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Determination of elastolytic activity with elastin-rhodamine
ANALYTICAL
74, 419-429
BIOCHEMISTRY
(196)
Determination of Elastolytic with Elastin-Rhodamine’
Activity
PAUL F. HUEBNER Department of Internal Medicine, Pulmonary School of Medicine, 4550 Scott Avenue,
Division, Washington St. Louis, Missouri
University 63110
Received November 17, 1975; accepted May 3, 1976 Elastin covalently labeled with rhodamine-B-isothiocyanate has been found to be a sensitive substrate for analysis of elastolytic activity in comparison to elastin-orcein and elastin-Congo red. Using elastin-rhodamine in a 2@min test, elastase concentrations as low as 0.1 pg/ml are accurately assayed. Variations in analytical parameters such as temperature, pH, and particle size of the substrate greatly influence the rate of elastolysis.
Methods for the determination of elastolytic activity of pancreatic elastase most often rely upon solid elastin labeled with a chromophore or a fluorophore (1). Some of these methods contain analytical drawbacks. Ionic bonded labels, such as orcein and Congo red, are readily eluted from elastin during incubation by nonspecific protein resulting in erroneous elastolytic response (1,2). Additionally, these substrates lack the sensitivity necessary to quantitate minute amounts of elastase unless the period of incubation is excessively long. Within the current methods, certain properties such as pH and temperature of the incubation medium and particle size of the substrate remain unexplored. We describe in this communication a method for the quantitative analysis of elastolytic activity using as substrate elastin covalently labeled with rhodamine-B-isothiocyanate. The method is rapid, sensitive, and reproducible, and the label being covalently linked with elastin strongly resists elution by nonspecific protein. Also described are kinetic properties of the enzyme-substrate complex, conditions for assay, and advantages in comparison with elastin labeled with orcein and Congo red. Procedures based upon similar concepts were first developed by Rinderknecht et al. (1) in which elastin was labeled with fluorescein isothiocyanate or remazol brilliant blue. We selected rhodamine-B-isothiocyanate as label due to its high fluorescent intensity (3), ease of synthesis, and readability of the soluble colored products of elastolysis either in a spectrophotometer or fluorometer. ‘This work was supported in part by a grant from the National Institute, No. 5PO1 HL 16118-02. 419 Copyright All
rights
0 of
1976 reproduction
by
Academic in any
Press,
Inc.
form
reserved.
Heart and Lung
420
PAUL F. HUEBNER
MATERIALS
Trypsin l-300, TAME,2 BTEE, and HPA were obtained from Nutritional Biochemicals Corp. HA was purchased from SchwarziMann. Orcein and Congo red dyes, DEAE-cellulose, buffer salts, and dialysis tubing of 3500MW cutoff were obtained from Fisher Scientific Co. M-diethylaminophenol and 4-nitrophalic acid were purchased from Eastman Chemical Co. Thiophosgene and 5% rhodium on charcoal were obtained from Pfaltz and Bauer. Sieves used to fractionate the elastin powders were constructed of brass wire cloth and were Tyler screen scale 100,150,200, and 400 mesh series. Two times crystallized porcine elastase was prepared from trypsin l-300 at 5°C according to the method of Lewis et al. (4). The crystallized elastase was purified to the water-soluble elastase by a procedure similar to that of Baumstark et al. (5). DEAE-cellulose was washed before use as described by Wintersberge (6) and equilibrated by batch washing on a medium porosity f&ted-glass Buchner funnel with 0.043 M Na&O,-HCl buffer, pH 8.8. Crystallized elastase (100 mg) was suspended in carbonate buffer ~2m~rnl) and stirred for 60 min with about 20 g of wet cellulose. The suspension was suctioned to a damp pad with slight (2-5 psi) negative pressure and washed four times with 50-ml aliquots of buffer. All filtrates were combined and dialyzed against deionized water until the dialysate was free of chloride ion as shown by lack of reaction with 5% AgN03. The contents of the dialysis bags were reduced to 114 vol by pervaporation at room temperature under a forced stream of air. The pervaporated solutions were filtered through a l-cm layer of washed Hyflo Super Cel on a fine porosity, f&ted-glass funnel and lyophilized. The yield of lyop~lized elastase was usually 30% of the starting weight of crystallized elastase. The lyophilized elastase showed no activity for proteolytic enzymes when assayed for trypsin and chymotrypsin (7), for carboxypeptidase A (8), and for carboxypeptidase B (9). The preparation was homogenous on 7% polyacrylamide-gel electrophoresis at pH 9.3 (10). Dete~ination of elastolytic units by the method of Sachar et al. (11) yielded 95.5 units/mg of protein. A lyophilized quantity of the enzyme, dissolved in 0.2 M Tris buffer, pH 8.8, yielded an extinction coefficient of EzsO1% = 19.61 in IO-mm cells which was used to calculate the protein concentration of elastase solutions. Rhodamine-B-isothiocyanate (RB-SCN) was prepared by modifications of the procedure described in Chemical Memorandum No. 8 (12) for the preparation of fluorescein isothiocyanate. Twice the molar quantity of M-diethylaminophenol was fused with *Abbreviations used: TAME,p-toluene-sulfonyl-L-arginine methyl ester: BTEE, benzoylL-tyrosine ethyl ester; HPA, hippuryl-L-phenyl-alanine; HA, hippuryk-arginine; RB-SCN, rhodamine-B-isothiocyanate; DEAE, diethylaminoethyl.
