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Studies on the digestive enzymes of the blowfly Calliphora erythrocephala. II. Kinetic constants of the larval gut proteinase
EXPERIMENTAL
Studies
PARASITOLOGY
7,
69-81 (1958)
on the Digestive Enzymes of the Blowfly CuZZiphora erythrocephala. II. Kinetic Constants of the Larval Gut Proteinase W. A. L. Evans
Department
of Zoology
and Comparative Anatomy, Great Britain
(Submitted for publication,
University
College,
Car&#
21 March 1957)
The marked proteolytic activity shown by extracts of larval blowfly alimentary tracts has been established for several species.Apart from the effect of pH on activity little information is available on the kinetic properties of these enzymes. Hence it was thought possible that the determination of such kinetic constants as the activation energy and Michaelis constant for larval Calliphora gut proteinase, besides being new data, would provide a basis for comparison for the proteinases of other animals, particularly those of invertebrates, for which information is lacking. The introduction by Charney and Tomarelli (1947) of a diazo derivative of casein as a substrate for proteinase activity has provided a method which can be readily modified for different animals so that some of the kinetic properties of the enzymes can be ascertained. Day and Powning (1949) have used azocaseinfor their studies on certain properties of cockroach proteinase with satisfactory results. METHODS
Preparation of the Enzyme Extract Mature larvae, bred in the manner described by Evans (1956) were placed in dry sawdust for 24 hr. After dissection under distilled water the guts (midgut and hindgut) were placed on filter paper to remove excess water, transferred to petri dishes and then dried in a desiccator. The dried guts were ground and stored at room temperature, 2000 larvae yielding 2.9 g dried gut powder. Proteinase was obtained from the gut powder by 69
70
EVANS
extracting 0.1 g with 10 ml distilled water at room temperature. After the addition of 1 ml toluene, shaking and subsequent dialysing against running tap water for approx. 24 hr, the supernatant clear aqueous layer in the dialysing sack was used as the source of enzyme. Dialysed extracts showed approx. 25 % greater proteinase activity than non-dialysed extracts. Measurement of Protein Hydrolysis The substrate, sulfanilamide-azocasein, was prepared according to the method of Charney and Tomarelli (1947). These workers obtained a solution of the azocasein suitable for their study of mammalian tryptic digestion by dissolving in 1% sodium carbonate solution and then adjusting to pH 8.3. Since the pH optimum of larval blowfly proteinase is somewhat lower it was convenient to dissolve the substrate in 0.1 IM buffer solution at pH 7.8 heated to approx. 70°C. In experiments designed to have reaction mixtures at different pH values, the substrate was dissolved in 0.1 N sodium hydroxide and the solution neutralised by adding 0.1 N hydrochloric acid. The reaction mixtures (subsequently abbreviated to r.m.) comprised buffer, substrate and enzyme extract in proportions suitable for particular experiments. Either borate or phosphate buffers were used since it was shown that increased concentrations of these ions in r.m. did not alter the rate of azocasein hydrolysis. Two-ml r.m. samples were added to 3-ml trichloracetic acid and the whole filtered. After adding 2 ml 0.5 N sodium hydroxide to an equal volume of filtrate the color density of the solution was measured with an absorptiometer using a 430 rnp filter. It was found that the optical density values of azocasein solutions did not maintain a linear relationship with the concentration of azocasein above an optical density value of 0.5, (see Table I). Consequently it was necessary to dilute intensely colored solutions so that they gave an optical density reading below 0.5. As in the case of most calorimetric analyses care must be taken to consider any extraneous color present in the r.m. R.M. samples were always taken at zero incubation time, and the optical density value of the treated samples subtracted from the optical density values of treated samples which were subsequently removed. This procedure takes into account any color resulting from the presence of enzyme extract, and also of any loosely adsorbed colored particles in the azocasein preparation. Extraneous color due to the latter source must also be estimated when determining the original substrate concentration in the r.m. Charney
71
PROPERTIES OF BLOWFLY PROTEINASE TABLE
I
Optical Density Values of Azocasein Solutions of Diflerent Strength Optical density Strength of azocasein solution
0.2% 0.1% 0.05% 0.025% 0.0125%
Untreated solution
T.C.A. filtrate
0.85 0.70 0.47 0.245 0.13
0.03 0.02 0.015 0.01
0.82 0.68 0.455 0.235 0.12
0.01
0.041 0.068 0.091
0.090 0.095
and Tomarelli (1947) did not consider this possible source of error, but it is clear from the figures shown in Table I that for the azocasein preparation used here a blank estimation is necessary, because the color in trichloroacetic acid filtrates of azocasein solutions has a small optical density value which increases with increased azocasein concentration. The Use of the Velocity Constant, K, as a Measure of Enzyme Activity One of the chief advantages of the azocasein method is the ease with which values of Cl and Cz can be obtained for the equation K = f log;
2
so that K, the velocity constant, can be determined. C1 is obtained by measuring the optical density of the r.m. at zero time, while CZ is calculated, after t min incubation, by subtracting the optical density value of the trichloracetic acid filtrate of the r.m. from C1 . This procedure is permissible because the optical density values of azocasein before hydrolysis and after complete hydrolysis (no precipitate with trichloracetic acid) are the same. The velocity constant K is a very convenient index of proteinase activity as long as the reaction proceeds in accordance with 1st order kinetics. This was shown to be so for blowfly proteinase for a period of at least 435 hr incubation since the plot of log CJC, against time showed the expected straight line relationship. RESULTS AND DISCUSSION
The E$ect of pH on Activity Figure 1 shows that for the experimental period of 2 hr the rate of azocasein hydrolysis is greatest at pH 7.8, while on either side of this
72
EVANS
5.0
“*
4.5
Y “, 4.0 .z F : 3.5
6
6
7
9
PH
FIQ. 1. pH activity-curve teinase.
