43-50 (1979)

A New and Highly Sensitive Fluorescence Assay for Collagenase-Like Peptidase Activity KOHICHI KOJIMA, HIDEKO KINOSHITA, TAKESHI KATO, TOSHIHARU KATSUMITAKADA,* ANDSHUMPEISAKAKIB.~RA* Laboratory


of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227, Japan, and *Peptide Institute, Protein Research Foundation, Minoh, Osaka 562, Japan

Received February 28, 1979 A highly sensitive fluorescence assay for collagenase-like peptidase (CL-peptidase) has been developed using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)4-methylcoumaryl-7-amide (Sue-GPLGP-MCA). Sue-GPLGP-MCA was hydrolyzed at the Leu-Gly bond by CL-peptidase, (Gly-Pro)-4-methylcoumaryl-7-amide liberated by the enzyme was immediately hydrolyzed to Gly-Pro and 7-amino-4-methylcoumarin (AMC) by an excess of an auxiliary enzyme, X-prolyl dipeptidyl-aminopeptidase, and the fluore’scence intensity of the AMC was measured at 460 nm with excitation at 380 nm. When assayed by this method, CL-peptidase partially purified from chick embryo showed a pH optimum at 8.0 and a K, value of 4.0 x 10e4 M toward Sue-GPLGP-MCA. Under the optimum condition, the reaction proceeded linearly up to 4 h. The CL-peptidase activity was found in normal human sera by this method and the mean and standard deviation of the activity was 0.59 r 0.10 nmotimin/ml of serum (n = 10). This assay was also applicable for the CL-peptidase in human liver and kidney. The results suggest that the CL-peptidase assayed by this new substrate may be different from the “PZ-peptidase” which cleaves a synthetic substrate for collagenase-like peptidase, 4-phenylazobenzyloxycarbonyl (PZ)Pro-Leu-Gly-Pro-D-Arg (PZ-peptide). The new peptide, Sue-GPLGP-MCA, was found not to be a substrate for specific collagenase from tadpole.

4 - Phenylazobenzyloxycarbonyl (PZ) - L Pro-L-Leu-Gly-~-Pro-D-At-g, commonly abbreviated as PZ-peptide ,l has been widely used in searching for animal proteases with collagenase-like specificity (1-13).2 This substrate has the advantage that the chromogenic product, PZ-Pro-Leu, is not water soluble and can be assayed after extraction

into organic solvent. However, the method using PZ-peptide as substrate has also a few disadvantages. First, it is not highly sensitive because of the calorimetry. Second, as the substrate also has the same absorption spectrum as PZ-Pro-Leu liberated enzymatically, the product has to be separated from the substrate by extraction, and the procedure is time consuming. Many fluorogenic substrates have been used for highly sensitive enzyme assays. For example, Reinharz and Roth (14) reported that, when the octapeptide substrate, benzyloxycarbonyl-Pro - Phe - His - Leu Leu-Val-Tyr-Ser-/3-naphthylamide, is incubated with renin and an excess of an auxiliary enzyme, aminopeptidase M, the fluorescent P-naphthylamide is liberated at

’ Abbreviations used: PZ, 4-phenylazobenzyloxycarbonyl; PZ-peptide, PZ-t.-Pro-r-Leu-Gly-L-Pro-D-Arg; X-Pro DAP, X-prolyl dipeptidyl-aminopeptidase; GlyPro-MCA, Gly-L-Pro-4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; Sue-GPLGP-MCA, succinyl-Gly-L-Pro-L-Leu-Gly-L -Pro) -4 -methylcoumaryl-7-amide; CL-peptidase, collagenase-like peptidase; BOC, butoxycarbonyl; DMF, dimethylformamide; DCC, dicyclohexylcarbodiimide. * The enzyme hydrolyzing PZ-peptide is designated PZ-peptidase or collagenase-like peptidase. 43

0003-2697179/170043-08$02.00/0 Copyright All rights

0 1979 by Academic Press. Inc. of reproduction in any form reserved.




