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Effect of ultrasound on activation of serine proteases precursors
Ultrasonics Sonochemistry 12 (2005) 219–223 www.elsevier.com/locate/ultsonch
Effect of ultrasound on activation of serine proteases precursors S.L. Ovsianko a,*, E.A. Chernyavsky a, V.T. Minchenya b, I.E. Adzerikho c, V.M. Shkumatov a a
Research Institute for Physical and Chemical Problems, Belarusian State University, Leningradskaya St., 14, Lab 602, Minsk 220050, Belarus b Belarusian Polytechnical Academy, Minsk, Belarus c Department of Cardiology, Belarusian, Medical Academy of Postgraduate Education, Minsk, Belarus Received 19 January 2003; accepted 22 October 2003 Available online 9 December 2003
Abstract The effect of ultrasound (US) (26.4 kHz, 26 W/cm2 ) on the activation process of a mixture of chymotrypsinogen and trypsinogen was studied. US led to a significant decrease in proteolytic activity, as well as inhibition of the activation process in general. It was shown that inhibition of proteinase activity under US influence was a consequence of inhibition of chymotrypsinogen–chymotrypsin transformation and the complete proteolytic trypsin degradation in the proenzymes mixture. 2003 Elsevier B.V. All rights reserved. Keywords: Low-frequency ultrasound; Proenzymes; HPLC; Activation of serine proteases
1. Introduction The combination of ultrasound (US) and plasminogen activators offers a useful approach for blood vessel recanalization [1–4]. However, the effects of US on the structure and function of coagulation, and fibrinolysis enzymes was not investigated. Identification of such changes may enable us to determine threshold modes of US treatment and the administration sequence of fibrinolysis proteins activators, whilst minimizing side effects of US. The processes of coagulation and fibrinolysis enzyme activation (for example prothrombin, plasminogen) are known to proceed according to the mechanism of limited proteolytic modification of proteins-precursors. The active forms of these serine proteases are classified among the chymotrypsin family [5]. An appropriate structure-function model for the investigation of US effect on serine proteases precursors activation is a mixture of chymotrypsinogen and trypsinogen. a-Chymotrypsin forms by the release of two internal dipeptides and trypsin by the release of the N-terminal
*
Corresponding author. Tel./fax: +375-17-209-5461. E-mail address: [email protected] (S.L. Ovsianko).
1350-4177/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2003.10.012
hexapeptide from chymotrypsinogen and trypsinogen respectively [6]. In the present work, we have studied the US effect upon the process of proteolytic autoactivation of the serine proteases precursors––chymotrypsinogen and trypsinogen.
2. Materials and methods 2.1. Ultrasound exposure The source of ultrasound is the ultrasonic device for thrombus destruction ‘‘Pulsar’’ APT-YH1 developed at the Belarusian Polytechnical Academy, Belarus. The device consists of an ultrasound generator with frequency of 26.4 kHz and adjustable intensity of 1–40 W/ cm2 , an ultrasound oscillatory system including piezoelectrical transducer and flexible acoustic-horn system of variable section with a length of 560 mm to concentrate US oscillations on a working instrument. The diameter of the final acoustic-horn step is 0.8 mm, the diameter of the emitting surface of the working instrument is 1.8 mm. To achieve maximum ultrasound radiation intensity the stepwise joints of the flexible acoustic-horn act
220
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223 35
30
Intensity, W/cm 2
25
20
15
10
5
0 1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
Axial distance, mm
Fig. 1. US field measured along the center axis of the acoustic horn.
