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Fluorescence polarization assay of plasmin, plasminogen, and plasminogen activator
ANALYTICAL
BIOCHEMISTRY
Fluorescence
KAZUO
104, 15-22 (1980)
Polarization Assay of Plasmin, Plasminogen Activator KINOSHITA,*
HIROSHI
MAEDA,?,’
Plasminogen,
AND YORIO
and
HINUMA?
Received July 5. 1979 We describe two assay methods for plasminogen activator (urokinase) employing the fluorescence polarization technique. One method utilizes fluoroscein isothiocyanate-labeled plasminogen as the substrate for urokinase (direct method), and the other utilizes the new plasmin generated from plasminogen by urokinase, for which fluoroscein isothiocyanatelabeled fibrinogen was employed as the substrate (indirect method). Also in the assay of trypsin and plasminogen, fluoroscein isothiocyanate-labeled fibrinogen was used as the substrate. Proteolysis by the enzymes (urokinase. plasmin. and trypsin) yielded smaller peptide fragments from the labeled substrates. resulting in a decreased fluorescence polarization value. Urokinase activity determined by the present method correlated well with the data obtained by the known method with a synthetic chromogenic substrate. Enzyme levels as low as 1.5 mIU/ml (direct method) or 0.11 IUiml (indirect method) of urokinase and 0.5 millicasein unit/ml and 0.012 &ml of plasminogen and trypsin. respectively, were quantified within I to 2 h
also been shown useful for the assay of proteolysis by trypsin, papain, pepsin, and Pronase using FITC-labeled substrates (6.7). When many of the fluorochrome-labeled substrates are subjected to digestion by the various proteases, a decrease in the P value of the substrates becomes apparent (7). Their enzyme specificity was confirmed with respective inhibitors (7). Based on this principle, we have extended the method for the assay of plasminogen and plasminogen activator (urokinase) as well as of trypsin using FITC-labeled fibrinogen and plasminogen as the substrates. The plasminogen activator is not only an important clinical parameter but it is increasingly becoming a critical parameter in the process of cell transformation (8- 10) and tumorigenesis ( 11.12). Furthermore, its level in serum manifests an activated state of macrophage function (13). Thus a rapid and sensitive assay of the plasmin activator will facilitate analysis
In order to obtain kinetic and equilibrium information for the protein-protein interaction, the fluorescence polarization method has been shown to be useful (1,2). The fluorescence polarization value (P value)? changes according to the size of the protein molecule which is labeled with fluorochrome due to the difference in the Brownian motion. The fluorescence polarization method has been applied to many of the antigenantibody reactions (3-5). Quantification of antigens or antibodies becomes possible by measuring the increase in the P value of the complex being formed. Recently. the fluorescence polarization method has ’ To whom requests for reprints should be addressed. ” Abbreviations used: P value, fluorescence polarization value in arbitrary units; FITC-, fluorescein isothiocyanate-labeled; cu. casein unit; MCA, methylcoumarin amide: TPCK, tosylphenylalanine chloromethyl ketone: CTA unit, unit defined by the Committee on Thrombolytic Agents: PBS. phosphatebuffered solution, pH 7.0. IS
0003-2697!80/0700 IS-08$0?.00/0 Cupynght All r,ghf*
< 1980 by Acxkmx F’re\\. Inc. of reproductm ,n any form rewrved
16
KINOSHITA.MAEDA.ANDHINUMA
of the host’s response. MATERIALS
defense
mechanism
and
AND METHODS
Itzstruments. The fluorescence spectropolarimeter. Model MAC-2 Type HR-I (Japan Immunoindustries Co. Ltd., Takasaki) was employed for the measurement of fluorescence polarization, The instrument is equipped with a primary polarizer, an excitation filter for 490 nm, a cell housing, an analyzer-polarizer that is driven by a synchronous motor at 1800 rpm, an emission filter for 520 nm, and a photomultiplier with an amplifier system. Neither the background of protein fluorescence, which is primarily due to aromatic amino acid residues and is located in the near-uv region, nor the light scattering of excitation light by protein will affect the measurement because of the efficient cutoff filter at 520 nm (Ditric Optics Inc., 247R Maple St., Marlboro, Mass.). The instrument can detect a fluorescein concentration of 1.5 pmol/ml at or above pH 7.0. A built-in microcomputer is also furnished in the which calculates P values instrument, before printout every 82 s. Each P value printed out is an integral average of 50 measurements in 50 s. All P values obtained in the present experiments are a multiple of about 2.0 x 1O:j of the real value and are expressed as changes in the percentage of the P value. The temperature of the cuvette was kept constant (within *O.I”C of error) during the operation with a “Ucool” circulation unit (Neslab Inst. Inc., Portsmouth, N. H.). Chemids. Substrates and enzymes used were obtained from the following sources: plasminogen-free bovine fibrinogen and bovine plasminogen (specific activity 9.2 cu) were from Daiichi Pure Chemicals Company Ltd. (Tokyo); human plasminogen from AB KABI (Stockholm; 15 cu/mg protein) and Worthington Biochemical Corporation (Freehold, N. J.: 174 Uimg):
TPCK-treated trypsin ( 183. I U/mg) from Worthington Biochemical Corporation: human urokinase, free of plasmin, was a generous gift from Mochida Pharmaceutical Company (Tokyo). FITC (isomer I) with a purity of more than 90% was obtained from Dojin Chemical Company (Kumamote). Phenylmethylsulfonyl fluoridine, an inhibitor of serine protease, was from Calbiochem (San Diego, Calif.). GlutarylGly-Arg-MCA was from Peptide Institute Inc. (Osaka). Urine specimens were obtained from the clinical department. All other chemicals were obtained from commercial sources. FITC Iuhrling. The labeling of bovine fibrinogen was carried out as follows. A 25mg sample of plasminogen-free bovine fibrinogen and 2.5 mg of FITC were dissolved in 5 ml of 0.1 M borate buffer, pH 8.0. After reacting for 3 h at 37°C in the darkness.‘j the reaction mixture was applied to a column (1.5 x 52 cm) of Sephadex G-50 which had been equilibrated with 0.1 M borate buffer, pH 8.0. The FITC-fibrinogen which came out first was collected and then dialyzed in the cold (4-8°C) against 0.01 M phosphate-buffered 0.15 M NaCl solution (PBS), pH 7.0, containing I mM ethylenediamine tetraacetate (EDTA) for about 3 days with a few changes of the dialysis buffer each day. By this procedure a complete removal of nonspecifically bound or unbound fluorochrome was ascertained based on the P value and the fluorescence intensity of the dialysis buffer. The solution of FITCfibrinogen was stored at -20°C as a stock solution. The substrate solutions for the assay were prepared by diluting the stock solution 50-fold with 0.1 M Tris-HCI buffer. pH 8.5. A 1.5-ml aliquot of this substrate solution was stored at -20°C until use. Both stock and substrate solutions of FITC,’ We jugates fluorescent can
have observed that exposure of the conto intensive light liberates an unidentified low molecular weight compound. which
be removed
by dialyG\.