DETERMINATION
OF ELASTOLYTIC
421
ACTIVITY
4nitrophalic acid at 170°C for 4 hr on a hot plate. The purple melt, composed of mixed isomers of nitro-rhodamine B, was purified by repeated precipitation from saturated acetone solution by dropwise addition into 10 vol of cold, rapidly stirring water. Two hundred milliliters of reprecipitated nitro-rhodamine B in dry methanol (2%, w/v) and 2 g of 5% rhodium on charcoal were shaken in a hydrogen atmosphere at 40 psi for 3 hr. The catalyst was removed by filtration and the filtrate evaporated at 60°C to r/4 vol. The reduced compound (amino-rhodamine-B) was precipitated by adding the methanol solution to 10 vol of cold water, filtered, and then dried at 80°C in a vacuum oven. The isothiocyanate derivative was prepared by dropwise addition of 50 ml of 5% thiophosgene (w/v in dry acetone) to a filtered solution of 1.5 g of amino-rhodamine B in 200 ml of dry acetone. The reaction mixture was refluxed for 2 hr and evaporated at 50°C to approximately 50 ml. The evaporation from 200 ml of acetone was repeated twice in order to remove excess thiophosgene. The semisolid residue was suspended in 300 ml of acetone and dissolved by refluxing for 2 hr. The solution was then evaporated to l/10 vol and added to 10 vol of rapidly stirring cold water, and the pH was adjusted to 5.2 with saturated NaHCO,. The red precipitate of RB-SCN was collected on a f&ted-glass Buchner funnel, dried over silica gel, and stored at 4°C over desiccant. The elastin powder used to prepare the following labeled substrates was purified from ox ligamentum nuchae by the alkaline extraction method of Lowry et al. (13). The purified elastin was repeatedly passed through a Wiley mill until the particle size was 100-200 mesh. Elastin-rhodamine.3 Fifty grams of elastin powder was suspended in 100 ml of acetone and mixed with 900 ml of 1 M NaHCO,. A filtered solution of 5 g of RB-SCN in 800 ml of acetone was added, and the suspension was stirred at 4°C for 18 hr. The labeled elastin suspension was suctioned onto a Buchner funnel and washed in sequence with 2 liters of 50% 0.1 M NaHCO,-acetone (v/v), 50% acetone in water. Washing was continued with 500-ml portions of acetone then water until the filtrates were colorless. The washed elastin-rhodamine was lyophilized and ground in a Wiley mill until all particles passed through a 200-mesh screen. The powder was then shaken on a 400-mesh screen which retained the bulk of the powder. Yield: 37.5 g of particle size 200-400 mesh and 10 g of particle size passing the 400-mesh screen. Elastin -orcein. Elastin-orcein was prepared according to the method of Sachar ef al. (11). Fifty grams of elastin powder (100-200 mesh) were stirred for 18 hr at 4°C in 300 ml of 70% ethanol containing 3 ml of concentrated hydrochloric acid and 3 g of orcein dye. The labeled elastin was washed on a Buchner funnel with alternate l-liter vol of 70% ethanol and water until the filtrates were colorless. The elastin-orcein was dried at 3 Elastin-rhodamine
is commercially
available
from
Elastin
Products
Co. St. Louis.