NHzM,;, ab




step of the procedure of Morales and Woessner (lo), dialyzed against 0.5 mM Tris-HCl buffer, pH 7.0, and stored at -80°C. The concentration was 519 &ml. X-Pro DAP of human submaxillary gland (18) was purified homogeneously according to the method described elsewhere (K. Kojima, T. Hama, T. Kato, and T. Nagatsu, manuscript in preparation), dialyzed against 2 mM acetate buffer, pH 6.0, and stored at -80°C. The specific activity toward Gly-Pro-p-nitroanilide (16) was 57.4 ~moYmin/mg protein, and the concentration was 9.84 &ml. Pure collagenase from tadpole (19) was kindly supplied by Dr. Yutaka Nagai (Tokyo Medical and Dental University, Tokyo); the enzyme solution (24 pg/ml) contained 0.05 M Tris-HCI (pH 7.6)/l M NaClI5 mM CaClz/ 0.02% sodium azide, and was stored at 0°C. The specific activity against collagen was 8.42 mg/min/mg protein. Human blood samples were collected by venipuncture, and after clotting, the serum was separated by cent~fuging at 3000 rpm for 15 min. Human submaxillary gland, liver, and kidney were obtained at autopsy and homogenized in water. Synthesis u~~uc-GPLGP-~CA. This material was synthesized following a scheme in Fig. 1. Compound II was obtained by coupling Boc-Pro with 7-amino-4-methylcoumarin using the mixed anhydride method following a published procedure (20-22); mp 215°C (decomp.); [a];” -68.3” (c 2, DMF). Anal. Calcd for C&,H,,N,O,:C, 64.50; H, 6.50; N, 7.52%. Found: C, 64.55; H, 6.50; N, 7.39%. Compound I (22) was converted to the Nhydroxysuccinimide ester using DCC as the reagent, and the product was further coupled with compound II in a mixture of tetrahydrofuran and DMF (2:l v/v) after removal of the Boc group using p-toluenesulfonic acid as the reagent. The product was dissolved in AcOEt:MeOH (3:l v/v), and the solution was washed with 5% NaHCO,, water, 1 M HCl, and water, successively;






Z-Gly-Pro-Leu-Gly-Pro-MCA 4 Hz /Pd 4 Succlnic

Ulll Anhydride

SW-Gly-Pro-Leu-Gly-Pro-MCI\ (SW-GPLGP-MCA)

FIG. 1. Scheme for the synthesis of (succinyl-GlyPro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (SucGPLGP-MCA). M.A., mixed anhydride method; DCC: dicyclohexylcarbodiimide; HONSu: N-hydroxysuccin~ide; Boc, butoxyc~~nyl.

a rate related to renin activity. We have recently developed a highly sensitive fluorescence assay for the activity of X-prolyl dipeptidyl-aminopeptidase (X-Pro DAP) (15, 16) with (Gly-Pro)-Cmethylcoumaryl-7amide (Gly-Pro-MCA) as substrate (17). X-Pro DAP specifically hydrolyses Gly - ProMCA to Gly-Pro and 7-amino-4-methylcoumarin (AMC). By using a newly synthesized substrate, (succinyl-Gly-L-Pro-L-LeuGly-L-Pro)-4-methylcoumaryl-7-amide (SucGPLGP-M~A), and the X-Pro DAP as an auxiliary enzyme, we have now developed a highly sensitive assay method for collagenase-like peptidase (CL-peptidase) activity. In the present paper, we provide the new assay method for the CL-peptidase, and also describe the presence and measurement of a CL-peptidase activity in human serum from normal subjects and in human liver and kidney. MATERIALS