for the hydrolysis
of azocasein by larval gut pro
pH value there was relatively slow decreasein activity. This result agrees closely with the findings of Hobson (1931) for the digestion of gelatin by gut extracts of Lu&a sericata larvae. In this species the pH optimum is approx. 7.6-7.8 and again activity falls off slowly on either side of this value. The midgut of Culliplaora larvae resembles that of other blowfly larvae insofar as it can be divided into 3 regions; 1) anterior midgut with contents at a pH 7.2-7.4, 2) mid-midgut at a pH 3.5-3.7 and 3) posterior midgut at a pH 7.6-7.8. The pH optimum of CuZZiphora larval gut proteinase can be closely correlated with the pH of the gut contents in the anterior and posterior midgut where rapid digestion of proteins must take place. The proteinase of blowfly larvae is of the alkaline type. As in Gasterophdus larvae (Roy, 1937) and Cordylobia larvae (Blacklock et al, 1930) there is no acid proteinase to correspond with the mammalian peptic enzyme. This was established for Culliphora larvae by estimating the hydrolysis of gelatin at pH 1.4 and 3.75 using the copper method of Pope and Stevens (1939) for measuring the concentration of amino acids produced. At these pH values there was negligible activity while at pH 7.65 there was marked hydrolysis. Substrate Concentration and Activity
The result shown in Fig. 2 indicates that the velocity-substrate concentration relationship for the larval proteinase is typical and that the Michaelis-Menten equation VMS v=K,+
PROPERTIES
OF
BLOWFLY
73
PROTEINASE
is applicable. From the curve the Michaelis constant, K, , is calculated to be in the region of 5.0 mg/‘7 ml r.m. K,,, cannot be expressedin terms of molar concentration of substrate because this is unknown. Since it is desirable to express K, in more standard terms the unit mg substrate/ 100 ml r.m., has been adopted, giving a value for K, , from Fig. 3, of 7.14 x 10-Z. The value of K,,, obtained from the velocity-substrate-concentration curve can only be approximate because it, is difficult to determine the value of the limiting velocity V, . However several methods have been devised so that straight line plots can be drawn after suitable transposition of the Michaelis-Menten equation. Table II summarizes different methods for obtaining K, and the precise value obtained in each case has been calculated. The curves drawn for the different, equations all show good straight line relationship except the point corresponding to the lowest substrate concentration used. (See Fig. 3.) Such marked discrepancies indicate that the analytical procedure is unsatisfactory for measuring the low rate of enzyme activity in r.m. with low substrate concentrations. ?-
“0>”
2I I IO
1 20
I 30
I 40
I 50
I 60
I 70
I 80
1 9%
S FIG. 2. The velocity-substrate concentration curve for azoeasein hydrolysis; v measured as the optical density of treated reaction mixtures and s as the concentration of azocasein in mg/7 ml reaction mixture.
74
EVANS
TABLE
Lineweaver and Burk (1934)
II
of K, for Azocasein Hydrolysis
The Determination
by Different
Transposed MichaelisMenten equation
Plot
1 1 1 K, -V = ; ’ v, + j?,
1 1 - against V
Methods
7.03 x 10-Z
S
(The double reciprocal plot) Dixon (1953)
1 1 --z-e V s
K,,,
1
1 1 - against -
v+v, M
V
6.97 X lo-*
S
and extrapolation of the line to the abscissa. At intercept -- 1 =- 1 s Ken Lineweaver and Burk (1934) Hofstee (1953) Eadie (1942, 1953)
S _ = ?V
. s + !!z
VM
_sagainst s
VM
v against v
VM = f . K,,, + v v = VM - v. S
K,
7.38 x lo-2
V
Slope = -&
7.33 x lo-2
1
The ‘Eadie-Hofstee’ equations and plot have several advantages; viz: 1) The points are more evenly scattered along the line than in the Lineweaver-Burk plots, where several points are close together near the abscissa. 2) The determination of Km being based on the slope of the line is more accurate than methods depending upon extrapolation as in all the other methods referred to. 3) The form of the ‘Eadie-Hofstee’ equation is such that the slope need not be calculated graphically, but a much more satisfactory statistical method can be used. The slope of the best straight line drawn through the points is given by the regression coefhcient which is equal to (S xy)/(S x2), where x and y are the deviations from the means for values of v/s and v respectively. The Michaelis constant K,,, besides corresponding to the substrate concentration at half maximum velocity also represents a reaction constant for the reactions involved in enzyme catalysis. The original concept of Michaelis and Menten that K,,, was a fundamental kinetic constant because its reciprocal value represented the affinity of the enzyme
PROPERTIES
OF
BLOWFLY
PROTEINASE
75
+ x10’ FIG. 3. The ‘Eadie-Hofstee’
Plot for azocasein hydrolysis.
