Casein Interference in Bovine Plasmin Assays Using a Synthetic Substrate' ERIC
D. BASTIAN, RODNEY J. BROWN, and C. ANTHON ERNSTROM Department of Nutrition and Food Sciences Utah State University Logan 84322-87m
ABSTRACT
Bovine plasmin (EC 3.4.21.7) activity on H-D-valyl-L-leucyl-L-lysyl4nitroanilide was measured by determining the change in absorbance at 405 nm. Initial rates of reactions were estimated at all combinations of the following substrate concentrations [.4, 4, and 40 times the substrate concentration at one-half maximum velocity (Vmax) (Km)]and casein concentrations [.068, .68, and 6.8 times the inhibitor constant for competitive inhibition (&)]. By nonlinear least squares fitting of the data to an equation that described reversible enzyme kinetics, steady state kinetic parameters, maximum velocity (V-), substrate concentration at one-half maximum velocity (V-) (Km),inhibitor constant for competitive inhibition (KI),and inhibitor constant for uncompetitive inhibition (K;) were estimated. casein fit the equation as a competitive inhibitor of bovine plasmin. This enzyme has a catalytic constant (Kcat) of .0158 change in absorbance at 405 n d m i n per nM, substrate concentration at one-half maximum velocity ) ,V( (Kd of .lo7 mM substrate, and inhibitor constant for competitive inhibition (KJ) of .86 mg/ml of casein. Bovine plasmin activity can be measured directly in bovine milk without interference from casein. (Key words: plasmin, inhibition, casein, enzyme kinetics)
tion, K = time at one-half Amax,Kcat = catalytic constant, K I = inhibitor constant for com, petitive inhibition, K , = inhibitor constant for uncompetitive inhibition, K , = substrate concentration at one-half maximum velocity, MW = molecular weight, S = substrate concentration, VLLN = H-D-valyl-L-leucyl-L-lysyl4nitroanilide. V, = maximum velocity. INTRODUCTION
Plasminogen [molecular weight (MW) 88,0921 is a blood enzyme that enters the mammary gland as plasminogen and causes hydrolysis of &- and pcaseins in milk (4, 14). Its concentration and activity are increased in both mastitic milk (13) and late lactation milk (7, 12, 13). Plasmin activity has been measured in dairy products using synthetic chromogenic (5, 10, 13, 15) or fluorogenic (7, 9, 12) substrates. Lysine peptides linked to chromogenic or fluorogenic compounds are useful substrates because plasmin has high affiiity for lysine residues and cleaves Lys-X bonds (2). N-
Succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-
amido-4-methyl coumarin does not fluoresce, but plasmin hydrolysis of this compound releases 7-amido-4-methyl coumarin, a fluorescent compound. Rate of fluorescence increase is proportional to plasmin activity. Plasmin hydrolysis of the chromogenic substrate H-Dvalyl-L-leucyl-L-lysyl4Ntroanilide (VLLN) releases 4-nitroaniline, which absorbs light at 405 nm. Both methods are rapid, sensitive, specific, and require little sample preparation. However, the latter method may be more conAbbreviation key: A = absorbance, A, = venient because a spectrophotometer can be maximum absorbance, I = inhibitor concentra- used. Using a fluorogenic substrate, Richardson (7) found that plasmin concentrations in early and late lactation milk were .15 and .37 mg/L Received December 10, 1990. in Jersey milk and .27 and .53 mg/L in HolAccepted May 31, 1991. stein milk, suggesting that plasmin activity is 'Conhibution Number 4094 of the Utah Agricultural dependent on breed of cow and stage of lactaExperiment Station. Approved by the director. 1991 J Dairy Sci 74:4115L4124
41 19
4120
BASTIAN ET AL.