MO.
PAUL F. HUEBNER
422
0.2
0.4
0.6
m g SUBSTRATE
0.8
1.0
/ml
FIG. 1. Comparison of total absorbance of solubilized elastin-rhodamine (R) read at 550 nm; elastin-orcein (0) read at 590 nm; elastin-Congo red (C) read at 500 nm; and elastin (E) read at 275 nm. Fluorescent intensity (-----) of elastin-rhodamine.
80°C and ground in the Wiley mill, and the powder was fractioned on 200 and 400-mesh screens. &&z-Congo red. Elastin-Congo red was prepared by a method similar to that of Hall (14). Fifty grams of elastin (100-200 mesh) were stirred for 18 hr at 4°C in 2 liters of a filtered aqueous solution of Congo red. The labeled elastin was washed with 10% (v/v) ethanol until the filtrate was colorless, lyophilized, ground in the Wiley mill, and fractionated on 200 and 400-mesh screens. Suspensions of the substrates (20mg/ml) were prepared by adding the dry powders to gently stirring 0.2 M Tris-HCl, pH 8.8, containing 0.01% Triton X-100. Addition of detergent was necessary in order to wet the substrate. Attempts to obtain homogenous suspensions without detergent were unsuccessful due to the hydrophobic nature of dry elastin. The suspensions were refrigerated overnight and then suctioned onto a medium porosity Buchner funnel using slight vacuum and washed with buffer (not containing Triton X- 100) until the filtrates were colorless. During washing the pad of substrate must always be covered by buffer to prevent clumping of the particles. After washing, the substrates were resuspended to 20 mg/ml in buffer and stored in the refrigerator. Two drops of octanol were added per 100 ml to suppress foaming. METHODS
The determination of elastolytic activity with the three substrates and nonlabeled elastin was performed under identical experimental conditions. Unless otherwise indicated the buffer used throughout was 0.2 M Tris-HCl,
DETERMINATION
OF ELASTOLYTIC TABLE
EXTINCTION
Chromophore RB-SCN Orcein Congo red
COEFFICIENTS
1 OF CHROMOPHORES
Chromophoreb (wdmg of elastin)
Wavelength (nm) 550 590
112 262 562
500
423
ACTIVITY
Binding capacity of chromophore’ 1.00 0.11
75.9 8.2 5.0
0.06
a Extinction coefficients were determined in 10 mm cells on solutions of the chromophores dissolved in buffer. * Micrograms of chromophore per milligram of elastin was calculated as the ratio of slope of solubilized substrate (Fig. 1) and the extinction coefficient times 10,000. c The binding capacity of RB-SCN was unity.
pH 8.8, containing 0.01% sodium azide. In general, buffer and elastase, totaling 2 ml vol, were separately pipetted into lo-ml conical flasks. Then 1 ml (20 mg) of substrate was pipetted into each flask from a rapidly stirring suspension in buffer. The flasks were loosely stoppered and incubated in a shaking water bath at 37°C for 20 min. The flasks were set in ice, adjusted to 10 ml with cold buffer, and filtered on Whatman No. 41 paper into test tubes. Absorbance and/or fluorescent intensity of the filtrates was determined against a blank that contained all reagents except elastase and was taken through the incubation and filtration procedure. The filtrates of elastin-rhodamine were read in lo-mm cuvettes at 550-nm wavelength in a Coleman Model 111 spectrophotometer. Fluorescent intensity was determined in 12-mm cuvettes in a Turner Model 111 fluorometer using filters No. 546 and No. 590 in the excitation and emission sources, respectively. Usually, a l/IO dilution of the filtrates was necessary TABLE RELEASE
OF LABEL
2
BY VARIOUS
TREATMENTS
Treatment” Substrate
0.1 N NaOH
Elastin-rhodamine Elastin-orcein Elastin-congo red
40.5 85.7
10.7b
0.1 N I-ICI
Buffer
Albumin
3.5 7.7 22.5
4.5 16.7 31.4
1.2 92.9 95.7
u Substrates were treated with boiling acid, base, and buffer and with albumin at 37°C. washed and lyophilized. Total absorbance was determined on completely solubilized quantities. b All values are percentages of label removed by treatments in comparison with nontreated substrates.
