ANALYTICAL BIOCHEMISTRY
50, 163-173 (1972)
A Sensitive and Specific Plate Test for the Quantitation
of Phospholipases
E. HABERMANN
AND
K. L. HARDT
Pharmakologisches Institut der Justus Liebig-Universitat, 63 Giessen, Germany
Received March 22, 1972; accepted May 22, 1972
1. Phospholipases A clear suspensions of egg yolk. This is also true when
the substrate is incorporated into agar agar or agarose gels. Round, cleared
areas develop from circumscribed enzyme depots. Their diameter is, over a
wide dose range, proportional to the logarithm of the enzyme concentration
applied. This parameter can be used for the measurement of phospholipases
A from bee venom, Crotalus terrificus venom, and pancreas. The optimal
reaction conditions have been worked out for the bee venom enzyme.
2. Under these conditions, 7.5--15 pg of the bee venom enzyme and 30-60
ng of pancreatic and Crotalus phospholipase A are still detectable. Incubation
at 50°C increases the cleared areas and eliminates simultaneously less resistant enzymes, such as trypsin and chymotrypsin.
3. The specificity of the test for phospholipase A can be further improved
by incorporation of human erythrocytes into the egg yolk plates. Sensitivity
remains unchanged.
4. Phospholipase C produces turbid areas in egg yolk plates. Their diameter
can be used for the quantitation of the enzyme. Low activities of phospholipase B, together with highly active phospholipase A, can be followed
in the egg yolk plate since phospholipase B renders the once cleared zones
centrally turbid. With lysolecithin plates, phospholipase B can be measured
by this dimming effect.
The substrates of phospholipases undergo heavy physical changes
upon hydrolysis. These changes can be utilized in the quantitation of the
enzyme. The splitting of the micellar lecithins and cephalins into the
solubilizing lyso compounds and fatty acids by phospholipases A has
been used as the basis for some already established methods, such as the
clearing of egg yolk suspensions (1-5). The production of lyso compounds also delays the coagulation of egg yolk induced by heat, a very
simple method for measuring phospholipases A (1,6). Clearing of lipoprotein suspensions, delay of heat coagulation of egg yolk, and hemolysis, altogether result from the detergent character of lysolecithin and
related compounds (17).
Phospholipases B catalyze the cleavage of lyso compounds into the
highly hydrophilic glycerophosphorylcholine and the very hydrophobic
163
Copyright© 1972 by Academic Press, Inc.
All rights of reproduction in any form reserved.
164
HABERMANN AND HARDT
fatty acids. This becomes manifest with time by an increasing turbidity
of the incubation mixture and by a progressive shortening of heat coagulation time of egg yolk previously treated with phospholipases A (Habermann, unpublished).
Phospholipase C releases lipophilic diglycerides as well as the hydrophilic phosphorylcholine. Thus it increases the turbidity of lipoprotein
suspensions, for instance diluted egg yolk. The flocculation of egg yolk
has proved useful for measurement of a-toxin of Clostridium perfringens,
and for checking its purity (7,8).
The turbidity tests mentioned are simple and very sensitive. However,
some drawbacks must be taken into account. First, the physical state of
the substrate is critical and may be badly reproducible. Second, a mixture of various phospholipases producing turbidity or clearing cannot
be analyzed.
During experiments on the localization of phospholipase A after disc
gel electrophoresis, we incorporated egg yolk into agar plates which
were then loaded with the sliced gel columns. Electrophoretic zones containing phospholipases A cleared the underlaying egg yolk. This
prompted us to study the reaction in detail in order to use it as a micro
method for phospholipases, especially phospholipase A. A detailed description is given in reference 9.
METHODS
Agar agar (0.24 gm) or agarose (for concentration see below) was
dissolved in 30 ml 0.05 M sodium acetate/sodium barbital buffer, pH
7.5, in a boiling water bath. The solution was cooled to 50°C. Then 26.0
ml was mixed with 0.25 ml egg yolk suspension (see "Materials") and 0.25
ml 0.01 M CaCl 2 solution and poured into plastic Petri dishes (13.6 cm
i.d., plain bottom) preheated to 50°0. After consolidation of the layer
we punched, (with the aid of a sharpened metallic tube) cylindrical
holes and emptied them by suction through an injection needle. The
layer was about 1.8 mm thick and the volume of the holes was 23 µl.