tion. Schaar (12) measured plasmin activity in milk from four breeds of dairy COWS and reported .112 mits of 7-amid04methyl coumarjn/ml of Holstein milk and .084 units/ ml of Jersey milk. When plasmin activity was adjusted for casein content, no difference was seen between Holstein and Jersey milk. Schaar (12; p. 375) concluded that “The negative correlation between casein content and plasmin activity. . . is probably due to competition between casein and the synthetic substrate for the enzyme.” Because casein is a natural substrate for plasmin in milk, it competes for the active site of plasmin during analysis with synthetic substrates. This type of interference can be considered reversible competitive inhibition because casein is bound and then released by plasmin. Casein has numerous sites that bind plasmin, many of which likely would not be cleaved during a 30-min assay. Pearse et al. (6) found that digestion of casein by plasmin at optimal pH and temperature takes several hours despite addition of extra plasmin to milk. Also, sites on plasmin that bind uncleaved casein may exist and act in a true inhibitory fashion. In a similar experiment, Wiman et al. (17) measured plasmin activity with VLLN substrate in presence of fibrinogen. They found that some fibrinogen sites are not cleaved by plasmin and that fibrinogen competitively inhibits the action of plasmin toward the substrate. Several kinds of reversible enzyme inhibition are described by Quation [l]:
VmaX
I + -
I S
l+A KI
Competitive inhibition increases the apparent value of K, by increasing KI;K;increases to a value much larger than I, making
4 insignifiKI
cant. So, for competitive inhibition,
In mixed inhibition, Km is increased by a factor of I 1-tKI I
’
1 + 7
Kl
and,V
is decreased by a factor of 1 I
1+K;
as in Equation [Z]. Uncompetitive inhibition decreases the apparent values of both Km and V,, by a factor of 1
I 1+-
K;
where v = initial velocity,, V = maximum velocity, K, = the substrate concentration at one-half V-, KI = inhibitor constant for competitive inhibition, S = substrate concen, tration, I = inhibitor concentration, and KI = inhibitor constant for uncompetitive inhibition. Different kinds of inhibition can be characterized by their effects on Equation [l] (1, 17). This is more easily seen if Equation [l] is rearranged to Quation [2]: Journal of Dairy Science Vol. 74, No. 12, 1991
but keeps their ratio constant; KIincreases to a I value much larger than I, making - insignifiKI
cant. So, for uncompetitive inhibition, Vmax
I
S
1+?
K,
[41
4121
PLASMIN INHIBITION
A rarely encountered fourth type of inhibition, noncompetitive, decreases the apparent value of,,V but has no effect on K,. This is only possible if KI= KI. Because the term noncompetitive is commonly used to include mixed inhibition, noncompetitive inhibition is often referred to as pure noncompetitive inhibition (3). Only competitive or pure noncompetitive inhibition can be overcome by increasing the substrate concentration relative to inhibitor concentration. Functionally, pure noncompetitive inhibition is only possible when the inhibitor molecule is very small, and it is not likely even then (3). The objective of this research was to determine whether casein inhibits the reaction of plasmin with a synthetic substrate and, if so, to determine the type of inhibition. If the effect of casein on plasmin assays using a synthetic substrate follows competitive inhibition kinetics, increasing substrate concentration or decreasing casein concentration would remove the inhibition. MATERIALS AND METHODS
min. Casein concentration of the supernatant was estimated by Kjeldahl nitrogen determination using a Kjeldahl factor of 6.36 (16). Using linear transformations of initial rate data (3) collected from reaction mixtures that represented several combinations of substrate and casein, preliminary estimates of Km and KI (competitive inhibition constant) were obtained. Three levels of substrate (.l, 1, and 10 x K& were combined with three levels of casein (.l, 1, and 10 x Ki, to prepare nine different reaction mixtures. Plasmin solution (150 pl) was added to each reaction mixture at time = 0 to give a final enzyme concentration of 26.8 nM (calculated using M W 88,092 and total volume 850 pl). Reported plasmin concentrations in skim milk vary from .14 to .73 mg/L (1.6 to 8.2 nM) (7, 9). Final substrate concentrations were .04,.40,and 4.00 mM, and final casein concentrations were .0585, 3 5 , and 5.85 mg/ml. Release of 4-nitroaniline was followed by measuring the increase in absorbance at 405 nm for 30 min using a Beckman DU-8B spectrophotometer (Beckman Instrument, Palo Alto, CA) equipped with a kinetics compuset module. The experiment was replicated.