PLASMIN,
PLASMINOGEN.
AND TABLE
SYSTEMS
System
USED
FOR.~HE
Enzyme to be assayed (sample = 50 ~1)
ASSAY
OF TRYPSIN,
Enzyme hydrolyzing FITCsubstrate
FITCsubstrate
Trypsin Plasminogen
Trypsin Plasmin
Fibrinogen Fibrinogen
3 4
Urokinase Urokinase
Urokinase Plasmin
Plasminogen Fibrinogen
‘I The reaction an approximate
mixture contains concentration
PLASMINOGEN,
4~~2
Activator in SO &I
UROKINASE”
Proenzyme in 50 ~1
Assay Direct Indirect
Urokinase (30 IU)
I .9 ml of a 0. I M phosphate buffer solution, of lo-50 pmol/ml of fluorescein equivalent
fibrinogen were stable over 1 year at -20°C based on P value. (A decrease in P value indicates autodigestion or degradation.) Labeling of human plasminogen (AB KABI) was carried out as follows. About 1 mg of plasminogen and 0.1 mg of phenylmethylsulfonyl fluoridine were dissolved in I ml of PBS. pH 7.6, and allowed to react for about 90 min in a reciprocal shaker (2 Hz) at room temperature. Subsequently, FITC (1.25 mg), dissolved in 0.5 ml of 0.1 M borate buffer, pH 8.0. was added to the mixture. These were reacted and purified in a manner similar to that described above for fibrinogen. The FITC-plasminogen was collected and then stored at -20°C until use. The FITC-fibrinogen and FITC-plasminogen had 17.2 and 2.2 mol of fluorochromes per mole of protein (F/P), respectively. F/P was determined spectroscopically. The molecular extinction coefficient of fluorescein at 490 nm was 6.76 x 10” at pH 8.0. For human fibrinogen and plasminogen, the molecular extinction coefficients used were 5.88 x 10” and 1.49 x 10” both at 280 nm, respectively. Enzynzr reactions. A test tube containing I .9 ml of 0.1 M phosphate buffer, pH 7.5, was placed in the U-cool circulator at 3o”C, after which FITC-substrate (100 or 50 ~1
17
ASSAY
I
I 2
have
UROKlNASE
Plasminogen (46 pg) pH 7.5. The labeled in each system.
Direct Indirect
substrates
of the appropriate substrate) was added in all assays. The enzyme reactions were then initiated by an addition of a varied amount of each enzyme, and, if required, a definite amount of the plasminogen or plasminogen activator (urokinase). In Table 1, the reagents utilized in four different assay systems are described. After incubation for a definite time period at 30°C in the U-cool circulator, the reaction mixture was placed in a cuvette and the P value was measured at 30°C. The concentration of the FITClabeled substrate in the cuvette was designed to yield a significant signal at a cathode voltage between 500 and 650 V in the photomultiplier which corresponds to 5- 10 pmoliml fluorescein. System 1 is designed for the assay of trypsin which was dissolved in 0.01 M HCI with 2 mM Ca?+. System 2 is designed to assay the amount of plasminogen in which a definite amount (about 15 W/ml) of urokinase is added to activate a varied amount of plasminogen to plasmin. A stock solution of plasminogen was dissolved in 0.25 M phosphate buffer, pH 7.5, containing 0.05 M acetate. As for the assay of urokinase (plasminogen activator), two types of assay systems are described. One is the direct method (system 3) using FITC-plasminogen as the substrate of urokinase which specifically hydrolyzes
18
KINOSHITA.
MAEDA.
AND
HINUMA
urine is added to the above-described action mixture in a cuvette for assay. RESULTS
re-
AND DISCUSSION
Enzyme activities were expressed as a percentage of the P value after reaction to that of the blank without the enzyme. The real P value corresponding to a P I ‘\ value of 100% is shown in the legends. ‘\ hi. Figure 1 was obtained for the varied amount 60 1 '\ ot t of trypsin added to the FITC-fibrinogen and 0 001 01 / 100 Concentrdion of ttypsiZgg/ml) digested in I h. This indicates a linear relationship between the decrease in P value FIG. 1. Relationship between the concentration of and the log of the amount of trypsin trypsin and the change in the fluorescence polarizain 0.012-100 &ml. This result indicates tion (P) value. The FITC-labeled fibrinogen, about 50 pmoliml fluorescein equivalent in 0.1 M phosthat it is more sensitive than that of the was digested by varied phate buffer, pH 7.5. radiolabeled synthetic substrate, benzoylamounts of trypsin for a period of I h and the P DL-arginine I:‘H]anilide which has a devalues obtained for each trypsin concentration are tection limit of 0.05 pg/ml ( 17). This tryptic plotted. Temperature: 30°C. P( lOO%c) = 0.118. activity was inhibited by various trypsin inhibitors (data not shown) (7). It should FITC-plasminogen; the change in the P be noted that a commercial preparation of value reflects the proteolytic cleavage of fibrinogen from different sources exhibited plasminogen by urokinase directly. The autolytic degradation as revealed by the P other is the indirect method in which a value. Treatment with diisopropyl fluorodefinite amount (22 E.cg/ml) of plasminogen phosphate was advantageous in such cases. is added to each test tube which is sub- Although the substrate solution is stable for sequently activated at different rates by a over 1 year at - 20°C. repeated freeze varied amount of urokinase in the sample. thawing tend to result in a gradual deThe activated plasminogen (plasmin) thus crease in the P value. degrades FITC-fibrinogen at different rates The FITC-fibrinogen was digested simdepending upon the amount of the gen- ilarly with plasmin. In Fig. 2A, the time erated plasmin. Therefore, the change in the course of fibrinogen digestion by plasminP value in this system reflects proteolysis ogen after activation by I5 IU/ml of caused by plasmin where the activation of urokinase is shown.” Urokinase activity plasminogen by urokinase is the rate-limitalone does not alter the P value, thus ing step. substantiating the substrate specificity. The Urokinase was also assayed using a results obtained for the varied amounts of synthetic chromogenic substrate as de- plasminogen are shown in Fig. 2B in scribed by Iwanaga and co-workers (14,15) which plasminogen was activated with 30 IU utilizing methylcoumarin amides of the of urokinase per test tube. A linear corresponding substrate peptide ( 16), glu- relationship between the decrease in P taryl-Gly-Arg-MCA, based on fluorescence value and the varied amount of plasminointensity. This and fluorescence polarization methods were tested for the quan4 It should be noted that an excess amount of tification of urokinase activity in the urokinase is present in this system. A high increase human urine which was used within 5 h of P value (about 30%) was always observed. This. did not interfere with the assay data. after obtaining the sample. About 50 ~1 of however.