Each hole was charged with 20 µl enzyme solution in saline containing
0.1 % bovine serum albumin. During incubation for 20 hr at 50°, the
enzyme diffused into the gel and either cleared the egg yolk (phospholipase A) or increased its turbidity (phospholipase C). The diameter of
these areas could be measured easily with dividers since the border lines
were always surprisingly sharp. For the measurement of phospholipase B,
400 µg lysolecithin/ml was incorporated into agarose plates and incubated at 37°.
To increase the specificity of the egg yolk plates toward phospholipase A, the sites of lysolecithin formation were also localized by in-
QUANTITATION OF PHOSPHOLIPASES
165
corporation of erythrocytes. Then 0.3 ml washed human erythrocytes
per plate was added. Under otherwise identical conditions, incubation
took place for 20 hr at 37°. The areas of hemolysis were well circumscribed and of approximately the same diameter as the cleared zones in
pure egg yolk plates.
MATERIALS
The substrate was standardized in the following way: 1 part fresh egg
yolk was mixed with 3 parts 0.85% (v/v) saline and centrifuged for 2
min at 2000 rpm (620g). Then 1 ml portions of the supernatant were
frozen by liquid air in small polyethylene vessels and stored at -30°C.
Under these conditions the suspension retained its properties for at least
6 months. Storage at +4° or after slow freezing proved to be less satisfactory.
Agar agar (purified and free from inhibitors, for bacteriological
purposes) was from E. Merck A.G. (Darmstadt), agarose from Serva,
Heidelberg. Phospholipase A2 was prepared from bee venom according
to reference 10. Phospholipase A from crotoxin1 was obtained according
to reference 11. Pancreatic phospholipase A2 was a gift of Professor
G. H. De Haas, Department of Biochemistry, Utrecht (12). As a source
of phospholipase C, we used a culture filtrate from C. perfringens (600
DLM 2 /ml), which was a gift of Behringwerke, Marburg. A lyophilized
saline extract from the stinging apparatus of hornets (Vespa crabro)
served as a mixture of phospholipases A and B. Crystallized trypsin and
chymotrypsin were from Boehringer, Mannheim.
RESULTS
(1) Dose-Response Relationship for Various Phospholipases A
The relationship between logarithm of concentration of bee venom
phospholipase A and diameter of the cleared areas was linear over a
very wide dose range (Fig. 1). Reproducibility is satisfactory, as can
be seen also from Fig. 1.
We also assessed reproducibility when the same amount of bee venom
phospholipase was applied to a series of egg yolk plates. Each of five
plates was incubated with five deposits of 2 ng enzyme as described under
"Methods." Means and standard deviations were calculated for the
values measured on the individual plates, and on randomly selected
values from the 5 plates (Table 1). It can be concluded that the re1
Crotoxin is a complex between phospholipase A and crotapotin, obtained from
crotalus terrificus venom.
• DLM = minimum lethal dose.
16fi.
HABERMANN AND HARDT
25
y
[mm]
20
15-
Y =9.07+3.84 log c
10
Y=8.89+3.88log c
5
10000
100
Frn. 1. Dose-response relationship for bee venom phospholipase A. Two sets of
experiments were performed on the egg yolk-agarose plate. Ordinate: diameter of
cleared zones (mm). Abscissa: concentration c (ng/ml). Values of regression lines
for the two experiments are given in the figure; for one line the standard deviation
Sy is also drawn.
producibility is the same whether the enzyme is applied to one or to
different plates.
Whereas the detection limit for bee venom phospholipase A is in the
range of 7.5-15 pg, the system responds much less sensitively to the
TABLE 1
Mean Values (y) and Standard Deviation (s,) as Calculated for 2 ng Bee Venom
Phospholipase A Applied to Different Plates
Five loadings per plate
y
(mm)
16_3
16_7
Sy
(mm)
0.11
16.5
16.5
0.22
0.21
0.35
16.2
0.16
Five loadings from five plates,
randomly combined -
y
(mm)
Sy
(mm)
16.5
16 ..5
0.35
0.37
16.4
16.4
0.10
16.3
0.13
0.18
167
QUANTITATION OF PHOSPHOLIPASES
mm
22·
18
14
10
JJ9/ml
6-t---"........-'--.--.--,-,-,-,"'"T'T~~-.--.--.--....~r-r-r"'T"
1
10
100
Frn. 2. Dose-response relationship for phospholipase A from Crotalus terrificus
(lower curve) and pancreas (upper curve) in egg yolk-agarose plate. Ordinate:
diameter of cleared zones (mm). Abscissa: concentration of solutions applied
(µg/ml).
pancreatic or Crotalus (rattle snake) enzyme. With these enzymes, it is
important to use agarose instead of agar, otherwise the dose-response
line will become uselessly flat. In any case, semilogarithmic linearity
applies for a restricted dose range only, and the sensitivity is at least
1000 times less than with bee venom enzyme. The detection limit lies
in the range of 30 ng for both Crotalus and pancreatic phospholipase
(Fig. 2).