Reagents The VLLN (catalog number V 7127), plasmin purified from bovine blood (.25 Sigma units/mg, catalog number P 7911), and Eamino-ncaproic acid (catalog number A 2504) were purchased from Sigma Chemical Company (St. Louis, MO). Purified casein was purchased Erom Fisher Scientific (Pittsburgh, PA). Kinetic Assays The assay buffer contained 50 mM Tris, 110 mM NaCl, and 3 mM e-amino-ncaproic acid and was adjusted to pH 7.4 with HCl(l3). Although E-amino-n-caproic acid is an inhibitor of plasmin, its KI is so large that it does not interfere with assays at this concentration. The E-amino-n-caproic acid is included in standard plasmin assay buffers to enhance activation of plasminogen. Enzyme solution contained 6.3 mg of plasmid25 ml of assay buffer. Also, VLLN was dissolved (5 mg/ml) in the buffer. Casein solution was prepared by dissolving 4 g in 100 ml of buffer, mixing 3 ml of this solution with 1 ml of .4 M sodium citrate, and centrifuging at 27,000 x g for 10
initial Rate Calculation Assuming that the early part of the absorbance-time curve approximates a rectangular hyperbola, initial reaction rates, v, for each mixture were determined by fitting absorbance-time data to Equation [5] using the NLIN procedure of SAS@(Marquardt method) (11).
A =
Amax x t K + t
[51
where A = absorbance, A ,, = the maximum absorbance approached by the curve, t = time, and K = time at one-half Amax.Least squares estimates of Aand K were obtained and used to determine initial rates from the derivative of m a t i o n [5]:
Determination of Kinetic Constants Kinetic constants were estimated by fitting experimental data to Equation [l]. Four kinetic Journal of Dairy Science Vol. 74. No. 12, 1991
4122
BASTIAN ET AL.
TABLE 1. Inhibition patterns as a function of inhibition constanlS (I).
Type of inhibition' None
I KI
I K;
0
0 0
>o
Competitive Uncompetitive Mixed
0
>o
>o
>o
TABLE 2. Estimates of kinetic constants for casein inhibition of plasmin.
constant'
Value'
SE
Kud Km
.0158 A.4 Mper m h
.01
.lo7 mM ,860 mghnl
.02
K! Kl I KI
'KI = Inhibitor constant for competitive inhibition; gI = inhibitor constant for uncompetitive inhibition.
.05
109 mg/ml
.68
I
10-10
I 'I and were calculated with I = .585 mghnl. KW = KI K,
inhibition was then-deduced from Tal% 1.
*AA =
Change in absorbance.
RESULTS AND DISCUSSION
A typical reaction curve for bovine plasmin activity toward VLLN in presence of casein is approach reduced the error caused by omitting shown in Figure 1. A line that shows the data from later parts of the reaction curves and statistically determined initial rate using Equa- estimated the slope at t = 0 using Equation [6] rather than at t = some short time. tion [5] has been added to the graph. This Richardson and Elston (8) reported plasmin method is more accurate than simply taking activity in commercially prepared caseins. The the slope of the line between the first two casein solution used in our experiment was points measured as the initial rate or subjec- assayed for residual plasmin activity. Absorbtively deciding the initial rate. This statistical ance increased .0027units over 30 min for the highest casein concentration used in this study. This increase was negligible compared with the increase of .3 absorbance units observed for the slowest reaction mixtures and, thus, did not influence reaction rates. Brown (1) proposed that enzyme inhibition patterns are functions of inhibition constants (Table 1) and that type of enzyme inhibition can be determined from estimates of KIand K;. If these parameters become large, the terms in which they are found in Equation [2] approach zero. If both KI and K , are large, there is no inhibition, and Equation [2]becomes the simple Michaelis-Menton equation for enzyme kinetics: 0
10
20
30
Time (min) Figure 1. Increase in absorbance during 30 min reaction of plasmin with .4 mM H-D-valyl-Lleucyl-Llysyl4-nitroanilide and ,585 ml~mlof casein. Initid rate or tangent line at zero time was determined using SAS estimates of maximum absorbance approached by the curve and the time at half the maximum absorbance. JoUmat of Dairy Science Vol. 74, No. 12, 1991
v = - v,
s
Km+S'
r71