PLASMIN,
PLASMINOGEN.
AND
UROKINASE
ASSAY
19
of plasminogen in the presence of urokinase. (A) Time course study of FIG. 2. Assay activated plasminogen. Solid and open circles indicate the P values of FITC-labeled fibrinogen with both in the presence of urokinase (30 IlJI2.l ml). or without plasminogen. respectively, Plasminogen: 0.744 casein unit/ml. Other conditions are similar to those in Fig. 1. P( 100%) = 0.155. See footnote 4. (B) Quantification of plasminogen after activation by urokinase (30 lUi2. I ml). P values (%) after 1 h incubation with varied amounts of plasminogen are plotted.
gen in log scale was also observed. Thus, the specificity of urokinase is limited to plasminogen only. This was also confirmed in a separate experiment (Fig. 3). The direct urokinase activity on plasminogen was determined, using FITC-plasminogen as the sole substrate of urokinase, from the P value as shown in Fig. 3. In this case 0.05% sodium dodecyl sulfate was added to the cuvette before measurement in order to dissociate unclarified molecular interactions. Without this detergent. the change in P value would be very small and would become progressively smaller above 20 mIUim1. In this system, the low P value reflects the liberated FITC-labeled N-terminal peptide of 8200 out of 87.000 daltons (18). This result indicates that the sensitivity is comparable to the radioisotope method using ‘Ylabeled casein (19). However, reliability is limited to the range 1.5 to 20 mIU/ml of urokinase activity. Above this limit the P values increase gradually, probably due to the molecular association of FITC-plasminogen and its proteolytic products or to an unknown mechanism. Attempts to dissociate the molecular association with O.Ol- 1.0% Triton X-100, and Sarkosyl,
2 M urea, 0.05-l mM ethylenediamine tetraacetate, higher ionic strength (such as 2 M KCI and 2 M LiCl), various buffers (Tris-HCl, borate, acetate, phosphate)
FIG. 3. Direct assay of urokinase. A correlation between the amount of urokinase and the P value of FITC-labeled plasminogen is shown. The labeled plasminogen is subjected to digestion by its activator (urokinase) and the P values after 1 h are plotted. Other conditions are similar to Fig. I. P( lOO”‘c) = 0.058.
20
KINOSHITA.
MAEDA.
PI%I
FIG. 4. Indirect assay of urokinase. Varied amounts of urokinase were added to test tubes containing 46 pg of plasminogen which, after activation, hydrolyzed FITC-labeled fibrinogen. Circles, triangles. and squares represent P values obtained after 1. 2, and 3 h of incubation. Conditions are similar to those in Fig. 1. P(lOO%) values without urokinase were 0.124, 0.118. and 0.113 all at 1, 2, and 3 h, respectively.
at various pHs (6.0, 7.0, 7.5, 8.0, 8.5) and perturbants (such as ethylene glycol) all resulted in failure. However, only sodium dodecyl sulfate had exhibited a limited value. Therefore, despite its extreme sensitivity, the direct method of urokinase assay has a limited applicability. Figure 4 shows the results of the assay
AND
HINUMA
of urokinase in which FITC-fibrinogen was digested by plasmin (22 pug/ml in each tube) which had been activated from the zymogen (plasminogen) by a varying amount of urokinase in the system. A linear relationship between the decrease in the P value and the log of the urokinase activity ranging from about 0.03 to 10 IUiml was obtained. A longer incubation time (3 h) exhibited a higher sensitivity (about fivefold) compared with 1 h incubation. In order to elaborate the specificity of urokinase, we have examined a correlation of urokinase activity determined by this and by the fluorometric method using a synthetic chromogenic substrate which is known to be specific to urokinase (14.15). The results indicate (Fig. 5A) a good correlation between the two methods, where the enzyme concentration was between 0.112 and 7.14 IUlml. Furthermore, results of assay for human urokinase in urine by the two methods show a fairly good correlation (I’ = 0.754) (Fig. 5B). Since the urine samples have undergone no pretreatment to separate other inhibitors and protein in urine, the result can be improved by such processing. When B APtlbl
y = 263 x - 1.18 r = 0.754 Ill- 19)
20
/ /
l
0 01
Relative
fluorescence
intensityt
I
MCA J
0 Rel&ive
l
8 0.05 fluorescence
0. 1 intensitytMCAl
FIG. 5. Correlation between the fluorometric assay using chromogenic substrate (abscissa) and the fluorescence polarization assay (ordinate). (A) Different levels of urokinase were used: 0.112. 0.446, 1.786, and 7.143 IU/ml final. Other results are similar to those in Fig. 4. (Bl Urokinase activity of human urine determined by the polarization or chromogenic substrate methods. Enzyme activity is expressed as AP(‘%) and fluorescence intensity, respectively.
PLASMIN.