as a Fourier concentrator, to minimize the tension on joints and vibration of acoustic horn. The emitting face of the instrument is a concave hemisphere a radius of at least 0.5 mm to generate axially directed radiation. Measurements of the ultrasound field were made using a piezoelectric element inserted horizontally within a defined distance into a quartz cuvet filled with buffer. The measured field in the axial direction is shown in the Fig. 1. US oscillations intensity was adjusted within the range of 4–30 W/cm2 . The exposure to sound was carried out at the US intensity of 26.4 W/cm2 unless otherwise stated. A cuvet containing insonified protein solutions in 0.05 M potassium-phosphate buffer (pH 7.4) was maintained at 37 C (under insonification, the temperature increased by not more than 0.5 C). 2.2. Isolation and activation of a mixture of chymotrypsinogen and trypsinogen Bovine pancreas was homogenized and extracted with 0.125 M sulfuric acid for 24 h and centrifuged (Biofuge 22R, Faust, Germany) at 15,000 g for 30 min. The supernatant was subjected to fractionation with dry ammonium sulfate, and the fraction precipitated in the range of 40–70% of saturation was collected. A precipitate was recovered by centrifugation (Biofuge 22R, Faust) at 15,000 g for 15 min, dissolved in 3 vol. of distilled water, dialyzed against 0.001 M hydrochloric acid for 24 h and dried by lyophilization. The content of individual proenzymes and minor amounts of active proteases in the mixture was estimated by high pressure liquid chromatography (HPLC). Since a low chymotrypsin content (<1%) is masked during HPLC with high
trypsinogen content [7], active centers of serine proteases were titrated with phenylmethylsulphonyl fluoride. Proenzyme activation was carried out at 37 C in 0.01 M potassium phosphate buffer (pH 7.4) for 5 h (control) and after treatment with US for 5, 20 and 40 min. Protease activity was determined by a modification of Anson’s method [8] at 37 C in 0.01 M potassium phosphate buffer (pH 7.2) using Na-caseinate as a substrate. One unit of protease activity is defined as the amount of enzyme that liberated 1 lg of tyrosine/min under above specified conditions. The residual trypsin activity was also analyzed with N -a-benzoyl-D L -arginine p-nitroanilide as a chromogenic substrate [9]. HPLC was performed on an LC-10AT chromatography system (Shimadzu, Kyoto, Japan) using an SPDM10A photodiode array UV/VIS-detector, and injector (Rheodyne, Cotati, Ca, USA) with a loop volume of 20 ll. A TSK Phenyl-5PW column (7.5 · 75 mm, Pharmacia Biotech, Uppsala, Sweden) was used. Hydrophobic interaction chromatography was performed under the following conditions: solutions A––0.02 M potassiumphosphate buffer (pH 6.0) and 1.75 M (NH4 )2 SO4 and solution B––0.02 M potassium phosphate buffer (pH 6.0). Reverse exponentional gradient of solution B during 23 min, flow rate 1 ml/min, sample volume––20 ll, k––280 nm. The CLASS VP software (Shimadzu) was used to set parameters of the process (gradient shape, elution rate) and to calculate peak areas [7]. Absorption spectra were recorded on a UV-1202 (Shimadzu) spectrophotometer.
3. Results and discussion A mixture of chymotrypsinogen and trypsinogen, obtained by ammonium sulfate fractionation of extract from bovine’s pancreas [10] was used for this study. The concentrations of proteins in the mixture were chymotrypsinogen––22.3 lM, trypsinogen––20.4 lM, chymotrypsin––0.2 lM, trypsin––0.1 lM. It was observed that there were no differences between the process of proenzymes activation under US at intensities of 4.5 and 10.4 W/cm2 and the control sample. However at an intensity of 26.4 W/cm2 a significant decrease in proteolytic activity was observed, as well as inhibition of the activation process in general (Fig. 2). A sigmoidal character of the curves indicated the autocatalytic character of the activation process. It is possible to identify three phases on the given curves: exponential (0–s1 ), linear increase of activity (s1 –s2 ) and the phase of stationary activity and inhibition (> s2 ). The experimental data were fitted with a kinetic equation of a first order reaction (0–s1 ), and the second phase to a kinetic equation of a zero order reaction (s1 –s2 ) [11]. Based on the data of sample insonification during 5, 20 and 40 min and consequent 5 h activation, rate
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223
221
relative activity, % 100 1
80 2
60
40
20
0 0
50
100
150
τ1
200
250 τ2
300
350
activation time, min
Fig. 2. Effect of US 26.4 W/cm2 upon proteolytic activation of proenzyme mixture. 1––initial proenzymes mixture, 2––proenzymes mixture after 40 min of effect of US.