Estimates of the kinetic parameters for this expriment are shown in Table 2. Because VKI was .68 mdml and l/Ki was mg/ml,
4123
PLASMIN INHIBITION
move interference of an inhibitor may vary. Because casein inhibition can be removed, plasmin activity in milk can be measured directly without interference from casein. ACKNOWLEDGMENTS
We thank The Jersey Cattle Club and the
Utah Agricultural Experiment Station for the funds provided to do this work. Figure 2. Effect of substrate and casein concentrations on velocity of plasmin reactions. Rates were calculated using estimates of substrate concentration at one-halfmaximum velocity (K,Jand inhibitor constant for competitive inhibition (KI) determined in this study using Equation [3]. [Substrate] from 0 to .04 and [casein] from 0 to .0585 arc extrapolated. AA = Change in absorbance.
Equation [2] was reduced to Equation [3]. Equation [3] describes competitive inhibition, so it was concluded that casein was competitively inhibiting the activity of bovine plasmin toward VLLN. Experimental data were fitted to Equation [3]. Estimates of the remaining kinetic parameters were not different within the precision of the experiments than those found by fitting the data to EQuation [2], although the residual sum of squares was lower. Reaction rates calculated using the estimates of Km and KI listed in Table 2 are plotted in Figure 2. &uation 131 was used for calculating reaction rates achieved when Concentrations of substrate and casein were varied. If casein concentration was increased while substrate concentration was constant, reaction rate decreased. By increasing the substrate concentration at a fixed casein concentration, inhibition was removed. CONCLUSIONS
Casein competitively inhibits plasmin in activity assays that use the synthetic chromogenic substrate VLLN. Adding sufficient substrate or decreasing inhibitory casein concentration negates inhibitor-enzyme binding and removes interference. Substrate concentration of .4 mM removed inhibition associated with 5 mg/ml of casein. Because inhibitor content in enzyme assays depends on sample preparation, amount of substrate needed to re-
REFERENCES
lBrown, R. J. 1980. Application of non-Iinear least squares fitting to analysis of inhibition in bipbasic kinetic systems. J. Them. Biol. 83527. 2 Collen, D., and H. R. Lijnen. 1985. The fibrinolytic system in man: an overview. Page 1 in Thrombolysis: biological and therapeutic properties of new thrombolytic agents. D. Collen, H. R. Lijnen, and M. Verstraete, ed. Churchill Livingstone, Edinburgh, Scotland. 3 Cornish-Bowden, A. 1979. Page 73 in Fundamentals of enzyme. kinetics. Butterworth and Co. Ltd., London, Engl. 4Eige1, W. N., C. J. Hofman, B.A.K. Chibber, J. M. Tomich, T. W. Keenan, and E. T. Mertz. 1979. Plasmin mediated proteolysis of casein in bovine milk. Roc. Natl. A d . Sci. USA 76:2244. 5Korycka-Dahl. M,B. R. Dumas, N. Chene, and J. Martal. 1983. Plasmin activity in milk. J. Dairy Sci. 6fk704. 6Pearse, M.J., P. M.Linklater, R. J. Hall,and A. G. Mackinlay. 1986. Extensive degradation of casein by plasmin does not impede subsequent curd formation and syneresis. J. Dairy Res. 53:477. 7 Riclmdson, B. C. 1983. Variation of the concentration of plasmin and plasminogen in bovine milk with lactation. N.Z. J. Dairy Sci. Technol. 18247. 8Richar&on, B. C., and P. D. Elston. 1984. Plasmin activity in commercial caseins and caseinates. N.Z. J. Dairy Sci. Technol. 1963. 9Richardson, B. C., and K. N. Pearce. 1981. The determination of plasmin m dauy products. NZ. J. Dairy Sci. Technol. 16209. IORollema, H. S., S. Visser, and J. K. Poll. 1983. Spectrophotometricassay of plasmin and plasminogen m bovine milk. Milchwissenschaft 38:214. 11 SAS@ User’s Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Caty, NC. 12 Schaar, J. 1985. Plasmin activity and proteosepeptone content of individual milks. J. Dairy Res. 52369. 13 Schaar, J., and H.m e . 1986. Effect of subclinical mastitis on milk plasminogen and plasmin compaed with that on sodium, antitrypsin and N-acetyl-PDglucosaminidase. J. Dairy Res. 53:515. 14 Schailer, J., P. W. Mow, G.A.K. Da~egger-Muller. J. Rosselet, U. Kampfer, and E. E. Rickli. 1985. Complete amino acid sequence of bovine plasminogen. Eur. J. Biochem. 149267.