General materials. SUC-GPLGP-MCA was newly synthesized in our laboratory (Peptide Institute, Protein Research Foundation) according to the method described in the next section. PZ-Peptide was purchased from Fluka (Switzerland). CL-Peptidase was purified from chick embryo up to the DEAE-cellulose column



the washed solution was dried and concentrated, The residue was dissolved in CHCI,:MeOH (4: 1 v/v), and reprecipitated with ether for purification; yield of product III was 79%; mp 150-170°C (sintered at 120°C); [cr]k5 -140.4” (c 0.5, DMF). The carbobenzoxy group of compound III was removed by catalytic hydrogenolysis in methanol in the presence of an equimolar amount of 0.4 M hydrochloric acid, The deprotected product was treated with 1.5 equivalents of succinic anhydride in methanol in the presence of an equivalent of t~ethylamine, and the succinated product was purified on a column of silica gel using CHCl,:MeOH: AcOH (95:5:3 v/v/v) as eluent. The final product was obtained in a 38% yield as a colorless amorphous powder after precipitation with ether; mp 170-190°C (sintered at 90°C); [a];” - 114” (c 0.86, DMF). Homogeneity of this material was confirmed by silica-gel thin-layer chromatography using the lower phase of a mixture, CHCI,: MeOH:AcOH:H*O (1O:lO:l:lO v/v/v/v), as solvent; Rf 0.59. Anal. Calcd for C34H44N6010+4H20; C, 53.11; H, 6.82: N, 10.93%. Found: C, 53.33; H, 6.70; N, 10.62%. This material was fairly soluble in water, methanol, or acetic acid, and less soluble in ordinary organic solvents. General methods. PZ-Peptidase activity was measured by a modification (9) of the method of Wtinsch and Heidrich (23). Protein was determined by the method of Lowry et al. (24) using bovine serum albumin as standard. Assay of CL-peptidase using Suc-GPLGPMCA as substrate. The principle of the assay for CL-peptidase activity using SucGPLGP-MCA as substrate is based on the fluorometric measurement of AMC liberated from the reaction product Gly-ProMCA by the second enzyme reaction with X-Pro DAP (Fig. 2). The standard incubation mixture (totai volume, 200 ~1) contained 50 ~1 of 0.2 M Tris-maleate buffer (pH 8.0) with 2.4 mM


CL-Peptidase I

Sue-Gly-ho-Leu-Gly-Pro-MCA Sue-Gly-Pro-Leu Gly-Pro-MCA





+ Gly-Pro-MCA Gly-Pro


FIG. 2. Principle of the fluorometric assay of collagenase-like peptidase with (succinyl-Gly-ProLeu-Gly-Pro)-4-methylcoumaryl-7-amide as substrate. SUC, succinyl; X-Pro DAP, X-prolyl dipeptidyl-aminopeptidase; MCA: 4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin.

Sue-GPLGP-M~A and 20 mM CaC&, 50 ~1 of X-Pro DAP (0.492 pg), and 100 ~1 of enzyme plus water. The blank and standard tubes contained water and 500 pmoi of AMC, instead of enzyme, respectively. The control tube without enzyme was run with each sample, All the tubes were incubated at 37°C for 1 h, and the reaction was stopped by adding 1.0 ml of 1 M sodium acetate buffer, pH 4.2. The same amount of enzyme was added to the control tubes after stopping the reaction. The fluorescence intensity of experimental (E), control (C), standard (S), and blank (B) was read at 460 nm with excitation at 380 nm, using a Shimadzu RF-500 spectrofluorophotometer. AMC liberated by the enzyme reaction was calculated as follows: E-C S-B

1 60 min

X 500 pm01 X ~

= 50(E - ‘) pmol/min,

6(S - B) It was also possible to assay the enzyme in two steps. At first, only the CL-peptidase reaction was done. Then after boiling the mixture for 5 min and briefly centrifuging, X-Pro DAP was added to the supernatant and incubated again for 30 min at 37”C, and the liberated AMC was assayed. Analysis of hydrolysates of substrate by CL-peptidase in the presence and absence of X-Pro DAP, The hydrolysis of SucGPLGP-MCA by CL-peptidase was carried out without or with X-Pro DAP. After the




400 450 WAVELENGTH hm)