PLASMINOGEN.
one wants to measure such activity in actual serum or plasma, the corresponding inhibitors (a,-antitrypsin, etc.) must be eliminated, for instance, by a small column of lysineSepharose which will separate fairly pure plasmin/plasminogen and plasminogen activator from the inhibitors. The usual method for a sensitive assay of these proteases involves radioisotopes which possess inherent drawbacks such as radiohazards and the limited half-life of the substrate, as well as being time consuming in case of radioimmunoassay. The recent development of a synthetic chromogenic substrate for the urokinase assay is becoming an alternative choice, which permits determination of amounts as small as 1 IUlml. Present results for the urokinase assay show that the fluorescence polarization method is advantageous in sensitivity (about IO-fold higher by the indirect and about 600-fold by the direct method than with the chromogenic substrate method with other types of fluorochrome) as well as in the use of natural substrates without isotopes. The present method, however, does not indicate the numbers of bonds hydrolyzed in a given time. Therefore, each assay requires a calibration curve as in the case employing precipitation by trichloroacetic acid. The reproducibility of the method is very satisfactory as compared to the previous report (5). The substrate FITC-fibrinogen. stored frozen at about -20°C and used for more than 1 year, always gives a constant P value of 0.118 at 30°C. However, depending upon the lot of the preparation, the P value varies about 5 to 10%. The reproducibility of the same sample and substrate within a day yielded a standard deviation of no more than ?5%. As shown in Fig. 5B, urine samples containing unknown amounts of urokinase were assayed using the two different methods. Each result correlated well and the present method seems satisfactory (y = 0.754).
AND
UROKINASE
21
ASSAY
Arbitrary minimum amounts of trypsin required for a reliable determination in the previously reported methods are as follows [amount of trypsin (assay method)]: 5 pg (caseinolytic, absorption at 280 nm) (20); 0.005 ,ug (radioactivity, Sepharosebound ““I-labeled casein) (19); 0.05 pg (radioactivity, benzoyl-DL-Arg-p-[“Hlanilide) (17); 0.012 pg (present fluorescence polarization), while those for plasminogen are: 0.03 cu (fibrin plate, clotting): 3.3 pg or 0.13 CTA unit (fluorescence) (21); 0.0004 cu or 0.05 pg (present fluorescence polarization method). The high sensitivity of the present method is partly due to the improved optical system. One of the main advantages is attributed to the elimination of the grating monochrometer; thus quantum input into fluorochrome is larger and upgraded. The fluorogenic substrate method can therefore be improved similarly. Another reason is that the fluorescein chromophore possesses a higher quantum yield than many other fluorochromes, and that it is in the wavelength range outside the spectroscopical interference of proteins. Therefore. the sensitivity of the present assay method of plasminogen and trypsin also appears to be comparable to that of the method utilizing radioisotopes. Thus. the present methods may be of potential value for applications in clinical chemistry. ACKNOWLEDGMENT We thank Professor M. Maeyama tinuous interest and encouragement.
for
his
con-
REFERENCES
?. Dandliker. W. B. (1977) ilz Immunochemistry of Proteins (Atassi, M. Z., ed.). Vol. 1, Chap. 3. pp. 131-269. Plenum. New York. 3. Dandliker, W. B.. Schapiro. H. C.. Meduski. J. W.. Alonso. R.. Feigen. G. A.. and Hamric. J. R.. Jr. (1964) f,,Imrrr~o~h~,misr~~ 1, l65- 191. 4. Harber, H., and Bennett, J. C. (1962) Pro<.. Nor. Ac~rd. SC;. USA 48, 1935-1943. 5. Maeda. H. (1978) Clirl. Chc,m. 24, 7139-2144.
22
KINOSHITA.
MAEDA,
6. Spencer, R. D., Toledo, F. B.. Williams. B. T., and Yoss. N. L. (1973) Clin. Chum. 19, 838-844. 7. Maeda, H. (1979) Anal. Biochem. 92, 222-227. 8. Ossowski. L., Unkeless. J. C.. Tobia, Quigley, J. B., Rifkin, D. B.. and Reich. (1973) J. E.rp. Med. 137, I12- 126.
A.. E.
9. Pollack, R.. Risser. R., Conlon. S., and Rifkin, D. (1974) Proc. Nut. Actrd. Sci. USA 71, 47924796. IO. Hanafusa, H. (1977) in Comprehensive Virology (Frankel-Conrat, H.. and Wagner, R. R., eds.). Vol. 10, pp. 436-458. Plenum. New York. 11. Rifkin, D. B., Loeb, J. N.. Moore, G.. and Reich, E. (1974)J. E.rp. Med. 139. 1317-1328. 12. Nagy. B., .I. Cancrr
Ban, J., and 19, 614-620.
Brder.
B.
(1977)
AND
14.
15. 16. 17. 18.
19. 20.
Inf.
13. Unkeless. J. C., and Gordon. S. (197.5) in Proteases and Biological Control (Reich, E., E.. eds.), Chap. Rifkin, D. B.. and Shaw,
21.
HINUMA
6, pp. 495-5 14. Cold Spring Harbor Laboratory. Cold Spring Harbor, N. Y. Morita. T., Kato, H.. Iwanaga. S., Takada. K.. Kimura. T., and Sakakibara. S. (1977) ./. Birxhem. 82, l495- 1498. Kanaoka. Y.. Takahashi. T., and Nakamura. H. (1977) Cir~m. Pharm. Bull. 25, 362-363. Zimmerman. M.. Yurewicz, E. C., and Patel, G. (1976) Anal. Biochrm. 7ll, 258-262. Roffman. S.. and Troll, W. (1974) Anul. Bioc~hrm. 61, l-5. Walther, P. J., Steinman. H. M.. Hill, R. L.. and McKee, P. A. (1974) ./. Biol. Clam. 249, 1173-1181. Sevier, E. D. (1976) Antrl. Biochrm. 74, 592-596. Robbins. K. C.. and Summaria, L. (1976) it1 Methods in Enzymology (Loland, L., ed.), Vol. 45, Part B. pp. 184- 199, Academic Press, New York. Bell. P. H.. Dziobkowski, C. T., and Englert, M. E. (1974) Anal. Bioc~hrm. 61, 200-208.