Journal of Dairy Science Vol. 74, No. 12, 1991
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15 Snoereo. T.H.M., and J.A.M. van Riel. 1979. Milk proteinase, its isolation and action on ~ 2 and casein. Milchwissenschaft 34528. 16 van Boekel, M.A.J.S.,and B. Ribadeau-Dumas. 1987. Addendum to the evaluation of the Kjeldahl factor for conversion of the oitrogen content of milk and milk
Journal of Dairy Science Vol. 74, No. 12, 1991
products to protein content. Neth. Milk Dairy J. 41: 281. 17 Wiman, B., H. R. Lijnan, and D. Collen. 1979. On &e specific interaction between the lysinebjnding sites in plasmin and complementary sites in Cfrpntiplasmin and in fibrinogen. Biochim. Biophys. Acta 579:142.
D. BASTIAN, RODNEY J. BROWN, and C. ANTHON ERNSTROM Department of Nutrition and Food Sciences Utah State University Logan 84322-87m
ABSTRACT
Bovine plasmin (EC 3.4.21.7) activity on H-D-valyl-L-leucyl-L-lysyl4nitroanilide was measured by determining the change in absorbance at 405 nm. Initial rates of reactions were estimated at all combinations of the following substrate concentrations [.4, 4, and 40 times the substrate concentration at one-half maximum velocity (Vmax) (Km)]and casein concentrations [.068, .68, and 6.8 times the inhibitor constant for competitive inhibition (&)]. By nonlinear least squares fitting of the data to an equation that described reversible enzyme kinetics, steady state kinetic parameters, maximum velocity (V-), substrate concentration at one-half maximum velocity (V-) (Km),inhibitor constant for competitive inhibition (KI),and inhibitor constant for uncompetitive inhibition (K;) were estimated. casein fit the equation as a competitive inhibitor of bovine plasmin. This enzyme has a catalytic constant (Kcat) of .0158 change in absorbance at 405 n d m i n per nM, substrate concentration at one-half maximum velocity ) ,V( (Kd of .lo7 mM substrate, and inhibitor constant for competitive inhibition (KJ) of .86 mg/ml of casein. Bovine plasmin activity can be measured directly in bovine milk without interference from casein. (Key words: plasmin, inhibition, casein, enzyme kinetics)
tion, K = time at one-half Amax,Kcat = catalytic constant, K I = inhibitor constant for com, petitive inhibition, K , = inhibitor constant for uncompetitive inhibition, K , = substrate concentration at one-half maximum velocity, MW = molecular weight, S = substrate concentration, VLLN = H-D-valyl-L-leucyl-L-lysyl4nitroanilide. V, = maximum velocity. INTRODUCTION
Plasminogen [molecular weight (MW) 88,0921 is a blood enzyme that enters the mammary gland as plasminogen and causes hydrolysis of &- and pcaseins in milk (4, 14). Its concentration and activity are increased in both mastitic milk (13) and late lactation milk (7, 12, 13). Plasmin activity has been measured in dairy products using synthetic chromogenic (5, 10, 13, 15) or fluorogenic (7, 9, 12) substrates. Lysine peptides linked to chromogenic or fluorogenic compounds are useful substrates because plasmin has high affiiity for lysine residues and cleaves Lys-X bonds (2). N-
Succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-