424
PAUL F. HUEBNER
FIG. 2. Relationship between absorbance (A) or fluorescence (B) and elastase concentration using elastin-rhodamine (R) read at 550 nm; elastin-orcein (0) read at 590 nm; and elastin-Congo red (C) read at 500 nm. Fluorescent intensity (-----) of elastin-rhodamine.
in order to obtain fluorescent readings within the range of the fluorometer. The absorbances of filtrates from assays with elastin-orcein, elastinCongo red, and elastin were determined at 590,500, and 275 nm, respectively. The latter was corrected for the absorbance of elastase included in the filtrates. The quantity of substrate digested was calculated by dividing the absorbance of the filtrate by the slope of the curve of total absorbance of solubilized substrate. Total absorbance of the substrates was determined after completely solubilizing known quantities (2-10 mg) by incubating at 37°C with elastase (150 pg) for about 2 hr. The incubates were brought to 10 ml with buffer, and absorbance or fluorescent intensity was determined. Substrate suspensions (10 mg/ml) were tested for release of the label by heating for 30 min on a boiling-water bath in acid (0.1 M HCl), in base (0.1 M NaOH), in buffer, and by incubation at 37°C with bovine serum albumin (5%, w/v, in buffer). After the various treatments the substrates were washed with water on No. 41 filter paper until the filtrates were colorless and lyophilized. Total absorbance of treated substrates was again determined and compared to values obtained for nontreated substrates. The protein assay of filtered incubates was determined by the microKjeldahl nitrogen method. Protein content was estimated using a factor of 16.34% nitrogen in nonlabeled elastin (15). Prior to nitrogen determination, the filtrates were dialyzed against three changes of 1000 vol of cold distilled water. The effect of particle size on the rate of elastolytic activity was determined using elastin-rhodamine sieved to particle size lOO- 150, 150-200, and 200-400 mesh and particle size passing the 400-mesh screen. The particles were suspended in buffer, and 1 ml (20 mg) of each suspension was incubated for 20 min with 2 ml of buffer containing 20 pg of elastase.
DETERMINATION
MESH
100-150
OF ELASTOLYTIC
ACTIVITY
zoo-400
400
150-200
425
FIG. 3. Relationship between the particle size of elastin-rhodamine and the rate of elastolysis. Incubation flasks contained 20 mg of elastin-rhodamine of particle size lOO- 1.50mesh (A); HO-200 mesh (B); 200-400 mesh (C); and particles passing the 400-mesh screen (D).
In kinetic experiments, 1 to 40 mg of 200-400-mesh elastin-rhodamine were incubated for 20 min with a constant amount of elastase (3, 10, or 20 pg) in 3 ml of buffer. Units for the rate of elastolysis, v, are milligrams of substrate solubilized per milliliter of incubation volume per minute of incubation time. In time-course experiments, 20 mg of substrate was incubated with and without shaking for 5 to 80 min in 3 ml of buffer containing 15 pg of elastase. Elastolysis was expressed as milligrams of substrate solubilized. The effect of temperature on elastolysis was determined by incubating 20 pg of elastase with 200-400-mesh elastin-rhodamine at 0 to 62°C for 20 min. The elastolytic activity at various pH’s of the Tris- HCl buffer was determined using 200-400-mesh elastin-rhodamine. In these determinations, the substrate was suspended in buffer of the pH under investigation, and absorbance read against a blank of the same pH. Elastolytic activity was expressed as milligrams of substrate solubilized per milligram of elastase. RESULTS AND DISCUSSION
The total absorbance of the three substrates and nondyed elastin after complete solubilization by elastase is shown in Fig. 1. The solubilized elastin-rhodamine exhibited approximately four times more light absorbed than either the solubilized elastin-orcein or the elastin-Congo red. The total absorbance of substrates would vary slightly between preparations. However, the absorbance of elastin-rhodamine was always highest among the three labeled substrates. Extinction coefficients indicated that solutions (l%, w/v, in buffer) of RB-SCN, orcein, and Congo red absorb light in the ratio of 1:2:5 (Table 1). Although orcein and Congo red have extinction coefficients greater than RB-SCN, the binding capacity of RB-SCN is 9 and 15 times greater than orcein and Congo red, respectively. Consequently, the solubilized products of elastin-rhodamine are four and
426
PAUL F. HUEBNER
20 40 TEMPERATURE
60 “c
6
8
10
12
PH
FIG. 4. Effects of temperature (A) and pH (B) on the rate of elastolysis of elastin-rhodamine (200-400 mesh, 20 mg/incubation flask).