(2) Some Important Parameters for Measurement of Phospholipase A
(a) Temperature. Phospholipases A belong to the relatively thermostable enzymes. Bee venom enzyme works better at 50°C than at 37°
or 60°. Since with increasing temperature the boundaries of the cleared
zones progressively lose their sharpness, incubation at 50° is a reasonable compromise. This temperature also improves the specificity of the
test. For instance, trypsin and chymotrypsin also brighten the egg yolk
suspension. Both enzymes lose most of their activity at 50°; at this
temperature, they are about 100,000 times less effective than bee venom
phospholipase A (Figs. 3 and 4).
168
HABERMANN AND HARDT
50°C
37°C
60°C
15
~
10
~
~
~
~
~
~
~
~
~
~
~
~
~
~
I.
~
~
~ 'W
~ It!
~
~
~
~
~
~
~
~ ~
~ ~
~
~
I
;;;;
Ii
I
i
i
i
i
~
~
~ :;.
~
~~ ;.::~ % ~~~
a b c d
III I
a b c d e
Iii
+ + • •
a b c d e
FIG. 3. Discrimination of trypsin and chymotrypsin from bee venom phospholipase A with different temperatures. Height of columns represents diameter of
cleared zones at different temperatures. (a) = 1 mg/ml chymotrypsin; (b) = 100
µg/ml chymotrypsin; (c) = 1 mg/ml trypsin; (d) = 100 µg/ml trypsin; (e) = 15.6
ng bee venom phospholipase A/ml. ( +) =no effect;
=slight, badly contoured clearing.
<•)
( b) Incubation time. The diameters of the cleared areas increase with
time, quickly within the first hours, then more slowly. Simultaneously,
the boundaries become more and more blurred. A 20 hr incubation is
the right compromise between sensitivity and readability of the test. The
dose-response curve is steeper after 20 hr incubation compared with
shorter times, which is an additional advantage of this incubation time.
(c) pH. We tried various buffers between pH 6.8 and 9.2. Although the
bee venom enzyme reaches its maximal activity between pH 8.0 and 8.4,
we preferred pH 7.5 because the turbidity of the incorporated egg yolk
and thus the contrast against the cleared zones decreases at higher pH
values.
(d) Ca++ ions. Since egg yolk already contains calcium ions, addition
of low amounts of calcium chloride (IQ- 4 to IQ-5 M final concentration)
failed to affect measurably the activity of the bee venom or pancreatic
enzyme; 10-3 M Ca++ partially inhibited both enzymes.
(e) Substrate concentration. Increasing amounts of egg yolk suspension
QUANTITATION OF PHOSPHOLIPASES
169
Frn. 4. Phospholipases A and proteases in egg yolk-agar plate at various
temperatures. Bee venom phospholipase A: (1) = 1 µg/ml, (2) = 250 ng/ml, (3) =
62.5 ng/ml, (4) = 15.6 ng/ml, (5) = 3.9 ng/ml, (6) = O.S7 ng/ml. Pancreatic phospholipase A: (7) = product of Sephadex G-50 chromatography according to (12).
Chymotrypsin: (Sa) = 1 mg/ml, (Sb) = 100 µg/ml, (Sc) = 10 µg/ml. Trypsin:
(9a) = 1 mg/ml, (9b) = 100 µg/ml, (9c) = 10 µg/ml. Crotalus phospholipase A:
(10) = 100 µg/ml. k = Control (0.1 % bovine serum albumin in saline). Arrows
denote shadows of holes due to illumination and not enzymic effects.
(see "Materials") were incorporated into the agar agar or agarose plates.
The plates were tested with concentrations of bee venom enzymes varying
between 1 µg/ml and 1 ng/ml with the other conditions kept constant.
The clearing zones were approximately equal in plates containing between
0.2 and 0.6 ml egg yolk suspension. However, it is inadvisable to apply
more than 0.4 ml because this results in loss of sharpness. In order to
gain optimal contrast, we finally used 0.25 ml substrate per agar plate,
and 0.4 ml substrate per agarose plate.
(f) Choice of supporting agents. Whereas bee venom phospholipase A
acted equally in 0.8% agar or 0.8% agarose, this was not the case with
pancreatic or Crotalus phospholipases A. Both enzymes were considerably
(by more than 50%) inhibited in agar when compared with agarose. Thus
the latter is preferable as a supporting agent.
