor of 50 immedi-
ately before assay. In the course of these experiments
it was observed that dilution of MDH results in a slow
decay in activity. This effect was too slow to be sig-
nificant during the time periods of the sonication ex-
periments and, in any case, would be corrected for by
the unsonicated control.
The method for sonication was similar to that used

1798 J. Acoust. Soc. Am. 67(5), May 1980 0001-4966/80/051798-04500.80 (D 1980 Acoustical Society of America 1798

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 by Williams et al. 9which utilizes a transversely driven
wire. This method has several advantages. First,
since the samples are degassed, it permits the effec-
tive sonication of the sample solution without the com-
plications of uncontrolled stable bubble activity or col-
lapse cavitation. Second, it allows the investigator to
continuously measure and control the displacement am-
plitude of the sound source. Knowledge of this para-
meter permits one to then use theory to calculate the
viscous stresses to which the enzymes were subjected.
Finally, while it does not utilize sonically driven bub-
bles, results obtained with this method have been
shown, by studies by Rooney, as well as Williams
et al. 9to yield results on the bioeffects of ultrasound
which can be related to ultrasonically excited bubble
activity.
Inour experiments a250 / diameter tungsten wire was
mechanically clamped to the tip of a step horn coupled
to a PZT-4 coaxial cylinder transducer which was driv-
en at 20 kHz by an oscillator amplifier combination.
The tip of the wire was rounded so that it was hemis-
pherical in shape with a radius of curvature equal to
that of the wire shank. The length of the wire was ad-
justed to be an odd multiple of a quarter-wavelength of
the transverse mode of the wire in order to achieve
maximum displacement amplitude of the tip. 9 In these
experiments this length was 1.1 cm.
The enzyme solutions were sonicated by placing 0.1
ml of the solution into a quartz vessel whose inner di-
mensions were I cm in height and 0.5 by 0.2 cm for its
base. The treatment vessel was mounted on a small
laboratory jack and positioned so that the tip of the
tungsten wire was in the central region of the solution
and the liquid surface was at a node in the oscillatory
pattern of the wire so that splashing or atomization of
the sample did not occur. These procedures were fol-
lowed in order to eliminate complications in the experi-
mental method as described by Sanders and Coakley. 2
The displacement amplitude of the tip of the wire was
measured and monitored during sonication using a mi-
croscope and calibrated eyepiece.
In this experiment 0.1 ml samples of the selected en-
zymes were sonicated for periods from 15 to 90 min at
wire tip displacement amplitudes ranging from 15 to
26 /. Control samples of the enzyme were sham soni-
cared, that is, placed in the apparatus and otherwise
treated identically, except for sonication, to the soni-
cared samples. After sonication the enzymes were as-
sayed spectrophotometrically as described above. Re-
sults were obtained for malate dehydrogenase at con-
centrationsof 185 /g/ml and3.7 /g/ml andfor cata-
lase at 6.7 /zg/ml. The experimentswere completed
at room temperature (27 C).
II. RESULTS AND DISCUSSION
Typical results for the effects of sound on the enzyme
activity as a function of the duration of treatment for a
displacement amplitude of 26 / are shown in Fig. 1.
Here the change in activity of the enzyme is based upon
a comparison of the sonicated sample to the sham-son-
1799 J. Acoust.Soc. Am., Vol. 67, No. 5, May 1980
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ioo
80
6o
<1:40
>- 5o
IO I I I I I I I
0 15 50 45 60 75 90 105
SONATION TIME t(min)
FIG. 1. Semilogarithmic plot of the percentage loss in
enzyme activity as a function of duration of sonation. Curve
C is for catalase and curves B and A are, respectively, for
malate dehydrogenase in concentrations of 185 and 3.7 /g/ml.
Data shown are for a displacement amplitude of 26
icated enzyme and is expressed as a percentage of the
sham's activity. The distribution of the data points is
indicative of the errors and reproducability in the ex-
periment which are the result of both errors in mea-
surement of the displacement amplitude and the enzyme
assay procedure.