three times more absorbant than elastin-orcein and elastin-Congo red, respectively. The release of label by hot chemical treatments and by incubation with albumin (Table 2) indicated that RB-SCN was least susceptible to removal from elastin. Resistance to removal of RB-SCN was attributed to the covalent linkage between the free amino groups of elastin and the -SCN moiety of the dye (1). Orcein and Congo red were readily removed by all treatments. Albumin removed most of the ionic bonded dyes, apparently by decreasing the electrostatic charge. This effect by soluble protein in eluting such ionic bonded dyes has been documented for both orcein (18) and Congo red (2). The combined effects of high binding capacity and resistance to elution contribute to the high sensitivity of the elastin-rhodamine substrate. Figure 2B shows that 0.1 pg/ml of elastase was easily detected by fluorescence using elastin-rhodamine as substrate. The absorbance responses with elastin-orcein and elastin-Congo red at this low level of enzyme concentration were barely detectable. Increased elastase concentration (Fig. 2A) yielded linear elastase activity with all labeled substrates, elastin-rhodamine having the highest response. Nitrogen determinations on the dialyzed filtrates showed that similar quantities of all substrate were solubilized. The sensitivity then, among the three labeled substrates, was directly related to absorbance of the label and the amount of label bound to elastin. Variations in the particle size of the elastin-rhodamine substrate significantly affected the rate of elastolysis during the 20-min incubation period (Fig. 3). The rate doubled as the particle size decreased. This
DETERMINATION
OF ELASTOLYTIC
C”r”e No. I
.Ob
Elk% 20
ACTIVITY
427
Kinetic Parameters Vmax Km (mghl /mm) mg/ml
.034 ,020 .oos
.70 .50 .70
FIG. 5. Relationship of substrate concentration and the rate of elastolysis using elastin-rhodamine (200-400 mesh) with three levels of elastase (3, i0, or 20 &incubation flask). The inset indicates kinetic parameters derived from each curve. V,,, was determined as the intercept of the ordinate with the flat portion of the curve. K, was determined as the intercept of the abscissa from M V,,, plotted on each curve.
relationship was due to the increase in reactive surface area made available for elastolysis. The effects of temperature and pH on the rate of elastolysis of elastin-rhodamine are shown in Fig. 4. The rate increased with temperature from 0 to 52°C then decreased at 57°C due to denaturing of the enzyme. The rate also increased with pH reaching a maximum between 9.5 and 11. Kinetic determinations were performed using elastin-rhodamine as the substrate. Rate of elastolysis was expressed as milligrams of substrate solubilized per milliliter of incubation volume per minute of incubation time. The rate was plotted against substrate concentration, S. The results of tests containing three levels of enzyme over a 40-fold concentration of substrate are shown in Fig. 5. The three curves indicated maximum rate of elastolysis when S exceeded 5 mg/ml and nearly constant K, values (ca. 0.70 mg/ml). The latter was approximately l/10 the K, value reported for elastin-Congo red (14). Maximum velocity (V,,,) was nearly linear with respect to enzyme concentration. Time-course experiments indicated linear elastolysis up to 20 min of incubation and a twofold increase in the rate of elastolysis when the incubates were shaken (Fig. 6). Presumably, shaking aids the reaction by
428
DETERMINATION
OF ELASTOLYTIC
ACTIVITY
FIG. 6. Relationship of elastolysis and time of reaction. Elastin-rhodamine (200-400 mesh) was incubated for various time periods with 15 pg of elastase with shaking (curve 1 at 80 oscillations/min) and without shaking (curve 2).