for the substrate has been superseded. K,,, is now generally regarded as representing a dissociation constant for an intermediate compound, or series of intermediate compounds, formed between the enzyme and substrate (Nielands and Stumpf, 1955). Whatever the theoretical significance of K,,, its practical determination is important because the most suitable substrate concentrations for investigating other properties of the enzyme can be readily ascertained. It is important that a very high substrate concentration be avoided in the case of azocasein hydrolysis, since a zero order reaction might well replace the first order reaction on which the whole method depends. Further, comparison of K, values of proteinases from different animals and reacting under similar conditions provides information to establish the extent to which the enzymes are similar or different . Temperature and Activity Figure 4 shows the relationship between proteinase activity and temperature at pH 7.75 and over a reaction period of 2 hr. Under these conditions azocasein hydrolysis is at a maximum at 44°C. It would be wrong to regard this “optimum temperature” for azocasein hydrolysis by larval proteinase as a true constant because its value depends on the prior heat treatment of the enzyme. The influence of temperature on enzyme activity is governed by two factors; 1) an increase in the rate of the enzyme catalysed reaction and 2) denaturing effects on the enzyme protein. The
76
EVANS
optimum temperature represents a point at which the rate of the catalysed reaction relative to the rate of enzyme denaturation is at a maximum. Clearly the denaturing effect will depend on the temperature plus the duration to which the enzyme is exposed during and before the experiment. At 44°C the rate of azocasein hydrolysis decreasedmuch more rapidly than at 25°C. From the curves shown in Fig. 5 the activity in the final 2 hr incubation period compared with the initial 2 hr period was 11.8 % while at 25°C it was 44.2 %. The fourfold decrease in activity at 44°C compared with that at 25’C can be attributed to denaturation of the enzyme protein.
T% 4. preparations FIQ.
Temperature of dialysed
and velocity gut extract.
of azocasein
hydrolysis
using two different
PROPERTIES
OF
BLOWFLY
77
PROTEINASE
The effect of keeping the dialysed enzyme extract at different temperatures for 24 hr before measuring its activity shows that there is a marked decrease in enzyme hydrolysis after being kept at 37°C and even more so at 44°C. (See Table III.) The values of the reaction consta.nt for the enzyme extract kept at 25°C are of the same order as those obtained for reactions over a 2hr incubation period without prior storage of the extract. It appears therefore that Calliphora larval gut proteinase is stable at 25°C for 24 hr but is slowly inactivated at 37°C and 44°C. On the basis of these results it follows that 25°C is a more suitable temperature for determining the kinetic constants for Calliphora proteinase. Indeed for any study of enzymes obtained from poikilotherms a suitable temperature for measuring activity should be established. It is unwise to assume that 37°C the 1.8
1.6 1.4
1.2 0 x
01 0” H ZI c 0 0 G >
1.0
.8 .6
.4
.2
I
I
I
I
2
4
6 HV.
8
I
IO
I
I2
FIG. 5. Relative activity of larval gut proteinase at 25°C (straight line) and 44°C (broken line).
78
EVANS
TABLE III of Proteinase After Exposure to Di$erent Temperatures for 24 hr
Thermal Inactivation
Prior exposure
temperature
Reaction
temperature
K (2 hr incubation)
25°C
25°C 37°C 44°C
1.65 X lO+ 4.17 X 10-d 5.37 x 10-4
37°C
25°C 37°C 44°C
0.82 x 10-d 2.08 x 10-d 3.55 x 10-a
44°C
25°C 37°C 44°C
0.48 x 10-d 1.22 x 10-4 2.72 X lo-”
“optimum temperature” for many mammalian enzymes necessarily applies to enzymes from other animal sources. Determination
of the Activation Energy
The plot of log velocity against the reciprocal of the absolute temperature shown in Fig. 6 and obtained from the data for the broken line curve
3.6
3.4 +A&
Fro. 6. Log velocity-temperature and for a 2 hr incubation period.