amido-4-methyl coumarin does not fluoresce, but plasmin hydrolysis of this compound releases 7-amido-4-methyl coumarin, a fluorescent compound. Rate of fluorescence increase is proportional to plasmin activity. Plasmin hydrolysis of the chromogenic substrate H-Dvalyl-L-leucyl-L-lysyl4Ntroanilide (VLLN) releases 4-nitroaniline, which absorbs light at 405 nm. Both methods are rapid, sensitive, specific, and require little sample preparation. However, the latter method may be more conAbbreviation key: A = absorbance, A, = venient because a spectrophotometer can be maximum absorbance, I = inhibitor concentra- used. Using a fluorogenic substrate, Richardson (7) found that plasmin concentrations in early and late lactation milk were .15 and .37 mg/L Received December 10, 1990. in Jersey milk and .27 and .53 mg/L in HolAccepted May 31, 1991. stein milk, suggesting that plasmin activity is 'Conhibution Number 4094 of the Utah Agricultural dependent on breed of cow and stage of lactaExperiment Station. Approved by the director. 1991 J Dairy Sci 74:4115L4124
41 19
4120
BASTIAN ET AL.
tion. Schaar (12) measured plasmin activity in milk from four breeds of dairy COWS and reported .112 mits of 7-amid04methyl coumarjn/ml of Holstein milk and .084 units/ ml of Jersey milk. When plasmin activity was adjusted for casein content, no difference was seen between Holstein and Jersey milk. Schaar (12; p. 375) concluded that “The negative correlation between casein content and plasmin activity. . . is probably due to competition between casein and the synthetic substrate for the enzyme.” Because casein is a natural substrate for plasmin in milk, it competes for the active site of plasmin during analysis with synthetic substrates. This type of interference can be considered reversible competitive inhibition because casein is bound and then released by plasmin. Casein has numerous sites that bind plasmin, many of which likely would not be cleaved during a 30-min assay. Pearse et al. (6) found that digestion of casein by plasmin at optimal pH and temperature takes several hours despite addition of extra plasmin to milk. Also, sites on plasmin that bind uncleaved casein may exist and act in a true inhibitory fashion. In a similar experiment, Wiman et al. (17) measured plasmin activity with VLLN substrate in presence of fibrinogen. They found that some fibrinogen sites are not cleaved by plasmin and that fibrinogen competitively inhibits the action of plasmin toward the substrate. Several kinds of reversible enzyme inhibition are described by Quation [l]:
VmaX
I + -
I S
l+A KI
Competitive inhibition increases the apparent value of K, by increasing KI;K;increases to a value much larger than I, making
4 insignifiKI
cant. So, for competitive inhibition,
In mixed inhibition, Km is increased by a factor of I 1-tKI I
’
1 + 7
Kl
and,V
is decreased by a factor of 1 I
1+K;
as in Equation [Z]. Uncompetitive inhibition decreases the apparent values of both Km and V,, by a factor of 1
I 1+-
K;
where v = initial velocity,, V = maximum velocity, K, = the substrate concentration at one-half V-, KI = inhibitor constant for competitive inhibition, S = substrate concen, tration, I = inhibitor concentration, and KI = inhibitor constant for uncompetitive inhibition. Different kinds of inhibition can be characterized by their effects on Equation [l] (1, 17). This is more easily seen if Equation [l] is rearranged to Quation [2]: Journal of Dairy Science Vol. 74, No. 12, 1991
but keeps their ratio constant; KIincreases to a I value much larger than I, making - insignifiKI
cant. So, for uncompetitive inhibition, Vmax
I
S
1+?