FIG. 3. Fluorescence spectrum of the incubation mixture for collagenase-like peptidase in chick embryo using (succinyl-Gly-Pro-Leu-Gly-Pro)-Qmethylcoumaryl-7-amide as substrate: control, no-enzyme incubation (1); experiment, collagenase-like peptidase partially purified from chick embryo (2). The standard incubation mixture was used. Incubation was carried out at 37°C for 1 h.

hydrolysis for 10 to 240 min at 37”C, the reaction was stopped by heating at 95°C for 5 min. This mixture was applied on a plastic sheet of silica gel 60 for thin-layer chromatography (precoated, Merck, Darmstadt) and subjected to ascending chromatography in n-ButOH:AcOH:H,O (2: 1: 1, v/v/v) as solvent. After drying, the sheet was stained with ninhydrin and the spots were analyzed both fluorometrically and colori-


metrically on a Shimadzu CS-910 dual-wavelength thin-layer chromatography scanner. Collagenuse assay. Sue-GPLGP-MCA was examined as a substrate for tadpole collagenase, as described by Hori and Nagai (19). The incubation mixture contained (total volume 200 ~1): 0.05 M TrisHCl buffer (pH 7.6), 0.15 M NaCI, 5 mM CaCl,, 0.02% bovine serum albumin, 0.6 mM Sue-GPLGP-MCA, X-Pro DAP (0.492 pg), and tadpole collagenase (2.4 pg). Incubation was carried out at 37°C for 2 or 16 h. The hydrolysis of the peptide substrate was examined both by the fluorometry of AMC and by thin-layer chromatography. RESULTS Assay of CL-peptidase activity using SucGPLGP-MCA. The standard assay proce-

dure was established mainly using SucGPLGP-MCA as substrate and a CL-peptidase partially purified from chick embryo and X-Pro DAP purified from human submaxillary gland. The substrate and the product, AMC, were highly fluorescent; the excitation/emission maxima were at 325 nm/385 nm and 345 run/445 nm, respectively. However, when the excitation and emission wavelengths of the spectro-

I 0TIME2hr)

FIG. 4. Time curve of collagenase-like peptidase activity using (succinyl-Gly-Pro-Leu-Gly-Pro)4 methylcoumaryl-7-amide as substrate. The standard incubation mixture was used with collagenase-like peptidase partially purified from chick embryo.

I 05



I 5

II 50

FIG. 5. Collagenase-like peptidase activity as a function of enzyme using (succinyl-Gly-Pro-LeuGly-Pro)4methylcoumaryl-7-amide as substrate. The standard incubation mixture was used with collagenase-like peptidase partially purified from chick embryo. Incubation was done at 37°C for 1 h.


1 7


, 8

I 9


FIG. 6. pH-Activity curve of hydrolysis of (succinyl-Gly-Pro-Leu-Gly-Pro)-6methylcoumaryMamide by collagenase-like peptidase partially purified from chick embryo. All incubations were done in the presence of 0.6 mM (succinyl-Gly-Pro-Leu-GlyPro)-4-methylcoumaryl-7-amide, 5 mM CaCl,, and 50 mM Tris-maleate buffer at the indicated pH values at 37°C for 1 h.

fluorophotometer were set at 380 and 460 nm, respectively, AMC was selectively measured, since the fluorescence of the fluorogenic substrate became very low. The fluorescence spectra of the experiment and control with the DEAE fraction of chick embryo enzyme is shown in Fig. 3. The enzyme reaction was found to be linearly related to time at 37°C for about 240 min (Fig. 4), and to the amount of enzyme from 0.2 to 20 pg (Fig. 5), using the partially purified chick embryo enzyme. Properties of CL-peptidase using SucGPLGP-~CA. Using Sue-GPLGP-MCA as substrate, CL-peptidase was optimally active at pH 8.0 in 50 mM Tris-maleate buffer at 37°C (Fig. 6). The K, of the enzyme toward Sue-GPLGP-MCA was 4.0 x 10V4 M at pH 8.0 in Tris-maleate buffer (Fig. 7). Analysis of the hydrolysates of SucGPLGP-~~A by CL-peptidase from chick embryo. Figure 8 shows the thin-layer chromatogram of the hydrolysates liberated from Sue-GPLGP-MCA by CL-peptidase with X-Pro DAP. The spots corresponding to AMC were quantitatively detected on a thin-layer chromatography scanner by fluorometric analysis, and the spots of Gly-Pro