BIOCHEMISTRY
Fluorescence
KAZUO
104, 15-22 (1980)
Polarization Assay of Plasmin, Plasminogen Activator KINOSHITA,*
HIROSHI
MAEDA,?,’
Plasminogen,
AND YORIO
and
HINUMA?
Received July 5. 1979 We describe two assay methods for plasminogen activator (urokinase) employing the fluorescence polarization technique. One method utilizes fluoroscein isothiocyanate-labeled plasminogen as the substrate for urokinase (direct method), and the other utilizes the new plasmin generated from plasminogen by urokinase, for which fluoroscein isothiocyanatelabeled fibrinogen was employed as the substrate (indirect method). Also in the assay of trypsin and plasminogen, fluoroscein isothiocyanate-labeled fibrinogen was used as the substrate. Proteolysis by the enzymes (urokinase. plasmin. and trypsin) yielded smaller peptide fragments from the labeled substrates. resulting in a decreased fluorescence polarization value. Urokinase activity determined by the present method correlated well with the data obtained by the known method with a synthetic chromogenic substrate. Enzyme levels as low as 1.5 mIU/ml (direct method) or 0.11 IUiml (indirect method) of urokinase and 0.5 millicasein unit/ml and 0.012 &ml of plasminogen and trypsin. respectively, were quantified within I to 2 h
also been shown useful for the assay of proteolysis by trypsin, papain, pepsin, and Pronase using FITC-labeled substrates (6.7). When many of the fluorochrome-labeled substrates are subjected to digestion by the various proteases, a decrease in the P value of the substrates becomes apparent (7). Their enzyme specificity was confirmed with respective inhibitors (7). Based on this principle, we have extended the method for the assay of plasminogen and plasminogen activator (urokinase) as well as of trypsin using FITC-labeled fibrinogen and plasminogen as the substrates. The plasminogen activator is not only an important clinical parameter but it is increasingly becoming a critical parameter in the process of cell transformation (8- 10) and tumorigenesis ( 11.12). Furthermore, its level in serum manifests an activated state of macrophage function (13). Thus a rapid and sensitive assay of the plasmin activator will facilitate analysis
In order to obtain kinetic and equilibrium information for the protein-protein interaction, the fluorescence polarization method has been shown to be useful (1,2). The fluorescence polarization value (P value)? changes according to the size of the protein molecule which is labeled with fluorochrome due to the difference in the Brownian motion. The fluorescence polarization method has been applied to many of the antigenantibody reactions (3-5). Quantification of antigens or antibodies becomes possible by measuring the increase in the P value of the complex being formed. Recently. the fluorescence polarization method has ’ To whom requests for reprints should be addressed. ” Abbreviations used: P value, fluorescence polarization value in arbitrary units; FITC-, fluorescein isothiocyanate-labeled; cu. casein unit; MCA, methylcoumarin amide: TPCK, tosylphenylalanine chloromethyl ketone: CTA unit, unit defined by the Committee on Thrombolytic Agents: PBS. phosphatebuffered solution, pH 7.0. IS
0003-2697!80/0700 IS-08$0?.00/0 Cupynght All r,ghf*
< 1980 by Acxkmx F’re\\. Inc. of reproductm ,n any form rewrved
16
KINOSHITA.MAEDA.ANDHINUMA
of the host’s response. MATERIALS
defense
mechanism
and
AND METHODS
Itzstruments. The fluorescence spectropolarimeter. Model MAC-2 Type HR-I (Japan Immunoindustries Co. Ltd., Takasaki) was employed for the measurement of fluorescence polarization, The instrument is equipped with a primary polarizer, an excitation filter for 490 nm, a cell housing, an analyzer-polarizer that is driven by a synchronous motor at 1800 rpm, an emission filter for 520 nm, and a photomultiplier with an amplifier system. Neither the background of protein fluorescence, which is primarily due to aromatic amino acid residues and is located in the near-uv region, nor the light scattering of excitation light by protein will affect the measurement because of the efficient cutoff filter at 520 nm (Ditric Optics Inc., 247R Maple St., Marlboro, Mass.). The instrument can detect a fluorescein concentration of 1.5 pmol/ml at or above pH 7.0. A built-in microcomputer is also furnished in the which calculates P values instrument, before printout every 82 s. Each P value printed out is an integral average of 50 measurements in 50 s. All P values obtained in the present experiments are a multiple of about 2.0 x 1O:j of the real value and are expressed as changes in the percentage of the P value. The temperature of the cuvette was kept constant (within *O.I”C of error) during the operation with a “Ucool” circulation unit (Neslab Inst. Inc., Portsmouth, N. H.). Chemids. Substrates and enzymes used were obtained from the following sources: plasminogen-free bovine fibrinogen and bovine plasminogen (specific activity 9.2 cu) were from Daiichi Pure Chemicals Company Ltd. (Tokyo); human plasminogen from AB KABI (Stockholm; 15 cu/mg protein) and Worthington Biochemical Corporation (Freehold, N. J.: 174 Uimg):
TPCK-treated trypsin ( 183. I U/mg) from Worthington Biochemical Corporation: human urokinase, free of plasmin, was a generous gift from Mochida Pharmaceutical Company (Tokyo). FITC (isomer I) with a purity of more than 90% was obtained from Dojin Chemical Company (Kumamote). Phenylmethylsulfonyl fluoridine, an inhibitor of serine protease, was from Calbiochem (San Diego, Calif.). GlutarylGly-Arg-MCA was from Peptide Institute Inc. (Osaka). Urine specimens were obtained from the clinical department. All other chemicals were obtained from commercial sources. FITC Iuhrling. The labeling of bovine fibrinogen was carried out as follows. A 25mg sample of plasminogen-free bovine fibrinogen and 2.5 mg of FITC were dissolved in 5 ml of 0.1 M borate buffer, pH 8.0. After reacting for 3 h at 37°C in the darkness.‘j the reaction mixture was applied to a column (1.5 x 52 cm) of Sephadex G-50 which had been equilibrated with 0.1 M borate buffer, pH 8.0. The FITC-fibrinogen which came out first was collected and then dialyzed in the cold (4-8°C) against 0.01 M phosphate-buffered 0.15 M NaCl solution (PBS), pH 7.0, containing I mM ethylenediamine tetraacetate (EDTA) for about 3 days with a few changes of the dialysis buffer each day. By this procedure a complete removal of nonspecifically bound or unbound fluorochrome was ascertained based on the P value and the fluorescence intensity of the dialysis buffer. The solution of FITCfibrinogen was stored at -20°C as a stock solution. The substrate solutions for the assay were prepared by diluting the stock solution 50-fold with 0.1 M Tris-HCI buffer. pH 8.5. A 1.5-ml aliquot of this substrate solution was stored at -20°C until use. Both stock and substrate solutions of FITC,’ We jugates fluorescent can
have observed that exposure of the conto intensive light liberates an unidentified low molecular weight compound. which
be removed
by dialyG\.