constants of activation reactions for exponential and linear increase of activity phase were determined (Table 1). US processing, irrespective of its duration, led to an increase of the exponential phase from 90 min for the control to 120 min for experimental samples. The value of s2 increased from 150 min (control) to 210–230 min for US treated samples. Rate constants for both phases (k1 and k2 ) decreased 1.6–2.9 fold under US processing. The increase of the exponential phase and the decrease in k1 and k2 values imply one of the proteins is more sensitive to the effect of US than the others. In order to determine which, the dynamics of the changes in all protein content was determined by HPLC. This method provided estimates of the concentrations of chymotrypsinogen, trypsinogen, chymotrypsin, trypsin and low-molecular autolysis products in the process of activation [7,10]. Chromatograms of initial mixture of proenzymes (a), control sample after 60 min of incubation in activation buffer without US treatment (b), the sample of 55 min incubation the proenzymes mixture after 5 min US treatment (c) and the 40 min incubation of sample after 20 min US treatment are presented in Fig. 3. In contrast to the control experiment (Fig. 3b), a significant decrease in trypsin content (about 0.1% of
Fig. 3. HPLC of initial proenzymes mixture (a) and after 60 min of activation without effect of US (b), the sample of 55 min incubation the proenzymes mixture after 5 min US treatment (c) and the 40 min incubation of sample after 20 min US treatment (d). 1––trypsinogen, 2––chymotrypsinogen, 3––chymotrypsin, 4––trypsin.
initial trypsinogen) was observed (Fig. 3c–d). Catalytic activity of chymotrypsin, isolated by HPLC from the US processed sample was close to the native protein values. Under the effect of US, this protein does not undergo covalent modifications or conformational changes, which may be accompanied by a decrease in activity. Integration of peaks on chromatograms allowed us to determine the relative molar content of different proteins in the process of activation (Fig. 4). For the control sample, trypsin showed a lower resistance to proteolytic degradation (maximum molar yield reached 30%) than chymotrypsin (molar yield 70–80%) (Fig. 4a). The effect of US hindered the rate of chymotrypsinogen
Table 1 Kinetic parameters of autoactivation process of trypsinogen and chymotrypsinogen mixture US effect (26.4 W/cm2 ), min
Amax a , %
s1 , min
0 5 20 40
100 91 74 72
90 120 120 120
a
k1 b , min
Amax ––maximum activity. k1 ––rate constant for exponential phase of a kinetic curve. c k2 ––rate constant for linear increase of activity phase of a kinetic curve. b
0.029 0.018 0.016 0.015
1
s2 , min 150 210 230 230
k2 c , unit/(mg · min) 0.044 0.022 0.017 0.015
222
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223 100
Percentage of protein, %
a 3
80 60 40
4 20 1 2
0 0
50
100
100
150
200
250
300
b
80 3
60 40 1
The inhibition of the activation processes of the studied zymogens under the conditions of the effect of US is of great importance for increasing the efficiency of acousto-enzymatic thrombolysis. Taking into account the autocatalytic mechanism of plasminogen activation caused during the first phase by conformational changes when it interacts with streptokinase [13], these results allow us to suppose that inactivation of plasmin located in close proximity to a thrombus may occur as a localized effect of US. Therefore, the application of streptokinase as a plasminogen activator before US treatment to obtain enough activated plasmin, and a US treatment time and intensity lower than 5 min and 26 W/cm2 creates the necessary prerequisites to minimize the effects of acysto-enzymatic thrombolysis in vitro.
2
20 4
0 0
50
100
150
200
250
300
Acknowledgements
activation time, min
Fig. 4. Kinetics of changes in molar content of trypsinogen (1), chymotrypsinogen (2), chymotrypsin (3) and trypsin (4) at activation of proenzymes mixture. (a)––control sample; (b)––US processing during 40 min.
transition to chymotrypsin: equimolarity of these proteins was observed at 80 min activation for control (Fig. 4a) and at 130 min activation for the sample after 40 min US processing (Fig. 4b). During the activation process, a decrease in trypsinogen concentration was observed without formation of trypsin (Fig. 4b, curves 1 and 4). The increase of the exponential phase of activation for the US processed sample compared with control (Fig. 2) may be caused, by US modified trypsin being attacked proteolytically by chymotrypsin, and partial autolysis of chymotrypsin. This hypothesis is strengthened by a decrease in the molar yield of trypsin (Fig. 4). In the initial studies of the effect of US on isolated proteins it has been demonstrated that chymotrypsin inactivation occurs under the effect of free radicals with possible destruction of Trp215 residue and salt bridge Asp194 -Ile196 , which maintains the catalytically active enzyme conformation [12]. These covalent or conformational modifications for chymotrypsinogen or chymotrypsin were not observed in our experiments for the dynamic model of proenzyme activation. This is associated with different threshold values of US treatment and higher initial proenzyme concentrations. The present work determined that the inhibition of proteinase activity under US influence (Fig. 2, Table 1) is a consequence of multiple processes. One of them is the inhibition of chymotrypsinogen–chymotrypsin transformation (Fig. 4), but probably the main contribution was caused by the almost complete proteolytic trypsin degradation in the proenzyme mixture after US processing (Figs. 3 and 4).