3.2
3.0
Xl03
curve for larval gut proteinase at pH 7.75
PROPERTIES
OF
BLOWFLY
PROTEINASE
79
in Fig. 4 indicates that a straight line relationship exists between 5°C and 35°C. A straight line relationship is to be expected if the Vant-Hoff-Arrhenius equation (d In Ic/dT) = (E,/RT2) is applicable to the data. In its integrated form the equation becomes log k = -2&$ where k is the reaction rate, R the gas constant, T the absolute temperature and E, the activation energy. From this equation it follows that the plot of log k against l/T” Abs yields a straight line with a slope equal to -E,/2.303 R. Therefore if the slope is known the activation energy can be calculated since E, = - (slope X 4.566). The slope of the curve, shown in Fig. 6, between the limits 5” and 35°C is best calculated statistically by determining the regression coefficient. The value obtained, taking into account that the abscissa values are multiplied by 103, is -2,860, so that the activation energy is calculated to be 13,060 Cal/mole. By adopting the statistical procedure the standard error, &, , of the regression coefficient, b, can be calculated and this enables a statistical significance test to be applied to the data. The numerical value of the rates b/& is 69.4. This value is so large that it becomes readily apparent by reference to statistical tables for the probability values of t( = b/L%) that it is extremely unlikely that the original experimental points show anything other than a straight line relationship. The activation energy, E, , is a constant which, from a thermodynamical viewpoint, can be regarded as the energy required to place the reacting molecules in an active state. If E, is large the rate of reaction increases rapidly in relation to an increased temperature. The EA value is particularly useful for comparative studies since it can be determined by using relatively crude enzyme extracts (Sizer, 1943). Table IV shows that the E, value for the hydrolysis of azocasein by larval gut proteinase falls within the range of values that has been obtained for trypsin and chymotrypsin using a variety of substrates (Butler, 1941), and for the tryptic digestion of casein over the temperature range 0”4O”C, (Sizer and Josephson, 1942). It is possible that in the gut of the blowfly larva, and hence in the aqueous extracts, there may be more than one proteinase present. However it seems unlikely that more than one enzyme is involved in the hydrolysis of the azocasein substrate because smooth curve relationships
80
EVANS TABLE Activation
Energies
IV
of Proteinases EA cal/mole
Substrate
Trypsin Trypsin Trypsin Trypsin Chymotrypsin Larval blowfly
Bensoyl-1-arginine Chymotrypsinogen Sturin Casein Pepsin Aeocasein
proteinase
amide
14,900 16,300 11,800 15,000 11,200 13,060
have been obtained with several kinds of experiment. If more than one enzyme were acting on the substrate then it is clear that they must have possessedvery similar properties in relation to hydrogen ion concentration, substrate concentration and temperature in order to give a resultant activity that was so uniform. 1. Sulfanilamide-azocasein is a suitable substrate for the quantitative estimation of larval blowfly gut proteinase. 2. The pa-activity curve is similar to that of other blowfly larvae and the pH optimum of 7.8 corresponds closely to the pH of anterior midgut and posterior midgut contents. 3. The velocity-substrate concentration relationship is typical. The K, value, obtained by a statistical and several graphical methods, corresponds to a substrate concentration of 7.19 X low2 g/100 ml. 4. Culliphora larval gut proteinase is partially inactivated after 24 hr at 37°C and 44”C, but for short periods of incubation (2 hr) the maximum activity is recorded at 44°C. 5. Over the range 5°C to 35°C the temperature-activity relationship follows the Vant-Hoff-Arrhenius equation. The activation energy, E, , is 13,060 Cal/mole. REFERENCES BUTLER, J. A. V. 1941. The molecular
kinetics of trypsin action. J. Am. Chem. Sot. 63, 2971-2974. BLACKLOCK, D. B., GORDON, R. M., AND FINE, J. 1930. Metasoan immunity. A report on recent investigations. Ann. Trop. Med. Parasitol. 24, 5-54. CHARNEY, J., AND TOMARELLI, R. M. 1947. A calorimetric method for the determination of proteolytic activity of duodenal juice. J. BioZ. Ghem. 171, 501505.
PROPERTIES
OF
BLOWFLY
PROTEINASE
81
DAY, M. F., AND POWNING, R. F. 1949. A study of the process of digestion in certain insects. Australian J. Sci. Research B2, 175-215. DIXON, M. 1953. The determination of enzyme inhibitor constants. Biochem. J. 66, 170-171. EADIE, G. S. 1942. The inhibition of cholinesterase by physostigmine and prostigmine. J. Biol Chem. 146, 85-93. EADIE, G. S. 1953. On the evaluation of constants VM and K, in enzyme reactions. Science 116, 688. EVANS, W. A. L. 1956. Studies on the digestive enzymes of the blowfly Calliphora erylhrocephala. 1. The carbohydrases. Exptl. Parasitol. 6, 191-206. HOBSON, R. P. 1931. Studies on the nutrition of blowfly larvae. 1. The structure and function of the alimentary tract. J. Esptl. Biol. 8, 109-123. HOFSTEE, B. H. J. 1953. On the evaluation of the constants VM and K, in enzyme reactions. Science 116.329-331. LINEWEAVER, H., AND BURH, D. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Sot. 66, 658-666. NIELANDS, J. B., AND STUMPF, P. K. 1955. Outlines of Enzyme Chemistry: John Wiley and Sons, Inc., New Pork. POPE, C. G., AND STEVENS, M. F. 1939. The determination of amino-nitrogen using a copper method. Biochem. J. 33, 1070-1077. ROY, D. N. 1937. The physiology of digestion in larvae of Gastrophilus equi. Parasitology 29, X0-162. SIZER, I. W. 1943. Effects of temperature onenzyme kinetics. Advances in Enzymology 3, 35-62. SIZER, I. W., AND JOSEPHSON,E. S. 1942. Kinetics as a function of temperature for lipase, trypsin and invertase activity from - 70°C to 50°C. Food Research 7, 201-209.