PLASMIN,
PLASMINOGEN.
AND TABLE
SYSTEMS
System
USED
FOR.~HE
Enzyme to be assayed (sample = 50 ~1)
ASSAY
OF TRYPSIN,
Enzyme hydrolyzing FITCsubstrate
FITCsubstrate
Trypsin Plasminogen
Trypsin Plasmin
Fibrinogen Fibrinogen
3 4
Urokinase Urokinase
Urokinase Plasmin
Plasminogen Fibrinogen
‘I The reaction an approximate
mixture contains concentration
PLASMINOGEN,
4~~2
Activator in SO &I
UROKINASE”
Proenzyme in 50 ~1
Assay Direct Indirect
Urokinase (30 IU)
I .9 ml of a 0. I M phosphate buffer solution, of lo-50 pmol/ml of fluorescein equivalent
fibrinogen were stable over 1 year at -20°C based on P value. (A decrease in P value indicates autodigestion or degradation.) Labeling of human plasminogen (AB KABI) was carried out as follows. About 1 mg of plasminogen and 0.1 mg of phenylmethylsulfonyl fluoridine were dissolved in I ml of PBS. pH 7.6, and allowed to react for about 90 min in a reciprocal shaker (2 Hz) at room temperature. Subsequently, FITC (1.25 mg), dissolved in 0.5 ml of 0.1 M borate buffer, pH 8.0. was added to the mixture. These were reacted and purified in a manner similar to that described above for fibrinogen. The FITC-plasminogen was collected and then stored at -20°C until use. The FITC-fibrinogen and FITC-plasminogen had 17.2 and 2.2 mol of fluorochromes per mole of protein (F/P), respectively. F/P was determined spectroscopically. The molecular extinction coefficient of fluorescein at 490 nm was 6.76 x 10” at pH 8.0. For human fibrinogen and plasminogen, the molecular extinction coefficients used were 5.88 x 10” and 1.49 x 10” both at 280 nm, respectively. Enzynzr reactions. A test tube containing I .9 ml of 0.1 M phosphate buffer, pH 7.5, was placed in the U-cool circulator at 3o”C, after which FITC-substrate (100 or 50 ~1
17
ASSAY
I
I 2
have
UROKlNASE
Plasminogen (46 pg) pH 7.5. The labeled in each system.
Direct Indirect
substrates
of the appropriate substrate) was added in all assays. The enzyme reactions were then initiated by an addition of a varied amount of each enzyme, and, if required, a definite amount of the plasminogen or plasminogen activator (urokinase). In Table 1, the reagents utilized in four different assay systems are described. After incubation for a definite time period at 30°C in the U-cool circulator, the reaction mixture was placed in a cuvette and the P value was measured at 30°C. The concentration of the FITClabeled substrate in the cuvette was designed to yield a significant signal at a cathode voltage between 500 and 650 V in the photomultiplier which corresponds to 5- 10 pmoliml fluorescein. System 1 is designed for the assay of trypsin which was dissolved in 0.01 M HCI with 2 mM Ca?+. System 2 is designed to assay the amount of plasminogen in which a definite amount (about 15 W/ml) of urokinase is added to activate a varied amount of plasminogen to plasmin. A stock solution of plasminogen was dissolved in 0.25 M phosphate buffer, pH 7.5, containing 0.05 M acetate. As for the assay of urokinase (plasminogen activator), two types of assay systems are described. One is the direct method (system 3) using FITC-plasminogen as the substrate of urokinase which specifically hydrolyzes
18
KINOSHITA.
MAEDA.
AND
HINUMA
urine is added to the above-described action mixture in a cuvette for assay. RESULTS
re-
AND DISCUSSION
Enzyme activities were expressed as a percentage of the P value after reaction to that of the blank without the enzyme. The real P value corresponding to a P I ‘\ value of 100% is shown in the legends. ‘\ hi. Figure 1 was obtained for the varied amount 60 1 '\ ot t of trypsin added to the FITC-fibrinogen and 0 001 01 / 100 Concentrdion of ttypsiZgg/ml) digested in I h. This indicates a linear relationship between the decrease in P value FIG. 1. Relationship between the concentration of and the log of the amount of trypsin trypsin and the change in the fluorescence polarizain 0.012-100 &ml. This result indicates tion (P) value. The FITC-labeled fibrinogen, about 50 pmoliml fluorescein equivalent in 0.1 M phosthat it is more sensitive than that of the was digested by varied phate buffer, pH 7.5. radiolabeled synthetic substrate, benzoylamounts of trypsin for a period of I h and the P DL-arginine I:‘H]anilide which has a devalues obtained for each trypsin concentration are tection limit of 0.05 pg/ml ( 17). This tryptic plotted. Temperature: 30°C. P( lOO%c) = 0.118. activity was inhibited by various trypsin inhibitors (data not shown) (7). It should FITC-plasminogen; the change in the P be noted that a commercial preparation of value reflects the proteolytic cleavage of fibrinogen from different sources exhibited plasminogen by urokinase directly. The autolytic degradation as revealed by the P other is the indirect method in which a value. Treatment with diisopropyl fluorodefinite amount (22 E.cg/ml) of plasminogen phosphate was advantageous in such cases. is added to each test tube which is sub- Although the substrate solution is stable for sequently activated at different rates by a over 1 year at - 20°C. repeated freeze varied amount of urokinase in the sample. thawing tend to result in a gradual deThe activated plasminogen (plasmin) thus crease in the P value. degrades FITC-fibrinogen at different rates The FITC-fibrinogen was digested simdepending upon the amount of the gen- ilarly with plasmin. In Fig. 2A, the time erated plasmin. Therefore, the change in the course of fibrinogen digestion by plasminP value in this system reflects proteolysis ogen after activation by I5 IU/ml of caused by plasmin where the activation of urokinase is shown.” Urokinase activity plasminogen by urokinase is the rate-limitalone does not alter the P value, thus ing step. substantiating the substrate specificity. The Urokinase was also assayed using a results obtained for the varied amounts of synthetic chromogenic substrate as de- plasminogen are shown in Fig. 2B in scribed by Iwanaga and co-workers (14,15) which plasminogen was activated with 30 IU utilizing methylcoumarin amides of the of urokinase per test tube. A linear corresponding substrate peptide ( 16), glu- relationship between the decrease in P taryl-Gly-Arg-MCA, based on fluorescence value and the varied amount of plasminointensity. This and fluorescence polarization methods were tested for the quan4 It should be noted that an excess amount of tification of urokinase activity in the urokinase is present in this system. A high increase human urine which was used within 5 h of P value (about 30%) was always observed. This. did not interfere with the assay data. after obtaining the sample. About 50 ~1 of however.