We thank Dr. N. Gromak (Sir William Dunn School of Pathology, University of Oxford, UK) and Mr. C. McConnell (University of Liverpool, UK) for their useful remarks during preparation of this article. This work was supported in part by grants of the Belarusian Republican Foundation for Fundamental Research (# BOO-259 and BOOM-027).
References [1] V.N. Suchkova, R.B. Baggs, C.W. Francis, Effect of 40-kHz ultrasound on acute thrombotic ischemia in a rabbit femoral artery thrombosis model: enhancement of thrombolysis and improvement in capillary muscle perfusion, Circulation 101 (19) (2000) 2296–2301. [2] I.E. Adzerikho, A.G. Mrochek, V.V. Dmitriev, O.A. Lukyanchenko, A.I. Kulak, Ultrasound fibrin clot destruction in vitro in the presence of fibrinolytic agent, Ultrason. Sonochem. 8 (3) (2001) 315–318. [3] S. Behrens, M. Daffershofer, D. Spiegel, M. Hennerici, Lowfrequency, low-intensity ultrasound accelerates thrombolysis through the skull, US Med. Biol. 25 (2) (1999) 269–273. [4] C.W. Francis, A. Blinc, S. Lee, et al., Ultrasound accelerates transport of recombinant tissue plasminogen activator into clots, US Med. Biol. 21 (1995) 419–424. [5] N.D. Rawlings, A.J. Barret, Families of serine peptidases, Methods Enzymol. 244 (1994) 19–61. [6] M.M. Krem, T. Rose, E. Di Cera, Sequence determinants of function and evolution in serine proteases, Trends Cardiovasc. Med. 10 (4) (2000) 171–176. [7] S.L. Ovsyanko, E.A. Chernyavskii, T.L. Yurkshtovich, V.M. Shkumatov, F.N. Kaputskii, A comparative analysis of various commercial a-chymotrypsin preparations by HPLC under nondenaturing conditions, Pharm. Chem. J. 35 (10) (2001) 573–576. [8] M.L. Anson, The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin, J. Gen. Physiol. 22 (1939) 79–91. [9] V. Guyonnet, F. Tluscik, P.L. Long, A. Polanowski, J. Travis, Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken, J. Chromatogr. A 852 (1) (1999) 217–225.
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223 [10] V.M. Shkumatov, S.L. Ovsyanko, A.L. Rudoi, et al., Hydrophobic and affinity interactions during obtaining high-purity alpha chymotrypsin, Proc. Natl. Acad. Sci. Belarus (in Russian) 2 (2000) 30–34. [11] M.J. Minch, Experiments in Biochemistry: Projects and Procedures, Prentice Hall, Englewood Cliffs, New Jersey, 1989, pp. 195– 201.
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[12] A.M. Klibanov, K. Martinek, I.V. Berezin, Action of ultrasonics on alpha-chymotrypsin. A new approach to the study of conformational changes (transitions) in the active centers of enzymes, Biochemistry (Mosc) 39 (4) (1974) 878–887. [13] K.C. Young, G.Y. Shi, D.H. Wu, et al., Plasminogen activation by streptokinase via a unique mechanism, J. Biol. Chem. 273 (5) (1998) 3110–3116.