by calorimetric analysis. The amounts of Gly-Pro and AMC, as measured by the peak area of each spot, almost linearly increased with the incubation time. No other fragments could be detected by fluorometric analysis and ninhydrin reaction. When Sue-GPLGP-MCA was incubated with CL-peptidase without X-Pro DAP, Gly-Pro-MCA and Suc-GPLGPMCA were detected. aspect of tadpule co~lagenuse on SucGPLGP-MCA. Sue-GPLGP-MCA was examined as a substrate for mammalian collagenase from tadpole. When the peptide was incubated with tadpole collagenase (specific activity: 8.42 mg collagen hydrolyzedlminlmg protein, 2.4 pg) for 2 or 16 h at 37°C no hydrolysis of the peptide was observed either by the fluorometry of AMC or by thin-layer chromatographic analysis. CL-Peptidase activity in normal human sera and some human tissues using SucGPLGP-MCA and PZ-peptide as substrates. The enzyme activities in normal human sera are shown in Table 1. The mean value of serum CL-peptidase activity was 0.59 t 0.10 (SD) and 0.61 + 0.28 {SD) nmollminlml of serum using Suc-GPLGPMCA and PZ-peptide as substrates, respectively. When human serum alone was used




5 i/s

IO (mMI-’





FIG. 7. Lineweaver-Burk plots of (succinyl-GlyPro-Leu-Gly-Pro)4methylcoumaryl-7-arnide against the activity of collagenase-like peptidase partially purified from chick embryo. The activity was measured in 50 mM Tris-maleate buffer at pH 8.0. The K, value was calculated to be 4.0 x IO+ M.







03 l/3












B 43


Q so

3, 8,.

1.5 (hr)








o @ Wcylproline






FIG. 8. Time course of the hydrolysis of (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7amide by collagenase-like peptidase. (Succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoum~l-7-~ide was incubated with collagenase-like peptidase partially purified from chick embryo at 37°C for l/6- to 4-h periods, and the reaction products were analyzed by thin-layer chromatography. n-ButOH:AcOH: H,O (2: 1: 1, v/v/v) was used as solvent. 4C and ST denote the control (incubation without CL-peptidase) at 4 h and authentic Gly-Pro as standard, respectively. The numbers at the right of each spot indicate the relative amount of the product as a percentage of the value at 4 h, which was determined by a Shimadzu CS-910 dual-wavelength thin-layer chromatography scanner. 7-Amino-Cmethylcoumarin was assayed fluorometrically at 450 nm with excitation at 360 nm, and glycylproline was assayed photometrically at 350 and 700 nm.

as enzyme with Sue-GPLGP-MCA as substrate, AMC was also identified on the chromatogram. This result indicates that Sue-GPLGP-MCA may be hydrolyzed preferentially by a CL-peptidase in human serum first to produce succinyl-Gly-ProLeu and the Gly-Pro-MCA, which is further hydrolyzed to Gly-Pro and AMC by X-Pro DAP in human serum. As compared with the actively obtained by the standard incubation mixture including exogenous X-Pro DAP the activity using serum alone was 90%. The CL-peptidase activities of some tissues are shown in Table 2. DISCUSSION