Studies
PARASITOLOGY
7,
69-81 (1958)
on the Digestive Enzymes of the Blowfly CuZZiphora erythrocephala. II. Kinetic Constants of the Larval Gut Proteinase W. A. L. Evans
Department
of Zoology
and Comparative Anatomy, Great Britain
(Submitted for publication,
University
College,
Car&#
21 March 1957)
The marked proteolytic activity shown by extracts of larval blowfly alimentary tracts has been established for several species.Apart from the effect of pH on activity little information is available on the kinetic properties of these enzymes. Hence it was thought possible that the determination of such kinetic constants as the activation energy and Michaelis constant for larval Calliphora gut proteinase, besides being new data, would provide a basis for comparison for the proteinases of other animals, particularly those of invertebrates, for which information is lacking. The introduction by Charney and Tomarelli (1947) of a diazo derivative of casein as a substrate for proteinase activity has provided a method which can be readily modified for different animals so that some of the kinetic properties of the enzymes can be ascertained. Day and Powning (1949) have used azocaseinfor their studies on certain properties of cockroach proteinase with satisfactory results. METHODS
Preparation of the Enzyme Extract Mature larvae, bred in the manner described by Evans (1956) were placed in dry sawdust for 24 hr. After dissection under distilled water the guts (midgut and hindgut) were placed on filter paper to remove excess water, transferred to petri dishes and then dried in a desiccator. The dried guts were ground and stored at room temperature, 2000 larvae yielding 2.9 g dried gut powder. Proteinase was obtained from the gut powder by 69
70
EVANS
extracting 0.1 g with 10 ml distilled water at room temperature. After the addition of 1 ml toluene, shaking and subsequent dialysing against running tap water for approx. 24 hr, the supernatant clear aqueous layer in the dialysing sack was used as the source of enzyme. Dialysed extracts showed approx. 25 % greater proteinase activity than non-dialysed extracts. Measurement of Protein Hydrolysis The substrate, sulfanilamide-azocasein, was prepared according to the method of Charney and Tomarelli (1947). These workers obtained a solution of the azocasein suitable for their study of mammalian tryptic digestion by dissolving in 1% sodium carbonate solution and then adjusting to pH 8.3. Since the pH optimum of larval blowfly proteinase is somewhat lower it was convenient to dissolve the substrate in 0.1 IM buffer solution at pH 7.8 heated to approx. 70°C. In experiments designed to have reaction mixtures at different pH values, the substrate was dissolved in 0.1 N sodium hydroxide and the solution neutralised by adding 0.1 N hydrochloric acid. The reaction mixtures (subsequently abbreviated to r.m.) comprised buffer, substrate and enzyme extract in proportions suitable for particular experiments. Either borate or phosphate buffers were used since it was shown that increased concentrations of these ions in r.m. did not alter the rate of azocasein hydrolysis. Two-ml r.m. samples were added to 3-ml trichloracetic acid and the whole filtered. After adding 2 ml 0.5 N sodium hydroxide to an equal volume of filtrate the color density of the solution was measured with an absorptiometer using a 430 rnp filter. It was found that the optical density values of azocasein solutions did not maintain a linear relationship with the concentration of azocasein above an optical density value of 0.5, (see Table I). Consequently it was necessary to dilute intensely colored solutions so that they gave an optical density reading below 0.5. As in the case of most calorimetric analyses care must be taken to consider any extraneous color present in the r.m. R.M. samples were always taken at zero incubation time, and the optical density value of the treated samples subtracted from the optical density values of treated samples which were subsequently removed. This procedure takes into account any color resulting from the presence of enzyme extract, and also of any loosely adsorbed colored particles in the azocasein preparation. Extraneous color due to the latter source must also be estimated when determining the original substrate concentration in the r.m. Charney
71
PROPERTIES OF BLOWFLY PROTEINASE TABLE
I
Optical Density Values of Azocasein Solutions of Diflerent Strength Optical density Strength of azocasein solution
0.2% 0.1% 0.05% 0.025% 0.0125%
Untreated solution
T.C.A. filtrate
0.85 0.70 0.47 0.245 0.13
0.03 0.02 0.015 0.01
0.82 0.68 0.455 0.235 0.12
0.01
0.041 0.068 0.091
0.090 0.095
and Tomarelli (1947) did not consider this possible source of error, but it is clear from the figures shown in Table I that for the azocasein preparation used here a blank estimation is necessary, because the color in trichloroacetic acid filtrates of azocasein solutions has a small optical density value which increases with increased azocasein concentration. The Use of the Velocity Constant, K, as a Measure of Enzyme Activity One of the chief advantages of the azocasein method is the ease with which values of Cl and Cz can be obtained for the equation K = f log;
2
so that K, the velocity constant, can be determined. C1 is obtained by measuring the optical density of the r.m. at zero time, while CZ is calculated, after t min incubation, by subtracting the optical density value of the trichloracetic acid filtrate of the r.m. from C1 . This procedure is permissible because the optical density values of azocasein before hydrolysis and after complete hydrolysis (no precipitate with trichloracetic acid) are the same. The velocity constant K is a very convenient index of proteinase activity as long as the reaction proceeds in accordance with 1st order kinetics. This was shown to be so for blowfly proteinase for a period of at least 435 hr incubation since the plot of log CJC, against time showed the expected straight line relationship. RESULTS AND DISCUSSION
The E$ect of pH on Activity Figure 1 shows that for the experimental period of 2 hr the rate of azocasein hydrolysis is greatest at pH 7.8, while on either side of this
72
EVANS
5.0
“*
4.5
Y “, 4.0 .z F : 3.5
6
6
7
9
PH
FIQ. 1. pH activity-curve teinase.