Effect of ultrasound on activation of serine proteases precursors S.L. Ovsianko a,*, E.A. Chernyavsky a, V.T. Minchenya b, I.E. Adzerikho c, V.M. Shkumatov a a
Research Institute for Physical and Chemical Problems, Belarusian State University, Leningradskaya St., 14, Lab 602, Minsk 220050, Belarus b Belarusian Polytechnical Academy, Minsk, Belarus c Department of Cardiology, Belarusian, Medical Academy of Postgraduate Education, Minsk, Belarus Received 19 January 2003; accepted 22 October 2003 Available online 9 December 2003
Abstract The effect of ultrasound (US) (26.4 kHz, 26 W/cm2 ) on the activation process of a mixture of chymotrypsinogen and trypsinogen was studied. US led to a significant decrease in proteolytic activity, as well as inhibition of the activation process in general. It was shown that inhibition of proteinase activity under US influence was a consequence of inhibition of chymotrypsinogen–chymotrypsin transformation and the complete proteolytic trypsin degradation in the proenzymes mixture. 2003 Elsevier B.V. All rights reserved. Keywords: Low-frequency ultrasound; Proenzymes; HPLC; Activation of serine proteases
1. Introduction The combination of ultrasound (US) and plasminogen activators offers a useful approach for blood vessel recanalization [1–4]. However, the effects of US on the structure and function of coagulation, and fibrinolysis enzymes was not investigated. Identification of such changes may enable us to determine threshold modes of US treatment and the administration sequence of fibrinolysis proteins activators, whilst minimizing side effects of US. The processes of coagulation and fibrinolysis enzyme activation (for example prothrombin, plasminogen) are known to proceed according to the mechanism of limited proteolytic modification of proteins-precursors. The active forms of these serine proteases are classified among the chymotrypsin family [5]. An appropriate structure-function model for the investigation of US effect on serine proteases precursors activation is a mixture of chymotrypsinogen and trypsinogen. a-Chymotrypsin forms by the release of two internal dipeptides and trypsin by the release of the N-terminal
*
Corresponding author. Tel./fax: +375-17-209-5461. E-mail address: [email protected] (S.L. Ovsianko).
1350-4177/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2003.10.012
hexapeptide from chymotrypsinogen and trypsinogen respectively [6]. In the present work, we have studied the US effect upon the process of proteolytic autoactivation of the serine proteases precursors––chymotrypsinogen and trypsinogen.
2. Materials and methods 2.1. Ultrasound exposure The source of ultrasound is the ultrasonic device for thrombus destruction ‘‘Pulsar’’ APT-YH1 developed at the Belarusian Polytechnical Academy, Belarus. The device consists of an ultrasound generator with frequency of 26.4 kHz and adjustable intensity of 1–40 W/ cm2 , an ultrasound oscillatory system including piezoelectrical transducer and flexible acoustic-horn system of variable section with a length of 560 mm to concentrate US oscillations on a working instrument. The diameter of the final acoustic-horn step is 0.8 mm, the diameter of the emitting surface of the working instrument is 1.8 mm. To achieve maximum ultrasound radiation intensity the stepwise joints of the flexible acoustic-horn act
220
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223 35
30
Intensity, W/cm 2
25
20
15
10
5
0 1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
Axial distance, mm
Fig. 1. US field measured along the center axis of the acoustic horn.
as a Fourier concentrator, to minimize the tension on joints and vibration of acoustic horn. The emitting face of the instrument is a concave hemisphere a radius of at least 0.5 mm to generate axially directed radiation. Measurements of the ultrasound field were made using a piezoelectric element inserted horizontally within a defined distance into a quartz cuvet filled with buffer. The measured field in the axial direction is shown in the Fig. 1. US oscillations intensity was adjusted within the range of 4–30 W/cm2 . The exposure to sound was carried out at the US intensity of 26.4 W/cm2 unless otherwise stated. A cuvet containing insonified protein solutions in 0.05 M potassium-phosphate buffer (pH 7.4) was maintained at 37 C (under insonification, the temperature increased by not more than 0.5 C). 2.2. Isolation and activation of a mixture of chymotrypsinogen and trypsinogen Bovine pancreas was homogenized and extracted with 0.125 M sulfuric acid for 24 h and centrifuged (Biofuge 22R, Faust, Germany) at 15,000 g for 30 min. The supernatant was subjected to fractionation with dry ammonium sulfate, and the fraction precipitated in the range of 40–70% of saturation was collected. A precipitate was recovered by centrifugation (Biofuge 22R, Faust) at 15,000 g for 15 min, dissolved in 3 vol. of distilled water, dialyzed against 0.001 M hydrochloric acid for 24 h and dried by lyophilization. The content of individual proenzymes and minor amounts of active proteases in the mixture was estimated by high pressure liquid chromatography (HPLC). Since a low chymotrypsin content (<1%) is masked during HPLC with high