A new fluorogenic substrate, Suc-GPLGPMCA, made it possible to assay the activity of CL-peptidase in tissues even with very low activities such as human serum. This

substrate is hydrolyzed into two products, succinyl-Gly-Pro-Leu and Gly-Pro-MCA, by a CL-peptidase, and then the Gly-ProMCA is further hydrolyzed to Gly-Pro and AMC by X-Pro DAP. This stepwise reaction was identified by thin-layer chromatography (Fig. 8). The fluorescence of AMC alone can be measured at 460 nm without the interference by the substrate when the excitation light is set at 380 nm. Thus, no separation procedure for the product from the substrate is required unlike the assay with PZ-peptide as substrate. Therefore, the present assay method for CL-peptidase with Suc-GPLGPMCA as substrate is simple, rapid, and highly sensitive. In the present report, the CL-peptidase reaction was carried out in the presence of X-Pro DAP, and quantitative hydrolysis of the product Gly-Pro-MCA was obtained during the CL-peptidase incubation. It is





OF NORMAL HUMAN SERUM" peptidase activity (nmol/min/ml) Ratio Sue-GPLGP-MCA l PZ-peptide

Substrate Serum (Expt No.)


1 2 3 4 5 6 7 8 9 10 Mean SD








0.62 0.68 0.44 0.62 0.53 0.76 0.58 0.62 0.66 0.43 0.59 0.10

0.84 1.16 0.61 0.40 0.57 0.60 0.71 0.71 0.20 0.29 0.61 0.28

0.74 0.56 0.72 1.55 0.93 1.23 0.82 0.87 3.30 1.48 1.22 0.80

n Activities were measured at pH 8.0 in 50 mM Tris-maleate buffer, and at a substrate concentration of 0.6 mM. The activity with Sue-GPLGP-MCA as substrate was assayed by the standard incubation mixture containing X-Pro DAP.

possible to assay CL-peptidase with twostep reactions as described under Methods, and essentially the same results were obtained with the simultaneous reaction method. The simultaneous reaction method is simple and regularly used. The partially purified enzyme from chick embryo had a K, of 4.0 x 1O-4 M toward Sue-GPLGP-MCA. When PZ-peptide was used as a substrate, the enzyme from chick embryo had a K, of 2.0 x 10e4 M (10). TABLE COLLAGENASE-LIKE





The CL-peptidase from chick embryo had a pH optimum at 8.0, and X-Pro DAP from human submaxillary gland had a pH optimum at 8.7 (18, Kojima et al., manuscript in preparation). In this new assay method, the CL-peptidase activity was determined at pH 8.0, where it was most active, and X-Pro DAP still had an enough activity for the assay at this pH. When human serum was used as an enzyme source, AMC was liberated from 2 OF HUMAN

Human liver Human kidney Chick embryo


peptidase activity (nmoliminlmg

Substrate Sue-GPLGP-MCA



0.53 3.13 88.7

’ Activities were measured at pH 8.0 in 50 mM Tris-maleate 0.6 mM. Tissues were homogenized in water.

0.40 0.52 29.0





Ratio i Sue-GPLGP-MCA \ PZ-peptide

, I

1.33 6.02 3.06

buffer, and at a substrate concentration




Sue-GPLGP-MCA even without addition of X-Pro DAP. This result indicates that SucGPLGP-MCA may be hydrolyzed by a CLpeptidase first to produce succinyl-GlyPro-Leu and the Gly-Pro-MCA, which is further hydrolyzed to Gly-Pro and AMC by X-Pro DAP in human serum. Since the activity with serum alone was 90% of that of exogenous X-Pro DAP, it is not necessary to add X-Pro DAP for routine chnical study of the serum enzyme. This assay for CL-peptidase using the new fluorogenic substrate may be useful in unders~nd~g the roles of CL-peptidase in degradation of collagen under physiological and pathological c~cumst~ces. It was noted that the ratio between the CL-peptidase assayed by this method and PZ-peptidase was variable in sera from different subjects and in human liver and kidney, suggesting that the CL-peptidase assayed with SW-GPLGP-MCA as substrate may be different from PZ-peptidase. Apparently, the PZ-peptide and SucGPLGP-MCA peptide are cleaved by different enzymes. The PZ-peptide was first synthesized to be a substrate for mamm~i~ collagenase substrate (25). The Sue-GPLGP-MCA peptide synthesized here was found not to be a substrate for tissue collagenase, either. Further measurement of the CL-peptidase activity from human serum and other human and animal tissues as well as its comparison with PZ-peptidase are currently under investigation. ACKNOWLEDGMENT We are grateful to Dr. Yutaka Nagai (Tokyo Medical and Dental University, Tokyo) for his generous suppiy of pure tadpole collagenase.