PLASMIN,
PLASMINOGEN.
AND
UROKINASE
ASSAY
19
of plasminogen in the presence of urokinase. (A) Time course study of FIG. 2. Assay activated plasminogen. Solid and open circles indicate the P values of FITC-labeled fibrinogen with both in the presence of urokinase (30 IlJI2.l ml). or without plasminogen. respectively, Plasminogen: 0.744 casein unit/ml. Other conditions are similar to those in Fig. 1. P( 100%) = 0.155. See footnote 4. (B) Quantification of plasminogen after activation by urokinase (30 lUi2. I ml). P values (%) after 1 h incubation with varied amounts of plasminogen are plotted.
gen in log scale was also observed. Thus, the specificity of urokinase is limited to plasminogen only. This was also confirmed in a separate experiment (Fig. 3). The direct urokinase activity on plasminogen was determined, using FITC-plasminogen as the sole substrate of urokinase, from the P value as shown in Fig. 3. In this case 0.05% sodium dodecyl sulfate was added to the cuvette before measurement in order to dissociate unclarified molecular interactions. Without this detergent. the change in P value would be very small and would become progressively smaller above 20 mIUim1. In this system, the low P value reflects the liberated FITC-labeled N-terminal peptide of 8200 out of 87.000 daltons (18). This result indicates that the sensitivity is comparable to the radioisotope method using ‘Ylabeled casein (19). However, reliability is limited to the range 1.5 to 20 mIU/ml of urokinase activity. Above this limit the P values increase gradually, probably due to the molecular association of FITC-plasminogen and its proteolytic products or to an unknown mechanism. Attempts to dissociate the molecular association with O.Ol- 1.0% Triton X-100, and Sarkosyl,
2 M urea, 0.05-l mM ethylenediamine tetraacetate, higher ionic strength (such as 2 M KCI and 2 M LiCl), various buffers (Tris-HCl, borate, acetate, phosphate)
FIG. 3. Direct assay of urokinase. A correlation between the amount of urokinase and the P value of FITC-labeled plasminogen is shown. The labeled plasminogen is subjected to digestion by its activator (urokinase) and the P values after 1 h are plotted. Other conditions are similar to Fig. I. P( lOO”‘c) = 0.058.
20
KINOSHITA.
MAEDA.
PI%I
FIG. 4. Indirect assay of urokinase. Varied amounts of urokinase were added to test tubes containing 46 pg of plasminogen which, after activation, hydrolyzed FITC-labeled fibrinogen. Circles, triangles. and squares represent P values obtained after 1. 2, and 3 h of incubation. Conditions are similar to those in Fig. 1. P(lOO%) values without urokinase were 0.124, 0.118. and 0.113 all at 1, 2, and 3 h, respectively.
at various pHs (6.0, 7.0, 7.5, 8.0, 8.5) and perturbants (such as ethylene glycol) all resulted in failure. However, only sodium dodecyl sulfate had exhibited a limited value. Therefore, despite its extreme sensitivity, the direct method of urokinase assay has a limited applicability. Figure 4 shows the results of the assay
AND
HINUMA
of urokinase in which FITC-fibrinogen was digested by plasmin (22 pug/ml in each tube) which had been activated from the zymogen (plasminogen) by a varying amount of urokinase in the system. A linear relationship between the decrease in the P value and the log of the urokinase activity ranging from about 0.03 to 10 IUiml was obtained. A longer incubation time (3 h) exhibited a higher sensitivity (about fivefold) compared with 1 h incubation. In order to elaborate the specificity of urokinase, we have examined a correlation of urokinase activity determined by this and by the fluorometric method using a synthetic chromogenic substrate which is known to be specific to urokinase (14.15). The results indicate (Fig. 5A) a good correlation between the two methods, where the enzyme concentration was between 0.112 and 7.14 IUlml. Furthermore, results of assay for human urokinase in urine by the two methods show a fairly good correlation (I’ = 0.754) (Fig. 5B). Since the urine samples have undergone no pretreatment to separate other inhibitors and protein in urine, the result can be improved by such processing. When B APtlbl
y = 263 x - 1.18 r = 0.754 Ill- 19)
20
/ /
l
0 01
Relative
fluorescence
intensityt
I
MCA J
0 Rel&ive
l
8 0.05 fluorescence
0. 1 intensitytMCAl
FIG. 5. Correlation between the fluorometric assay using chromogenic substrate (abscissa) and the fluorescence polarization assay (ordinate). (A) Different levels of urokinase were used: 0.112. 0.446, 1.786, and 7.143 IU/ml final. Other results are similar to those in Fig. 4. (Bl Urokinase activity of human urine determined by the polarization or chromogenic substrate methods. Enzyme activity is expressed as AP(‘%) and fluorescence intensity, respectively.
PLASMIN.