After our first stock of agarose had been exhausted, we were unable to
reproduce our previous results with fresh batches. The clearing zones
became asymmetrical and the dose dependence of the diameters was flat.
However, when we reduced the agarose concentration by 0.1-0.2% only,
the original performance of the plates could be restored. Therefore, 0.6%
agarose was the final concentration of choice.
0.1 % bovine serum albumin should always be incorporated into the
solvent used for preparation of enzyme dilutions. Otherwise more than
50% losses of activity may occur. This was true for the three phospholipases A tested, and might be due to absorption when the extreme dilu-
1.70
HABERMANN AND HARDT
tions are made. Subsequent addition of albumin to the enzyme samples
already filled into the holes did not restore the original activities.
(3) Preliminary Experiments with Phospholipases B and C
The plate tests were initiated for measuring phospholipases A. In
preliminary experiments, similar tests proved to be useful for the
determination of phospholipases B and C too.
(a) Hornet venom (phospholipase A and B). Egg yolk plates, when
incubated with the venom at 37° and 50°C, yield rosette-like areas
with a peripheral clear ring and a turbid center. The clearing is due to
phospholipase A since it is larger at 50° than at 37°. In egg yolk
erythrocyte plates, the cells are hemolyzed over the whole diameter of
the rosettes. The central turbidity is smaller at 50° than at 37°. Zones
of corresponding size and turbidity also appeared in the otherwise clear
lysolecithin plates, which were incubated at 37°. Both bee venom phospholipase A and C. perfringens culture filtrate failed to dim the lysolecithin plates.
mm
50°C
25
20
15
10
Filt ratverd unnung
FIG. 5. Dose-dependent turbidity of egg yolk plates at 50°C or egg yolkerythrocyte plates at 37°, caused by phospholipase C. Ordinate: diameter of turbid
areas. Abscissa: dilution of culture filtrate from C. perfringens.
171
QUANTITATION OF PHOSPHOLIPASES
(b) Culture filtrate of Clostridium perfringens (phospholipase C).
Phospholipase C from C. perfringens is known to increase the turbidity
of lipoprotein suspensions and to hemolyze washed red cells [for review
see (16)]. Dilutions of the filtrate were applied onto plates containing
egg yolk (incubated at 50°C), egg yolk and erythrocytes, and erythrocytes alone (both incubated at 37° for 20 hr). In the plates containing
egg yolk, sharp-edged zones of increased turbidity appeared whose
diameters were proportional to the log dose (Fig. 5). Hemolysis was not
visible on the plate containing egg yolk with erythrocytes, probably
because the more prominent turbidity covered the hemolytic clearing.
The latter was well demonstrable on the plates containing erythrocytes
alone. Thus phospholipase C can be quantified easily by our system.
DISCUSSION
Simplicity and extreme sensitivity are the advantages of the new
plate test for phospholipases A. A practically infinite number of samples
can be applied and compared at the same time. The sensitivity for the
bee venom enzyme is in a range usually reserved for radiological methods.
Only peroxidases can be measured in comparable concentrations (13).
Despite the simplicity of the test, some factors are to be considered.
Originally, the test was devised for bee venom phospholipase A. The
lower sensitivity for pancreatic and Crotalus terrificus phospholipase A
may be due to the fact that we used our system unchanged for these
enzymes. Second, the bee venom enzyme possesses a much higher specific
activity than the other two phospholipases A when measured in the
autotitrator (Table 2). Third, we avoided the addition of the strongly
activating deoxycholate for reasons given below.
The supporting gel merits special attention. Whereas both agar agar
TABLE 2
Turnover Rate (Moles Fatty Acids Liberated per Mole Enzyme) for
Various Phospholipases Aa
Substrate solution
Phospholipase A from
Bee venom
Pancreas
C. terrificus venom
Without additives
With 22.5 mM CaCI2 and
6.0 mM deoxycholateb
55,000
<30
2,000
60,000
8,000
20,000
' 'i> Automated titratiOn with 0.025 M NaOH at 50°C and pH 8.0 for 5 min. Substrate:
2.0 ml egg yolk, dilution 1: 12.
b Final concentration.
172
HABERMANN AND HARDT
and agarose are suitable for the bee venom enzyme, agarose is superior
for the pancreatic and Crotalus phospholipase A, probably because both
are basic substances. The I.P. 3 of Crotalus phospholipase A is about pH
9.2 (18), and of pancreatic phospholipase A about pH 7.4 (12). They
might interact with the acidic groups of agar agar which should be
eliminated during purification of agarose. Recently (14), an agarose
was prepared which does not interact with basic proteins. By introducing
such supporting media, the plate test could be adapted to basic
phospholipases.