K,
[41
4121
PLASMIN INHIBITION
A rarely encountered fourth type of inhibition, noncompetitive, decreases the apparent value of,,V but has no effect on K,. This is only possible if KI= KI. Because the term noncompetitive is commonly used to include mixed inhibition, noncompetitive inhibition is often referred to as pure noncompetitive inhibition (3). Only competitive or pure noncompetitive inhibition can be overcome by increasing the substrate concentration relative to inhibitor concentration. Functionally, pure noncompetitive inhibition is only possible when the inhibitor molecule is very small, and it is not likely even then (3). The objective of this research was to determine whether casein inhibits the reaction of plasmin with a synthetic substrate and, if so, to determine the type of inhibition. If the effect of casein on plasmin assays using a synthetic substrate follows competitive inhibition kinetics, increasing substrate concentration or decreasing casein concentration would remove the inhibition. MATERIALS AND METHODS
min. Casein concentration of the supernatant was estimated by Kjeldahl nitrogen determination using a Kjeldahl factor of 6.36 (16). Using linear transformations of initial rate data (3) collected from reaction mixtures that represented several combinations of substrate and casein, preliminary estimates of Km and KI (competitive inhibition constant) were obtained. Three levels of substrate (.l, 1, and 10 x K& were combined with three levels of casein (.l, 1, and 10 x Ki, to prepare nine different reaction mixtures. Plasmin solution (150 pl) was added to each reaction mixture at time = 0 to give a final enzyme concentration of 26.8 nM (calculated using M W 88,092 and total volume 850 pl). Reported plasmin concentrations in skim milk vary from .14 to .73 mg/L (1.6 to 8.2 nM) (7, 9). Final substrate concentrations were .04,.40,and 4.00 mM, and final casein concentrations were .0585, 3 5 , and 5.85 mg/ml. Release of 4-nitroaniline was followed by measuring the increase in absorbance at 405 nm for 30 min using a Beckman DU-8B spectrophotometer (Beckman Instrument, Palo Alto, CA) equipped with a kinetics compuset module. The experiment was replicated.
Reagents The VLLN (catalog number V 7127), plasmin purified from bovine blood (.25 Sigma units/mg, catalog number P 7911), and Eamino-ncaproic acid (catalog number A 2504) were purchased from Sigma Chemical Company (St. Louis, MO). Purified casein was purchased Erom Fisher Scientific (Pittsburgh, PA). Kinetic Assays The assay buffer contained 50 mM Tris, 110 mM NaCl, and 3 mM e-amino-ncaproic acid and was adjusted to pH 7.4 with HCl(l3). Although E-amino-n-caproic acid is an inhibitor of plasmin, its KI is so large that it does not interfere with assays at this concentration. The E-amino-n-caproic acid is included in standard plasmin assay buffers to enhance activation of plasminogen. Enzyme solution contained 6.3 mg of plasmid25 ml of assay buffer. Also, VLLN was dissolved (5 mg/ml) in the buffer. Casein solution was prepared by dissolving 4 g in 100 ml of buffer, mixing 3 ml of this solution with 1 ml of .4 M sodium citrate, and centrifuging at 27,000 x g for 10
initial Rate Calculation Assuming that the early part of the absorbance-time curve approximates a rectangular hyperbola, initial reaction rates, v, for each mixture were determined by fitting absorbance-time data to Equation [5] using the NLIN procedure of SAS@(Marquardt method) (11).
A =
Amax x t K + t
[51
where A = absorbance, A ,, = the maximum absorbance approached by the curve, t = time, and K = time at one-half Amax.Least squares estimates of Aand K were obtained and used to determine initial rates from the derivative of m a t i o n [5]:
Determination of Kinetic Constants Kinetic constants were estimated by fitting experimental data to Equation [l]. Four kinetic Journal of Dairy Science Vol. 74. No. 12, 1991
4122
BASTIAN ET AL.
TABLE 1. Inhibition patterns as a function of inhibition constanlS (I).
Type of inhibition' None
I KI
I K;
0
0 0
>o
Competitive Uncompetitive Mixed
0
>o
>o
>o
TABLE 2. Estimates of kinetic constants for casein inhibition of plasmin.
constant'
Value'
SE
Kud Km
.0158 A.4 Mper m h
.01
.lo7 mM ,860 mghnl
.02
K! Kl I KI
'KI = Inhibitor constant for competitive inhibition; gI = inhibitor constant for uncompetitive inhibition.