for the hydrolysis
of azocasein by larval gut pro
pH value there was relatively slow decreasein activity. This result agrees closely with the findings of Hobson (1931) for the digestion of gelatin by gut extracts of Lu&a sericata larvae. In this species the pH optimum is approx. 7.6-7.8 and again activity falls off slowly on either side of this value. The midgut of Culliplaora larvae resembles that of other blowfly larvae insofar as it can be divided into 3 regions; 1) anterior midgut with contents at a pH 7.2-7.4, 2) mid-midgut at a pH 3.5-3.7 and 3) posterior midgut at a pH 7.6-7.8. The pH optimum of CuZZiphora larval gut proteinase can be closely correlated with the pH of the gut contents in the anterior and posterior midgut where rapid digestion of proteins must take place. The proteinase of blowfly larvae is of the alkaline type. As in Gasterophdus larvae (Roy, 1937) and Cordylobia larvae (Blacklock et al, 1930) there is no acid proteinase to correspond with the mammalian peptic enzyme. This was established for Culliphora larvae by estimating the hydrolysis of gelatin at pH 1.4 and 3.75 using the copper method of Pope and Stevens (1939) for measuring the concentration of amino acids produced. At these pH values there was negligible activity while at pH 7.65 there was marked hydrolysis. Substrate Concentration and Activity
The result shown in Fig. 2 indicates that the velocity-substrate concentration relationship for the larval proteinase is typical and that the Michaelis-Menten equation VMS v=K,+
PROPERTIES
OF
BLOWFLY
73
PROTEINASE
is applicable. From the curve the Michaelis constant, K, , is calculated to be in the region of 5.0 mg/‘7 ml r.m. K,,, cannot be expressedin terms of molar concentration of substrate because this is unknown. Since it is desirable to express K, in more standard terms the unit mg substrate/ 100 ml r.m., has been adopted, giving a value for K, , from Fig. 3, of 7.14 x 10-Z. The value of K,,, obtained from the velocity-substrate-concentration curve can only be approximate because it, is difficult to determine the value of the limiting velocity V, . However several methods have been devised so that straight line plots can be drawn after suitable transposition of the Michaelis-Menten equation. Table II summarizes different methods for obtaining K, and the precise value obtained in each case has been calculated. The curves drawn for the different, equations all show good straight line relationship except the point corresponding to the lowest substrate concentration used. (See Fig. 3.) Such marked discrepancies indicate that the analytical procedure is unsatisfactory for measuring the low rate of enzyme activity in r.m. with low substrate concentrations. ?-
“0>”
2I I IO
1 20
I 30
I 40
I 50
I 60
I 70
I 80
1 9%
S FIG. 2. The velocity-substrate concentration curve for azoeasein hydrolysis; v measured as the optical density of treated reaction mixtures and s as the concentration of azocasein in mg/7 ml reaction mixture.
74
EVANS
TABLE
Lineweaver and Burk (1934)
II
of K, for Azocasein Hydrolysis
The Determination
by Different
Transposed MichaelisMenten equation
Plot
1 1 1 K, -V = ; ’ v, + j?,
1 1 - against V
Methods
7.03 x 10-Z
S
(The double reciprocal plot) Dixon (1953)
1 1 --z-e V s
K,,,
1
1 1 - against -
v+v, M
V
6.97 X lo-*
S
and extrapolation of the line to the abscissa. At intercept -- 1 =- 1 s Ken Lineweaver and Burk (1934) Hofstee (1953) Eadie (1942, 1953)
S _ = ?V
. s + !!z
VM
_sagainst s
VM
v against v
VM = f . K,,, + v v = VM - v. S
K,
7.38 x lo-2
V
Slope = -&
7.33 x lo-2
1
The ‘Eadie-Hofstee’ equations and plot have several advantages; viz: 1) The points are more evenly scattered along the line than in the Lineweaver-Burk plots, where several points are close together near the abscissa. 2) The determination of Km being based on the slope of the line is more accurate than methods depending upon extrapolation as in all the other methods referred to. 3) The form of the ‘Eadie-Hofstee’ equation is such that the slope need not be calculated graphically, but a much more satisfactory statistical method can be used. The slope of the best straight line drawn through the points is given by the regression coefhcient which is equal to (S xy)/(S x2), where x and y are the deviations from the means for values of v/s and v respectively. The Michaelis constant K,,, besides corresponding to the substrate concentration at half maximum velocity also represents a reaction constant for the reactions involved in enzyme catalysis. The original concept of Michaelis and Menten that K,,, was a fundamental kinetic constant because its reciprocal value represented the affinity of the enzyme
PROPERTIES
OF
BLOWFLY
PROTEINASE
75
+ x10’ FIG. 3. The ‘Eadie-Hofstee’
Plot for azocasein hydrolysis.