As shown in the figure, there is no significant change
in the activity of catalase under these sonication con-
ditions. In contrast, the activity of the realate dehy-
drogenase decreased at an exponental rate which was
dependent on the concentration of the enzyme. From
Fig. I we can see that the activity A of realate dehydro-
genase as a function of duration of treatment t can be
approximated by the equation
A =Aoe't . (1)
Here A o is the initial or sham-treated enzyme activity
and k is the rate constant of inactivation. A useful pa-
rameter for comparison of the effect of ultrasound at
different enzyme concentrations is the time for 50%
inactivation T5o which is given by the equation
T5o= 0.693/k. (2)
As seen in Fig. 1, the lower concentration of realate
dehydrogenase(3.7 zg/ml)hasa T5oof 40 rainwhile
the more concentratedsamples(185 g/ml) havea
value of 15 min. The values for/e are, respectively,
0.017 rain ' and 0.006 rain '.
The results obtained for malate dehydrogenase dem-
onstrated that ultrasound can decrease enzyme activity
under the specified sonication conditions. This reduc-
tion in enzyme activity is not the result of collapse cav-
itation since earlier studies utilizing this sonication
procedure, as well as the present investigation, have
shown that collapse cavitation activity sufficient to pro-
duce free radicals is not present. 9,u Temperature in-
crease of the sonicated enzyme solution was monitored
throughout the experiment using a copper-constantan
thermocouple and the temperature was found not to in-
crease more than 2 C even for the longest sonication
period.
Since collapse cavitation and sonically induced tern-
Kakoolietal.' Effects
ofultrasound
onenzyme
systems 1799
 perature increases are not important in the inactivation
of MDH, we believe that acoustic microstreaming plays
an important role in the observed effect. The mecha-
nism for the inactivation may either be stirring-en-
couraged surface inactivation or the result of the
shearing stresses present in the boundary layers of the
fluid flow. Evidence for the former mechanism has
been described by Macritchie. xa The suggestionthat
the latter mechanism is important is based on two
types of evidence. First, shearing associated with mi-
crostreaming has been shown to be capable of causing
hemolysis.,n and functional changesin cells.x4 Second,
the shearing stresses associated with hydrodynamic
flow have been shownto be capableof causingenzyme
inactvation.Theselatter effectsonseveralenzyme
systems have been reviewed by Tirrell. Of particular
interest to the present study is the work of Charm and
Wongx6on the shear degradationof catalase. In studies
of the hydrodynamic shearing of enzyme solutions flow-
iqg t,hrough
capi!larytubestheyreportedthat the en-
zymatic activity of catalase was decreased when the
applied shear rates and stresses were as low as 91.5
s'x and 1.7 dyn/cm2, respectively.