.05
109 mg/ml
.68
I
10-10
I 'I and were calculated with I = .585 mghnl. KW = KI K,
inhibition was then-deduced from Tal% 1.
*AA =
Change in absorbance.
RESULTS AND DISCUSSION
A typical reaction curve for bovine plasmin activity toward VLLN in presence of casein is approach reduced the error caused by omitting shown in Figure 1. A line that shows the data from later parts of the reaction curves and statistically determined initial rate using Equa- estimated the slope at t = 0 using Equation [6] rather than at t = some short time. tion [5] has been added to the graph. This Richardson and Elston (8) reported plasmin method is more accurate than simply taking activity in commercially prepared caseins. The the slope of the line between the first two casein solution used in our experiment was points measured as the initial rate or subjec- assayed for residual plasmin activity. Absorbtively deciding the initial rate. This statistical ance increased .0027units over 30 min for the highest casein concentration used in this study. This increase was negligible compared with the increase of .3 absorbance units observed for the slowest reaction mixtures and, thus, did not influence reaction rates. Brown (1) proposed that enzyme inhibition patterns are functions of inhibition constants (Table 1) and that type of enzyme inhibition can be determined from estimates of KIand K;. If these parameters become large, the terms in which they are found in Equation [2] approach zero. If both KI and K , are large, there is no inhibition, and Equation [2]becomes the simple Michaelis-Menton equation for enzyme kinetics: 0
10
20
30
Time (min) Figure 1. Increase in absorbance during 30 min reaction of plasmin with .4 mM H-D-valyl-Lleucyl-Llysyl4-nitroanilide and ,585 ml~mlof casein. Initid rate or tangent line at zero time was determined using SAS estimates of maximum absorbance approached by the curve and the time at half the maximum absorbance. JoUmat of Dairy Science Vol. 74, No. 12, 1991
v = - v,
s
Km+S'
r71
Estimates of the kinetic parameters for this expriment are shown in Table 2. Because VKI was .68 mdml and l/Ki was mg/ml,
4123
PLASMIN INHIBITION
move interference of an inhibitor may vary. Because casein inhibition can be removed, plasmin activity in milk can be measured directly without interference from casein. ACKNOWLEDGMENTS
We thank The Jersey Cattle Club and the
Utah Agricultural Experiment Station for the funds provided to do this work. Figure 2. Effect of substrate and casein concentrations on velocity of plasmin reactions. Rates were calculated using estimates of substrate concentration at one-halfmaximum velocity (K,Jand inhibitor constant for competitive inhibition (KI) determined in this study using Equation [3]. [Substrate] from 0 to .04 and [casein] from 0 to .0585 arc extrapolated. AA = Change in absorbance.
Equation [2] was reduced to Equation [3]. Equation [3] describes competitive inhibition, so it was concluded that casein was competitively inhibiting the activity of bovine plasmin toward VLLN. Experimental data were fitted to Equation [3]. Estimates of the remaining kinetic parameters were not different within the precision of the experiments than those found by fitting the data to EQuation [2], although the residual sum of squares was lower. Reaction rates calculated using the estimates of Km and KI listed in Table 2 are plotted in Figure 2. &uation 131 was used for calculating reaction rates achieved when Concentrations of substrate and casein were varied. If casein concentration was increased while substrate concentration was constant, reaction rate decreased. By increasing the substrate concentration at a fixed casein concentration, inhibition was removed. CONCLUSIONS
Casein competitively inhibits plasmin in activity assays that use the synthetic chromogenic substrate VLLN. Adding sufficient substrate or decreasing inhibitory casein concentration negates inhibitor-enzyme binding and removes interference. Substrate concentration of .4 mM removed inhibition associated with 5 mg/ml of casein. Because inhibitor content in enzyme assays depends on sample preparation, amount of substrate needed to re-
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