for the substrate has been superseded. K,,, is now generally regarded as representing a dissociation constant for an intermediate compound, or series of intermediate compounds, formed between the enzyme and substrate (Nielands and Stumpf, 1955). Whatever the theoretical significance of K,,, its practical determination is important because the most suitable substrate concentrations for investigating other properties of the enzyme can be readily ascertained. It is important that a very high substrate concentration be avoided in the case of azocasein hydrolysis, since a zero order reaction might well replace the first order reaction on which the whole method depends. Further, comparison of K, values of proteinases from different animals and reacting under similar conditions provides information to establish the extent to which the enzymes are similar or different . Temperature and Activity Figure 4 shows the relationship between proteinase activity and temperature at pH 7.75 and over a reaction period of 2 hr. Under these conditions azocasein hydrolysis is at a maximum at 44°C. It would be wrong to regard this “optimum temperature” for azocasein hydrolysis by larval proteinase as a true constant because its value depends on the prior heat treatment of the enzyme. The influence of temperature on enzyme activity is governed by two factors; 1) an increase in the rate of the enzyme catalysed reaction and 2) denaturing effects on the enzyme protein. The
76
EVANS
optimum temperature represents a point at which the rate of the catalysed reaction relative to the rate of enzyme denaturation is at a maximum. Clearly the denaturing effect will depend on the temperature plus the duration to which the enzyme is exposed during and before the experiment. At 44°C the rate of azocasein hydrolysis decreasedmuch more rapidly than at 25°C. From the curves shown in Fig. 5 the activity in the final 2 hr incubation period compared with the initial 2 hr period was 11.8 % while at 25°C it was 44.2 %. The fourfold decrease in activity at 44°C compared with that at 25’C can be attributed to denaturation of the enzyme protein.
T% 4. preparations FIQ.
Temperature of dialysed
and velocity gut extract.
of azocasein
hydrolysis
using two different
PROPERTIES
OF
BLOWFLY
77
PROTEINASE
The effect of keeping the dialysed enzyme extract at different temperatures for 24 hr before measuring its activity shows that there is a marked decrease in enzyme hydrolysis after being kept at 37°C and even more so at 44°C. (See Table III.) The values of the reaction consta.nt for the enzyme extract kept at 25°C are of the same order as those obtained for reactions over a 2hr incubation period without prior storage of the extract. It appears therefore that Calliphora larval gut proteinase is stable at 25°C for 24 hr but is slowly inactivated at 37°C and 44°C. On the basis of these results it follows that 25°C is a more suitable temperature for determining the kinetic constants for Calliphora proteinase. Indeed for any study of enzymes obtained from poikilotherms a suitable temperature for measuring activity should be established. It is unwise to assume that 37°C the 1.8
1.6 1.4
1.2 0 x
01 0” H ZI c 0 0 G >
1.0
.8 .6
.4
.2
I
I
I
I
2
4
6 HV.
8
I
IO
I
I2
FIG. 5. Relative activity of larval gut proteinase at 25°C (straight line) and 44°C (broken line).
78
EVANS
TABLE III of Proteinase After Exposure to Di$erent Temperatures for 24 hr
Thermal Inactivation
Prior exposure
temperature
Reaction
temperature
K (2 hr incubation)
25°C
25°C 37°C 44°C
1.65 X lO+ 4.17 X 10-d 5.37 x 10-4
37°C
25°C 37°C 44°C
0.82 x 10-d 2.08 x 10-d 3.55 x 10-a
44°C
25°C 37°C 44°C
0.48 x 10-d 1.22 x 10-4 2.72 X lo-”
“optimum temperature” for many mammalian enzymes necessarily applies to enzymes from other animal sources. Determination
of the Activation Energy
The plot of log velocity against the reciprocal of the absolute temperature shown in Fig. 6 and obtained from the data for the broken line curve
3.6
3.4 +A&
Fro. 6. Log velocity-temperature and for a 2 hr incubation period.