REFERENCES I. Gries, G., Buresch, Hi., and Strauch, L. (1970) Ex~erienria 26. 31-33.

ET AL. 2. Aswauikumar, S. and Radhakrishnan, A. N. (1972) Biochim. Biophys. Acta 276,241-249. 3. Heidrich, H. G., Kronshnabl, O., and Hanning, K. (1973) Hoppe-Seyler’s Z. Physiol. Chem. 354, 1399- 1404. 4. Koren, E., and Mildov& S. (1973) J. Reprod. Fert. 32, 329-356. 5. Aswanikumar, S., and R~h~~shan, A. N. (1973) Biockim. Biophys. Acta 304,210-216. 6. Hino, M., Nakano, G., Harada, M., and Nagatsu, T. (1975) Arch. Oral Biol. 20, 19-22. 7. Korting, G. W., and Morshes, B. (1975) Arch. Germ. Forsh. 251, 191-197. 8. Aswanikumar, S., and Radhakrishnan, A. N. (1975) Biochim. Biophys, Acta 384, 194-202. 9. Hino, M., and Nagatsu, T. (1976) Biochim. Biophys. Acta 429, 555-563. 10. Morales, T. I., and Woessner, I. F., Jr. (1977)J. Biol. Chem. 252,4855-4860. 11. Ito, A., Naganeo, K., Mori, Y., Hirakawa, S., and Hayashi, M. (1977) Clin. Chim. Actu 78, 267270. 12. N~elsc~idt, hp., Unger, Th., and Struck, II., (1977) Wien. Klin. Wochenschr. 89, 735-738. 13. Lukac, J., and Koren, E. (1977) J. Reprod. Fert. 49,95-99. 14. Reinharz, A., and Roth, M. (1%9) Eur. J. Biochem. 7,334-339. 15. Hopsu-Havu, V. K., and Glenner, G. G. (1966) Histochemie 7, 197-201. 16. Nagatsu,T., Hino, M., Fuyamada, H., Hayakawa, T., Sakakibara, S., Nakagawa, Y,, and Takemoto, T. (1976) Anal. Biochem. 74,466-476. 17. Kato, T., Nagatsu, T., Kimura, T., and Sakakibara, S. (1978) Biochem. Med. 19, 351-359. 18. Oya, H., Nagatsu, I., and Nagatsu, T. (1972) Biochim. Biophys. Actu 25%,591-599. 19. Hori, A., and Nagai, Y. (1979) Biochim. Biophys. Acta 566,211-221. 20. Zimmerman, M., Yurewicz, E. C., and Pate& G. (1976) Anal. Biochem. 70,258-262. 21. Kanaoka, Y., Takahashi, T., and Nakamura, H. (1977) Chem. Pharm. B&l. 25,362-363. 22. Sakakibara, S., and Nagai, Y. (1960) Bull. Chem. Sot. Japan 33, 1537-1542. 23. Wiinsh, E., and Heidrich, H. G. (1963) HoppeSeyler’s Z. Ptiysiol. Chem. 333, 149- 151. 24. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randah, R. J. (1951) J. Biol. Chem. 193, 265-275. 25. Harper, E., and Gross, J. (1970) Biochim. Biophys. Acta 198, 286-292.

A new and highly sensitive fluorescence assay for collagenase-like peptidase activity.

ANALYTICAL BIOCHEMISTRY 100, 43-50 (1979) A New and Highly Sensitive Fluorescence Assay for Collagenase-Like Peptidase Activity KOHICHI KOJIMA, HI...
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