keeping the substrate particles homogenously dispersed throughout the elastase solution. The experimentation with the three labeled substrates has led us to advance the conclusion that elastin-rhodamine was the most effective substrate for the determination of elastolytic activity. The properties of low K,, high absorbance, and well-defined particle size have provided fairly conclusive evidence for its efficiency under the described conditions of pH 8.8 and 37°C incubation temperature. If the conditions were changed to optimum (pH 9.5 and 52°C) then the rate of elastolysis would double during the 20-min incubation period. The elastin-rhodamine substrate has been of value in other respects. Nanogram quantities of elastase are rapidly detected by incubation in the wells of a gel composed of agar and solubilized elastin-rhodamine. Additionally, the proteolytic activity of nonspecific enzymes of plant origin, such as crystalline papain, may be assayed when incubated with elastin-rhodamine at pH 6.2 in 0.1 M citrate-HCl buffer. REFERENCES 1. Rinderknecht, H., Geokas, M. C., Silverman, D., Lillard, Y., and Haverback, B. J. (1%8) Clin. Chim. Acra 19, 327-339. 2. Banga, I., and Ardelt, W. (1%7) Biochim. Biophys. Acta 146, 284-286. 3. Undenfriend, S. (1962) in Fluorescent Assay in Biology and Medicine (Kaplan, N. O., and Scheraga, H. A., ed.), p. 100, Academic Press, New York. 4. Lewis, U. J., Williams, D. E., and Brink, N. G. (1956)J. Biol. Chem. 222, 705-720. 5. Baumstark, J. S., Bardawil, W. A., Sbarra, A. J., and Hayes, N. (1%3) Biochim. Biophys.
Acta
77, 676-679.
6. Wintersberger, E., Cox, D. J., and Neurath, H. (1962) Biochemistry 1, 1069-1077. 7. Hummel, B. C. W. (1959) Canad. J. Biochem. Physiol. 37, 1393-1399. 8. Folk, J. E., and Schirmer, E. W. (1963) J. Biol. Chem. 238, 3884-3894.
DETERMINATION
OF ELASTOLYTIC
ACTIVITY
429
9. Folk, J. E., Piez, K. A., Carroll, W. R., and Gladner, J. (1960) J. Biol. Chem. 235, 2272-2277. 10. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1%2) Nature (London ) 195, 281-283. 11. Sachar, L. A., Winter, K. K., Sicher, N., and Frankel, S. (1955) Proc. Sot. Exp. Bio/. Med.
90, 323-326.
12. Chemical Memorandum No. 8, Technical Development Laboratories, Laboratory Division, National Communicable Disease Center, Savannah, Ga. 13. Lowry, 0. H., Gilligan, D. R., and Katersky, E. M. (1941) J. Biol. Chem. 139, 795-804. 14. Hall, D. A. (1966)Biochem. .Z. 101, 29-36. 15. Pierce, J. A., and Hocott, J. A. (1959)J. Clin. Invest. 39, 8-14. 16. Mallory, D. A., and Travis, J. (1975) Biochemistry 14, 722-730. 17. Feinstein, G., Hofstein, R., Koifmann, J., and Sokolovsky, M. (1974) Eur. J. Biochem. 43, 569. 18. Rinderknecht, H., Geokas, M. C., Silverman, P., and Haverback, B. J. (1968) C/in. Chim. Acta 21, 197-203.
74, 419-429
BIOCHEMISTRY
(196)
Determination of Elastolytic with Elastin-Rhodamine’
Activity
PAUL F. HUEBNER Department of Internal Medicine, Pulmonary School of Medicine, 4550 Scott Avenue,
Division, Washington St. Louis, Missouri
University 63110
Received November 17, 1975; accepted May 3, 1976 Elastin covalently labeled with rhodamine-B-isothiocyanate has been found to be a sensitive substrate for analysis of elastolytic activity in comparison to elastin-orcein and elastin-Congo red. Using elastin-rhodamine in a 2@min test, elastase concentrations as low as 0.1 pg/ml are accurately assayed. Variations in analytical parameters such as temperature, pH, and particle size of the substrate greatly influence the rate of elastolysis.
Methods for the determination of elastolytic activity of pancreatic elastase most often rely upon solid elastin labeled with a chromophore or a fluorophore (1). Some of these methods contain analytical drawbacks. Ionic bonded labels, such as orcein and Congo red, are readily eluted from elastin during incubation by nonspecific protein resulting in erroneous elastolytic response (1,2). Additionally, these substrates lack the sensitivity necessary to quantitate minute amounts of elastase unless the period of incubation is excessively long. Within the current methods, certain properties such as pH and temperature of the incubation medium and particle size of the substrate remain unexplored. We describe in this communication a method for the quantitative analysis of elastolytic activity using as substrate elastin covalently labeled with rhodamine-B-isothiocyanate. The method is rapid, sensitive, and reproducible, and the label being covalently linked with elastin strongly resists elution by nonspecific protein. Also described are kinetic properties of the enzyme-substrate complex, conditions for assay, and advantages in comparison with elastin labeled with orcein and Congo red. Procedures based upon similar concepts were first developed by Rinderknecht et al. (1) in which elastin was labeled with fluorescein isothiocyanate or remazol brilliant blue. We selected rhodamine-B-isothiocyanate as label due to its high fluorescent intensity (3), ease of synthesis, and readability of the soluble colored products of elastolysis either in a spectrophotometer or fluorometer. ‘This work was supported in part by a grant from the National Institute, No. 5PO1 HL 16118-02. 419 Copyright All
rights
0 of
1976 reproduction
by
Academic in any
Press,
Inc.