While it is difficult to distinguish between these two
possible mechanisms, the fact that a mean threshold
displacement amplitude of approximately 20 was
found to be necessary to cause inactivation in the pres-
ent series of experiments seems to imply that stirring-
encouraged surface inactivation is not the major mech-
anism. Since acoustic shearing stresses may be the
important mechanism, it is useful to compare the mag-
nitude and duration of them with the available data
hydrodynamic inactivation of enzymes. The magnitude
of the shear rate and stress for the treatment condi-
tions used to obtain the data shown in Fig. I can be cal-
culated using theory previously derived and dis-
cussed.. At 20 kHz in aqueoussolutions the bound-
ary layer thickness is approximately 4 . For a dis-
placement amplitude of 26 in such solutions the
shear rate is of the order of 1.7 x l0 s s ' and the shear
Stress is 1700 dyn/cm2. An estimationof the various
errors involved in the experimental procedure and the
approximations of the theory show that the maximum
uncertaintYin the magnitude of the shear stress is 400
dyn/cm. Thus, we observedno loss in enzymaticac-
tivity for catalase even though the magnitude of the
shear rates and stresses were three orders-of-magni-
tude greater than those used in the hydrodynamic stud-
ies of the inactivation of ca[alase. We believe that the
important difference betweenthese experiments is the
duration of the applied stress. For [he experiments of
Charm and Wong the stress was applied in a continuous
manner for durations as long as 90 min. The duration
of stress in the ultrasound case was estimated using
the appropriate theory to be 0.6 ms. The importance
of duration of shearing stress as a parameter has been
pointed out by Charm and Wong6 as well. It is worth
noting that importance of duration of shearing stress
has also been demonstrated for the shearing of erythro-
cytes.?,'8
Concentration dependence of enzyme inactivation,
such as shown in Fig. 1, has been observed previousIf
1800 J. Acoust.Soc.Am.,Vol. 67, No. 5, May1980
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and suggests that molecular dissociation is involved in,
some way in the inactivation mechanism. Since the
MDH sample is not pure, the possibility that there are
stabilizing interactions between MDH and another un-
identified component cannot be ruled out. However,
other data suggest that it is the dissociation of MDH,
which exists as a dimer at high concentration, to mo-
nomers which produces the effect. Shore9 has shown
that the fluorescence of labels covalently attached to
MDH changed reversibly as the sample is diluted and
has interpreted the data to indicate a monomer--dimer
dissociationconstantof 3.5 x 10'a gin/1. Resultsof
frontal elution chromatography in our laboratory gen-
erally confirm this result, although they indicate a dis-
sociation constant somewhat lower than that observed
by Shore, the difference perhaps attributable to the
fact that our protein was unmodified by attachment of
fluorescence labels. The uncertainty in the dissocia-
tion constant does not affect the conclusion that there
would be substantial differences, on the order of 50%,
in the degree of association between the more and less
concentrated MDH solutions. Since the rate constants
for inactivation differ by a similar order of magnitude,
a reasonable interpretation would be that sonication
primarily affects the more labile monomeric form of
the enzyme, producing conformational changes which
inactivateit and/or prevent its reassociation. This
interpretation would be consistent with the independent
observation that the threshold amplitude for MDH in-
activation was not dependent on concentration.
Discussion of the mechanism(s) involved in inactiva-
on tion for either the present ultrasound study or the re-
lated hydrodynamic experiments is difficult at the
present time. The inactivation is probably the result
of alteration of the tertiary structure of the molecule
and our results suggest that it is the monomer form
which is primarily susceptable to inactivation. It is
not clear from our experiments whether the sonication
produces an effect on the monomer which is not nor-
mally significant or whether the ultrasound treatment
accelerates the previously observed slow process
which leads to MDH inactivation at low concentration.
Finally, it is important to place the present work in
perspective with other ultrasound experiments on en-
zyme activity, particularly those which have carefully
examined the mechanisms involved in the inactivation.
We believe that two important differences in the exper-
iment permitted elucidation of the observed effects.
These are the small sample volume used and the ex-
istence in the sample volume of a highly efficient
source of acoustic streaming. In contrast, previous
studies had utilized test volumes which were at least an
order-of-magnitude greater than ours. Also, for the
studies conducted using noncavitating ultrasound, care
was made to degas the liquid thereby removing the sta-
ble bubbles or nuclei which could serve as sources of
active acoustic streaming. It should be noted that in
the study of Dunn and Macleod7 streaming was observed
within the degassed sample in their test vessel but it
was a type useful for stirring the contents of the vessel
and typically does not have high shear rates or stresses
associated with it. Thus, we were able to measure the
' Kashkooli
et al.: Effectsof ultrasound
on enzymesystems 1800
 observed effects on MDH because in the small volume lI. E. El'piner, Ult'asound:
Physical,Chemical
andBiologi-
used the enzyme molecules were caused to flow through cal Effects (Consultants Bureau, New York, 1964).
the region near the source of acoustic streaming many 2M.DeGroisandP. Baldo,Acustica22, 222-228 (1969).
times during the duration of the exposure. W. T. Coakley,R. C. Brown, andC. J James,Arch.