trypsinogen content [7], active centers of serine proteases were titrated with phenylmethylsulphonyl fluoride. Proenzyme activation was carried out at 37 C in 0.01 M potassium phosphate buffer (pH 7.4) for 5 h (control) and after treatment with US for 5, 20 and 40 min. Protease activity was determined by a modification of Anson’s method [8] at 37 C in 0.01 M potassium phosphate buffer (pH 7.2) using Na-caseinate as a substrate. One unit of protease activity is defined as the amount of enzyme that liberated 1 lg of tyrosine/min under above specified conditions. The residual trypsin activity was also analyzed with N -a-benzoyl-D L -arginine p-nitroanilide as a chromogenic substrate [9]. HPLC was performed on an LC-10AT chromatography system (Shimadzu, Kyoto, Japan) using an SPDM10A photodiode array UV/VIS-detector, and injector (Rheodyne, Cotati, Ca, USA) with a loop volume of 20 ll. A TSK Phenyl-5PW column (7.5 · 75 mm, Pharmacia Biotech, Uppsala, Sweden) was used. Hydrophobic interaction chromatography was performed under the following conditions: solutions A––0.02 M potassiumphosphate buffer (pH 6.0) and 1.75 M (NH4 )2 SO4 and solution B––0.02 M potassium phosphate buffer (pH 6.0). Reverse exponentional gradient of solution B during 23 min, flow rate 1 ml/min, sample volume––20 ll, k––280 nm. The CLASS VP software (Shimadzu) was used to set parameters of the process (gradient shape, elution rate) and to calculate peak areas [7]. Absorption spectra were recorded on a UV-1202 (Shimadzu) spectrophotometer.
3. Results and discussion A mixture of chymotrypsinogen and trypsinogen, obtained by ammonium sulfate fractionation of extract from bovine’s pancreas [10] was used for this study. The concentrations of proteins in the mixture were chymotrypsinogen––22.3 lM, trypsinogen––20.4 lM, chymotrypsin––0.2 lM, trypsin––0.1 lM. It was observed that there were no differences between the process of proenzymes activation under US at intensities of 4.5 and 10.4 W/cm2 and the control sample. However at an intensity of 26.4 W/cm2 a significant decrease in proteolytic activity was observed, as well as inhibition of the activation process in general (Fig. 2). A sigmoidal character of the curves indicated the autocatalytic character of the activation process. It is possible to identify three phases on the given curves: exponential (0–s1 ), linear increase of activity (s1 –s2 ) and the phase of stationary activity and inhibition (> s2 ). The experimental data were fitted with a kinetic equation of a first order reaction (0–s1 ), and the second phase to a kinetic equation of a zero order reaction (s1 –s2 ) [11]. Based on the data of sample insonification during 5, 20 and 40 min and consequent 5 h activation, rate
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223
221
relative activity, % 100 1
80 2
60
40
20
0 0
50
100
150
τ1
200
250 τ2
300
350
activation time, min
Fig. 2. Effect of US 26.4 W/cm2 upon proteolytic activation of proenzyme mixture. 1––initial proenzymes mixture, 2––proenzymes mixture after 40 min of effect of US.
constants of activation reactions for exponential and linear increase of activity phase were determined (Table 1). US processing, irrespective of its duration, led to an increase of the exponential phase from 90 min for the control to 120 min for experimental samples. The value of s2 increased from 150 min (control) to 210–230 min for US treated samples. Rate constants for both phases (k1 and k2 ) decreased 1.6–2.9 fold under US processing. The increase of the exponential phase and the decrease in k1 and k2 values imply one of the proteins is more sensitive to the effect of US than the others. In order to determine which, the dynamics of the changes in all protein content was determined by HPLC. This method provided estimates of the concentrations of chymotrypsinogen, trypsinogen, chymotrypsin, trypsin and low-molecular autolysis products in the process of activation [7,10]. Chromatograms of initial mixture of proenzymes (a), control sample after 60 min of incubation in activation buffer without US treatment (b), the sample of 55 min incubation the proenzymes mixture after 5 min US treatment (c) and the 40 min incubation of sample after 20 min US treatment are presented in Fig. 3. In contrast to the control experiment (Fig. 3b), a significant decrease in trypsin content (about 0.1% of
Fig. 3. HPLC of initial proenzymes mixture (a) and after 60 min of activation without effect of US (b), the sample of 55 min incubation the proenzymes mixture after 5 min US treatment (c) and the 40 min incubation of sample after 20 min US treatment (d). 1––trypsinogen, 2––chymotrypsinogen, 3––chymotrypsin, 4––trypsin.