PLASMINOGEN.
one wants to measure such activity in actual serum or plasma, the corresponding inhibitors (a,-antitrypsin, etc.) must be eliminated, for instance, by a small column of lysineSepharose which will separate fairly pure plasmin/plasminogen and plasminogen activator from the inhibitors. The usual method for a sensitive assay of these proteases involves radioisotopes which possess inherent drawbacks such as radiohazards and the limited half-life of the substrate, as well as being time consuming in case of radioimmunoassay. The recent development of a synthetic chromogenic substrate for the urokinase assay is becoming an alternative choice, which permits determination of amounts as small as 1 IUlml. Present results for the urokinase assay show that the fluorescence polarization method is advantageous in sensitivity (about IO-fold higher by the indirect and about 600-fold by the direct method than with the chromogenic substrate method with other types of fluorochrome) as well as in the use of natural substrates without isotopes. The present method, however, does not indicate the numbers of bonds hydrolyzed in a given time. Therefore, each assay requires a calibration curve as in the case employing precipitation by trichloroacetic acid. The reproducibility of the method is very satisfactory as compared to the previous report (5). The substrate FITC-fibrinogen. stored frozen at about -20°C and used for more than 1 year, always gives a constant P value of 0.118 at 30°C. However, depending upon the lot of the preparation, the P value varies about 5 to 10%. The reproducibility of the same sample and substrate within a day yielded a standard deviation of no more than ?5%. As shown in Fig. 5B, urine samples containing unknown amounts of urokinase were assayed using the two different methods. Each result correlated well and the present method seems satisfactory (y = 0.754).
AND
UROKINASE
21
ASSAY
Arbitrary minimum amounts of trypsin required for a reliable determination in the previously reported methods are as follows [amount of trypsin (assay method)]: 5 pg (caseinolytic, absorption at 280 nm) (20); 0.005 ,ug (radioactivity, Sepharosebound ““I-labeled casein) (19); 0.05 pg (radioactivity, benzoyl-DL-Arg-p-[“Hlanilide) (17); 0.012 pg (present fluorescence polarization), while those for plasminogen are: 0.03 cu (fibrin plate, clotting): 3.3 pg or 0.13 CTA unit (fluorescence) (21); 0.0004 cu or 0.05 pg (present fluorescence polarization method). The high sensitivity of the present method is partly due to the improved optical system. One of the main advantages is attributed to the elimination of the grating monochrometer; thus quantum input into fluorochrome is larger and upgraded. The fluorogenic substrate method can therefore be improved similarly. Another reason is that the fluorescein chromophore possesses a higher quantum yield than many other fluorochromes, and that it is in the wavelength range outside the spectroscopical interference of proteins. Therefore. the sensitivity of the present assay method of plasminogen and trypsin also appears to be comparable to that of the method utilizing radioisotopes. Thus. the present methods may be of potential value for applications in clinical chemistry. ACKNOWLEDGMENT We thank Professor M. Maeyama tinuous interest and encouragement.
for
his
con-
REFERENCES
?. Dandliker. W. B. (1977) ilz Immunochemistry of Proteins (Atassi, M. Z., ed.). Vol. 1, Chap. 3. pp. 131-269. Plenum. New York. 3. Dandliker, W. B.. Schapiro. H. C.. Meduski. J. W.. Alonso. R.. Feigen. G. A.. and Hamric. J. R.. Jr. (1964) f,,Imrrr~o~h~,misr~~ 1, l65- 191. 4. Harber, H., and Bennett, J. C. (1962) Pro<.. Nor. Ac~rd. SC;. USA 48, 1935-1943. 5. Maeda. H. (1978) Clirl. Chc,m. 24, 7139-2144.
22
KINOSHITA.
MAEDA,
6. Spencer, R. D., Toledo, F. B.. Williams. B. T., and Yoss. N. L. (1973) Clin. Chum. 19, 838-844. 7. Maeda, H. (1979) Anal. Biochem. 92, 222-227. 8. Ossowski. L., Unkeless. J. C.. Tobia, Quigley, J. B., Rifkin, D. B.. and Reich. (1973) J. E.rp. Med. 137, I12- 126.
A.. E.
9. Pollack, R.. Risser. R., Conlon. S., and Rifkin, D. (1974) Proc. Nut. Actrd. Sci. USA 71, 47924796. IO. Hanafusa, H. (1977) in Comprehensive Virology (Frankel-Conrat, H.. and Wagner, R. R., eds.). Vol. 10, pp. 436-458. Plenum. New York. 11. Rifkin, D. B., Loeb, J. N.. Moore, G.. and Reich, E. (1974)J. E.rp. Med. 139. 1317-1328. 12. Nagy. B., .I. Cancrr
Ban, J., and 19, 614-620.
Brder.
B.
(1977)
AND
14.
15. 16. 17. 18.
19. 20.
Inf.
13. Unkeless. J. C., and Gordon. S. (197.5) in Proteases and Biological Control (Reich, E., E.. eds.), Chap. Rifkin, D. B.. and Shaw,
21.
HINUMA
6, pp. 495-5 14. Cold Spring Harbor Laboratory. Cold Spring Harbor, N. Y. Morita. T., Kato, H.. Iwanaga. S., Takada. K.. Kimura. T., and Sakakibara. S. (1977) ./. Birxhem. 82, l495- 1498. Kanaoka. Y.. Takahashi. T., and Nakamura. H. (1977) Cir~m. Pharm. Bull. 25, 362-363. Zimmerman. M.. Yurewicz, E. C., and Patel, G. (1976) Anal. Biochrm. 7ll, 258-262. Roffman. S.. and Troll, W. (1974) Anul. Bioc~hrm. 61, l-5. Walther, P. J., Steinman. H. M.. Hill, R. L.. and McKee, P. A. (1974) ./. Biol. Clam. 249, 1173-1181. Sevier, E. D. (1976) Antrl. Biochrm. 74, 592-596. Robbins. K. C.. and Summaria, L. (1976) it1 Methods in Enzymology (Loland, L., ed.), Vol. 45, Part B. pp. 184- 199, Academic Press, New York. Bell. P. H.. Dziobkowski, C. T., and Englert, M. E. (1974) Anal. Bioc~hrm. 61, 200-208.