Some peculiarities of the plate tests manifest themselves in the doseresponse relationship. Whereas it is approximately linear for related
tests where the substrate is premixed with the egg yolk suspension (for
instance inhibition of egg yolk coagulation (1), or clearing of egg yolk
suspensions (4,5)), the plate test for phospholipase A yields a semilogarithmic plot. This is to be expected because of the e function in the
formula for radial diffusion ( 15) .
During the interactions between the diffusing enzyme and the substrate fixed in the gel, the resistance of the medium toward diffusion
might be altered in various ways, e.g., by diminution of substrate and
formation of solubilizing (lysolecithin) or insoluble (diglyceride) agents.
Finally the diffusion constant depends on both the size and shape of the
enzyme molecule. The variables mentioned cannot be separated experimentally. More astonishing is the fact that clear, distinct, reproducible
values are obtained.
Limitations of our method are evident:
(a) Not only the amount and the specific activity of a given phospholipase, but also its ability to diffuse into the gel, determine the diameters
of the cleared or dimmed areas. Therefore, quantitative comparison between enzyme preparations possessing different diffusion characteristics
will not be feasible. Structurally bound enzymes are to be solubilized
before measurement. If the enzyme solution contains substances interfering with the diffusion process either directly (by combination with
the enzyme) or indirectly (by interaction with the gel), the results may
become misleading.
(b) The enzymic reaction becomes visible by a decrease or increase of
turbidity. On the other hand, some phospholipases need additives in
order to achieve a maximal turnover rate (see Table 2). These additives,
however, clear the egg yolk suspension, thus rendering the measurements
more difficult. As yet, we have not tried solubilizers, for instance deoxycholate, in the plate tests. Perhaps a relation between substrate and
• I. P.
=
Isoelectric point.
QUANTITATION OF PHOSPHOLIPASES
173
solubilizers can be found which activates sufficiently without too much
clearing.
REFERENCES
1. HABERMANN, E., AND NEUMANN, W. (1954) Hoppe-Seyler's Z. Physiol. Chem.
297, 179-193.
2.
3.
4.
5.
6.
NEUMANN, W., AND HABERMANN, E. (1957) Nature 180, 1284.
HABERMANN, E. (1958) Z. Gesamte Exp. Med. 130, 19-23.
DorzAKI, W. M., AND ZrEVE, L. (1964) J. Lab. Clin. Med. 63, 524-536.
MARINETTl, G. V. (1964) Biochim. Biophys. Acta 98, 554-565.
FLECKENSTEIN, A., AND G:ERKHARDT, H. (1952) Naunyn-Schmiedebergs Arch.
Exp. Pathol. Pharmakol. 214, 135--146.
7. RoTH, F. B., AND PrLLEMER, L. (1953) J. Immunol. 70, 533-537.
8. HABERMANN, E. (1959) Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol.
235, 513-534.
9. HARDT, K. L., Inaugural Dissertation, Giessen, 1972.
10. HABERMANN, E., AND RErz, K. G. (1965) Biochem. Z. 341, 451-466.
11. RuBSAMEN, K., BREITHAUPT, H., AND HABERMANN, E. (1971) Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. 270, 274-288.
12. DE HAAS, G. H., PoSTEMA, N. M., NIEUWENHUIZEN, W., AND VAN DEENEN,
L. L. M. (1968) Biochim. Biophys. Acta 159, 103-117.
13. CHANCE, B., AND MAEHLY, A. C. (1955) Assay of catalase and peroxidase, in
"Methods in Enzymology," Vol. II (S. P. Colowick and N. 0. Kaplan, eds.),
pp. 764-765. Academic Press, New York/London.
14. HJERTEN, S. (1971) J. Chromatogr. 61, 73-80.
15. CRANK, J. (1956) "The Mathematics of Diffusion." Oxford Univ. Press,
London/New York.
16. lsPOLATOVSKAYA, M. V. (1971) Type A Clostridium perfringens toxin, in "Microbial Toxins," vol. II A (S. Kadis et al., eds.), pp. 109-158. Academic Press,
New York/London.
17. HABERMANN, E. (1958) Z. Gesamte Exp. Med. 130, 19--23.
18. BREITHAUPT, H., AND HABERMANN, E., Naunyn-Schmiedebergs Arch. Pharmacol.
(submitted for publication).