form
reserved.
Heart and Lung
420
PAUL F. HUEBNER
MATERIALS
Trypsin l-300, TAME,2 BTEE, and HPA were obtained from Nutritional Biochemicals Corp. HA was purchased from SchwarziMann. Orcein and Congo red dyes, DEAE-cellulose, buffer salts, and dialysis tubing of 3500MW cutoff were obtained from Fisher Scientific Co. M-diethylaminophenol and 4-nitrophalic acid were purchased from Eastman Chemical Co. Thiophosgene and 5% rhodium on charcoal were obtained from Pfaltz and Bauer. Sieves used to fractionate the elastin powders were constructed of brass wire cloth and were Tyler screen scale 100,150,200, and 400 mesh series. Two times crystallized porcine elastase was prepared from trypsin l-300 at 5°C according to the method of Lewis et al. (4). The crystallized elastase was purified to the water-soluble elastase by a procedure similar to that of Baumstark et al. (5). DEAE-cellulose was washed before use as described by Wintersberge (6) and equilibrated by batch washing on a medium porosity f&ted-glass Buchner funnel with 0.043 M Na&O,-HCl buffer, pH 8.8. Crystallized elastase (100 mg) was suspended in carbonate buffer ~2m~rnl) and stirred for 60 min with about 20 g of wet cellulose. The suspension was suctioned to a damp pad with slight (2-5 psi) negative pressure and washed four times with 50-ml aliquots of buffer. All filtrates were combined and dialyzed against deionized water until the dialysate was free of chloride ion as shown by lack of reaction with 5% AgN03. The contents of the dialysis bags were reduced to 114 vol by pervaporation at room temperature under a forced stream of air. The pervaporated solutions were filtered through a l-cm layer of washed Hyflo Super Cel on a fine porosity, f&ted-glass funnel and lyophilized. The yield of lyop~lized elastase was usually 30% of the starting weight of crystallized elastase. The lyophilized elastase showed no activity for proteolytic enzymes when assayed for trypsin and chymotrypsin (7), for carboxypeptidase A (8), and for carboxypeptidase B (9). The preparation was homogenous on 7% polyacrylamide-gel electrophoresis at pH 9.3 (10). Dete~ination of elastolytic units by the method of Sachar et al. (11) yielded 95.5 units/mg of protein. A lyophilized quantity of the enzyme, dissolved in 0.2 M Tris buffer, pH 8.8, yielded an extinction coefficient of EzsO1% = 19.61 in IO-mm cells which was used to calculate the protein concentration of elastase solutions. Rhodamine-B-isothiocyanate (RB-SCN) was prepared by modifications of the procedure described in Chemical Memorandum No. 8 (12) for the preparation of fluorescein isothiocyanate. Twice the molar quantity of M-diethylaminophenol was fused with *Abbreviations used: TAME,p-toluene-sulfonyl-L-arginine methyl ester: BTEE, benzoylL-tyrosine ethyl ester; HPA, hippuryl-L-phenyl-alanine; HA, hippuryk-arginine; RB-SCN, rhodamine-B-isothiocyanate; DEAE, diethylaminoethyl.