initial trypsinogen) was observed (Fig. 3c–d). Catalytic activity of chymotrypsin, isolated by HPLC from the US processed sample was close to the native protein values. Under the effect of US, this protein does not undergo covalent modifications or conformational changes, which may be accompanied by a decrease in activity. Integration of peaks on chromatograms allowed us to determine the relative molar content of different proteins in the process of activation (Fig. 4). For the control sample, trypsin showed a lower resistance to proteolytic degradation (maximum molar yield reached 30%) than chymotrypsin (molar yield 70–80%) (Fig. 4a). The effect of US hindered the rate of chymotrypsinogen
Table 1 Kinetic parameters of autoactivation process of trypsinogen and chymotrypsinogen mixture US effect (26.4 W/cm2 ), min
Amax a , %
s1 , min
0 5 20 40
100 91 74 72
90 120 120 120
a
k1 b , min
Amax ––maximum activity. k1 ––rate constant for exponential phase of a kinetic curve. c k2 ––rate constant for linear increase of activity phase of a kinetic curve. b
0.029 0.018 0.016 0.015
1
s2 , min 150 210 230 230
k2 c , unit/(mg · min) 0.044 0.022 0.017 0.015
222
S.L. Ovsianko et al. / Ultrasonics Sonochemistry 12 (2005) 219–223 100
Percentage of protein, %
a 3
80 60 40
4 20 1 2
0 0
50
100
100
150
200
250
300
b
80 3
60 40 1
The inhibition of the activation processes of the studied zymogens under the conditions of the effect of US is of great importance for increasing the efficiency of acousto-enzymatic thrombolysis. Taking into account the autocatalytic mechanism of plasminogen activation caused during the first phase by conformational changes when it interacts with streptokinase [13], these results allow us to suppose that inactivation of plasmin located in close proximity to a thrombus may occur as a localized effect of US. Therefore, the application of streptokinase as a plasminogen activator before US treatment to obtain enough activated plasmin, and a US treatment time and intensity lower than 5 min and 26 W/cm2 creates the necessary prerequisites to minimize the effects of acysto-enzymatic thrombolysis in vitro.
2
20 4
0 0
50
100
150
200
250
300
Acknowledgements
activation time, min
Fig. 4. Kinetics of changes in molar content of trypsinogen (1), chymotrypsinogen (2), chymotrypsin (3) and trypsin (4) at activation of proenzymes mixture. (a)––control sample; (b)––US processing during 40 min.
transition to chymotrypsin: equimolarity of these proteins was observed at 80 min activation for control (Fig. 4a) and at 130 min activation for the sample after 40 min US processing (Fig. 4b). During the activation process, a decrease in trypsinogen concentration was observed without formation of trypsin (Fig. 4b, curves 1 and 4). The increase of the exponential phase of activation for the US processed sample compared with control (Fig. 2) may be caused, by US modified trypsin being attacked proteolytically by chymotrypsin, and partial autolysis of chymotrypsin. This hypothesis is strengthened by a decrease in the molar yield of trypsin (Fig. 4). In the initial studies of the effect of US on isolated proteins it has been demonstrated that chymotrypsin inactivation occurs under the effect of free radicals with possible destruction of Trp215 residue and salt bridge Asp194 -Ile196 , which maintains the catalytically active enzyme conformation [12]. These covalent or conformational modifications for chymotrypsinogen or chymotrypsin were not observed in our experiments for the dynamic model of proenzyme activation. This is associated with different threshold values of US treatment and higher initial proenzyme concentrations. The present work determined that the inhibition of proteinase activity under US influence (Fig. 2, Table 1) is a consequence of multiple processes. One of them is the inhibition of chymotrypsinogen–chymotrypsin transformation (Fig. 4), but probably the main contribution was caused by the almost complete proteolytic trypsin degradation in the proenzyme mixture after US processing (Figs. 3 and 4).
We thank Dr. N. Gromak (Sir William Dunn School of Pathology, University of Oxford, UK) and Mr. C. McConnell (University of Liverpool, UK) for their useful remarks during preparation of this article. This work was supported in part by grants of the Belarusian Republican Foundation for Fundamental Research (# BOO-259 and BOOM-027).
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