3.2
3.0
Xl03
curve for larval gut proteinase at pH 7.75
PROPERTIES
OF
BLOWFLY
PROTEINASE
79
in Fig. 4 indicates that a straight line relationship exists between 5°C and 35°C. A straight line relationship is to be expected if the Vant-Hoff-Arrhenius equation (d In Ic/dT) = (E,/RT2) is applicable to the data. In its integrated form the equation becomes log k = -2&$ where k is the reaction rate, R the gas constant, T the absolute temperature and E, the activation energy. From this equation it follows that the plot of log k against l/T” Abs yields a straight line with a slope equal to -E,/2.303 R. Therefore if the slope is known the activation energy can be calculated since E, = - (slope X 4.566). The slope of the curve, shown in Fig. 6, between the limits 5” and 35°C is best calculated statistically by determining the regression coefficient. The value obtained, taking into account that the abscissa values are multiplied by 103, is -2,860, so that the activation energy is calculated to be 13,060 Cal/mole. By adopting the statistical procedure the standard error, &, , of the regression coefficient, b, can be calculated and this enables a statistical significance test to be applied to the data. The numerical value of the rates b/& is 69.4. This value is so large that it becomes readily apparent by reference to statistical tables for the probability values of t( = b/L%) that it is extremely unlikely that the original experimental points show anything other than a straight line relationship. The activation energy, E, , is a constant which, from a thermodynamical viewpoint, can be regarded as the energy required to place the reacting molecules in an active state. If E, is large the rate of reaction increases rapidly in relation to an increased temperature. The EA value is particularly useful for comparative studies since it can be determined by using relatively crude enzyme extracts (Sizer, 1943). Table IV shows that the E, value for the hydrolysis of azocasein by larval gut proteinase falls within the range of values that has been obtained for trypsin and chymotrypsin using a variety of substrates (Butler, 1941), and for the tryptic digestion of casein over the temperature range 0”4O”C, (Sizer and Josephson, 1942). It is possible that in the gut of the blowfly larva, and hence in the aqueous extracts, there may be more than one proteinase present. However it seems unlikely that more than one enzyme is involved in the hydrolysis of the azocasein substrate because smooth curve relationships
80
EVANS TABLE Activation
Energies
IV
of Proteinases EA cal/mole
Substrate
Trypsin Trypsin Trypsin Trypsin Chymotrypsin Larval blowfly
Bensoyl-1-arginine Chymotrypsinogen Sturin Casein Pepsin Aeocasein
proteinase
amide
14,900 16,300 11,800 15,000 11,200 13,060
have been obtained with several kinds of experiment. If more than one enzyme were acting on the substrate then it is clear that they must have possessedvery similar properties in relation to hydrogen ion concentration, substrate concentration and temperature in order to give a resultant activity that was so uniform. 1. Sulfanilamide-azocasein is a suitable substrate for the quantitative estimation of larval blowfly gut proteinase. 2. The pa-activity curve is similar to that of other blowfly larvae and the pH optimum of 7.8 corresponds closely to the pH of anterior midgut and posterior midgut contents. 3. The velocity-substrate concentration relationship is typical. The K, value, obtained by a statistical and several graphical methods, corresponds to a substrate concentration of 7.19 X low2 g/100 ml. 4. Culliphora larval gut proteinase is partially inactivated after 24 hr at 37°C and 44”C, but for short periods of incubation (2 hr) the maximum activity is recorded at 44°C. 5. Over the range 5°C to 35°C the temperature-activity relationship follows the Vant-Hoff-Arrhenius equation. The activation energy, E, , is 13,060 Cal/mole. REFERENCES BUTLER, J. A. V. 1941. The molecular
kinetics of trypsin action. J. Am. Chem. Sot. 63, 2971-2974. BLACKLOCK, D. B., GORDON, R. M., AND FINE, J. 1930. Metasoan immunity. A report on recent investigations. Ann. Trop. Med. Parasitol. 24, 5-54. CHARNEY, J., AND TOMARELLI, R. M. 1947. A calorimetric method for the determination of proteolytic activity of duodenal juice. J. BioZ. Ghem. 171, 501505.
PROPERTIES
OF
BLOWFLY
PROTEINASE
81
DAY, M. F., AND POWNING, R. F. 1949. A study of the process of digestion in certain insects. Australian J. Sci. Research B2, 175-215. DIXON, M. 1953. The determination of enzyme inhibitor constants. Biochem. J. 66, 170-171. EADIE, G. S. 1942. The inhibition of cholinesterase by physostigmine and prostigmine. J. Biol Chem. 146, 85-93. EADIE, G. S. 1953. On the evaluation of constants VM and K, in enzyme reactions. Science 116, 688. EVANS, W. A. L. 1956. Studies on the digestive enzymes of the blowfly Calliphora erylhrocephala. 1. The carbohydrases. Exptl. Parasitol. 6, 191-206. HOBSON, R. P. 1931. Studies on the nutrition of blowfly larvae. 1. The structure and function of the alimentary tract. J. Esptl. Biol. 8, 109-123. HOFSTEE, B. H. J. 1953. On the evaluation of the constants VM and K, in enzyme reactions. Science 116.329-331. LINEWEAVER, H., AND BURH, D. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Sot. 66, 658-666. NIELANDS, J. B., AND STUMPF, P. K. 1955. Outlines of Enzyme Chemistry: John Wiley and Sons, Inc., New Pork. POPE, C. G., AND STEVENS, M. F. 1939. The determination of amino-nitrogen using a copper method. Biochem. J. 33, 1070-1077. ROY, D. N. 1937. The physiology of digestion in larvae of Gastrophilus equi. Parasitology 29, X0-162. SIZER, I. W. 1943. Effects of temperature onenzyme kinetics. Advances in Enzymology 3, 35-62. SIZER, I. W., AND JOSEPHSON,E. S. 1942. Kinetics as a function of temperature for lipase, trypsin and invertase activity from - 70°C to 50°C. Food Research 7, 201-209.