Bioehem. Biophys. 159, 722-729 (1973).
However, it is difficult to quantitatively extrapolate 4V.Stefanovic,
I. Kostic,M. Bresjanac,andD. Zivanovic,
from these results obtained with a vibrating wire to the Bull. Soy. Chim.'Belg. 24, 175-178 (1959).
more usual situation of a sonicated enzyme solution V. Stefanovic,
A. Kjukanovic,K. Velasevic,andD. Zivanovic,
Experimentia 16, 486- 487 (1960).
which may contain a distribution of active stable bub-
6R.M. MaeleodandF. Dunn,J. Aeoust.Soc.Am. 40, 1202-
bles. The reason for this difficulty is that the displace- 1203 (1966).
ment amplitudes required for inactivation are suffi- ?F. DunnandR. M. Maeleod,J. Aeoust. Soe.Am. 44, 932-
ciently large so that the bubble motion in a similarly 940 (1968).
sonicated liquid would be very complicated, as des- 8R.F. BeersandI. W. Sizer, J. Biol. Chem.195, 133 (1952).
cribed by Nyborg.' In particular it is likely that-sur- 9A.R. Williams, E. E. Hughes,andW. L. Nyborg,Science
face wave activity would be present on the bubbles. The 169, $71-873 (1970).

presence of these surface waves would drastically

10j.A. Rooney,Science169, 869-871 (1970).
llj. A. Rooney,J. Acoust.Soc.Am. 52, 1718-1724(1972).
change the acoustic streaming pattern near the bubbles
12M.F. SandersandW. T. Coakley,Exp.Cell Res. 73, 410
making any quantitative statement of thresholds for ef- (1972).
fects in such a situation impossible. In spite of this 3Advanced
P'oteinChemist,y,editedby C. B. Anfinsin,J. T.
difficulty, the more general implications of the present Edsall, and F. M. Richards (Academic, New York, 1978),
work do have relevance to that sonication situation. ol. 33, p. 283.
The important fact is that the present experiment dem- lcj. A. Crowell,B. K. Kusserow,andW. L. Nyborg,Ultra-
sound Med. Biol. 3, 185-190 (1977).
onstrates that collapse cavitation is not necessary for
sM.V. Tittell, J. Bioeng.2, 183-193 (1978).
inactivation of at least one of the more labile enzymes. 16S.E. CharmandB. L. Wong,Biotechnol. Bioeng.12,
Thus, the suggestion of this experiment is that inacti- 1103-1109 (1970).
vation of labile enzymes may occur in more convention- ?J.A. Rooney,J. Biol. Phys.2, 26-40 (1976).
al ultrasonic fields for sufficiently high amplitudes and 8C.G. Nevaril, E. C. Lynch,C P. Alfrey, Jr., andJ. C.
durations of treatment. Hellurns, J. Lab. Clin. Med 71, 784-790 (1968).
19j.D. ShoreandS. K. Chakrabanti,Blocbern.15, 875 (1976).
ACKNOWLEDGMENTS 'The
Proteins,editedby H. NeurathandR. L. Hill (Academic,
New York, 1975).
We wish to thank Sharon Quirtnet for assistance with 2W.L. Nyborg,P'oceedings
FiniteAmplitudeWaveEffects
the enzyme assay procedures. This research was sup- in Fluids, Copenhagen 1974 (I. P. A. Press, Guildford,
England, 1975), p. 245.
ported in part by the American Heart Association-
Maine Affiliate and by NIH Grant AM 18852.

1801 J.Acoust.
Soc.Am.,Vol.67,No.5,May1980 Kashkooli
etal.: Effects
ofultrasound
onenzyme
systems 1801

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