DETERMINATION
OF ELASTOLYTIC
421
ACTIVITY
4nitrophalic acid at 170°C for 4 hr on a hot plate. The purple melt, composed of mixed isomers of nitro-rhodamine B, was purified by repeated precipitation from saturated acetone solution by dropwise addition into 10 vol of cold, rapidly stirring water. Two hundred milliliters of reprecipitated nitro-rhodamine B in dry methanol (2%, w/v) and 2 g of 5% rhodium on charcoal were shaken in a hydrogen atmosphere at 40 psi for 3 hr. The catalyst was removed by filtration and the filtrate evaporated at 60°C to r/4 vol. The reduced compound (amino-rhodamine-B) was precipitated by adding the methanol solution to 10 vol of cold water, filtered, and then dried at 80°C in a vacuum oven. The isothiocyanate derivative was prepared by dropwise addition of 50 ml of 5% thiophosgene (w/v in dry acetone) to a filtered solution of 1.5 g of amino-rhodamine B in 200 ml of dry acetone. The reaction mixture was refluxed for 2 hr and evaporated at 50°C to approximately 50 ml. The evaporation from 200 ml of acetone was repeated twice in order to remove excess thiophosgene. The semisolid residue was suspended in 300 ml of acetone and dissolved by refluxing for 2 hr. The solution was then evaporated to l/10 vol and added to 10 vol of rapidly stirring cold water, and the pH was adjusted to 5.2 with saturated NaHCO,. The red precipitate of RB-SCN was collected on a f&ted-glass Buchner funnel, dried over silica gel, and stored at 4°C over desiccant. The elastin powder used to prepare the following labeled substrates was purified from ox ligamentum nuchae by the alkaline extraction method of Lowry et al. (13). The purified elastin was repeatedly passed through a Wiley mill until the particle size was 100-200 mesh. Elastin-rhodamine.3 Fifty grams of elastin powder was suspended in 100 ml of acetone and mixed with 900 ml of 1 M NaHCO,. A filtered solution of 5 g of RB-SCN in 800 ml of acetone was added, and the suspension was stirred at 4°C for 18 hr. The labeled elastin suspension was suctioned onto a Buchner funnel and washed in sequence with 2 liters of 50% 0.1 M NaHCO,-acetone (v/v), 50% acetone in water. Washing was continued with 500-ml portions of acetone then water until the filtrates were colorless. The washed elastin-rhodamine was lyophilized and ground in a Wiley mill until all particles passed through a 200-mesh screen. The powder was then shaken on a 400-mesh screen which retained the bulk of the powder. Yield: 37.5 g of particle size 200-400 mesh and 10 g of particle size passing the 400-mesh screen. Elastin -orcein. Elastin-orcein was prepared according to the method of Sachar ef al. (11). Fifty grams of elastin powder (100-200 mesh) were stirred for 18 hr at 4°C in 300 ml of 70% ethanol containing 3 ml of concentrated hydrochloric acid and 3 g of orcein dye. The labeled elastin was washed on a Buchner funnel with alternate l-liter vol of 70% ethanol and water until the filtrates were colorless. The elastin-orcein was dried at 3 Elastin-rhodamine
is commercially
available
from
Elastin
Products
Co. St. Louis.
MO.
PAUL F. HUEBNER
422
0.2
0.4
0.6
m g SUBSTRATE
0.8
1.0
/ml
FIG. 1. Comparison of total absorbance of solubilized elastin-rhodamine (R) read at 550 nm; elastin-orcein (0) read at 590 nm; elastin-Congo red (C) read at 500 nm; and elastin (E) read at 275 nm. Fluorescent intensity (-----) of elastin-rhodamine.
80°C and ground in the Wiley mill, and the powder was fractioned on 200 and 400-mesh screens. &&z-Congo red. Elastin-Congo red was prepared by a method similar to that of Hall (14). Fifty grams of elastin (100-200 mesh) were stirred for 18 hr at 4°C in 2 liters of a filtered aqueous solution of Congo red. The labeled elastin was washed with 10% (v/v) ethanol until the filtrate was colorless, lyophilized, ground in the Wiley mill, and fractionated on 200 and 400-mesh screens. Suspensions of the substrates (20mg/ml) were prepared by adding the dry powders to gently stirring 0.2 M Tris-HCl, pH 8.8, containing 0.01% Triton X-100. Addition of detergent was necessary in order to wet the substrate. Attempts to obtain homogenous suspensions without detergent were unsuccessful due to the hydrophobic nature of dry elastin. The suspensions were refrigerated overnight and then suctioned onto a medium porosity Buchner funnel using slight vacuum and washed with buffer (not containing Triton X- 100) until the filtrates were colorless. During washing the pad of substrate must always be covered by buffer to prevent clumping of the particles. After washing, the substrates were resuspended to 20 mg/ml in buffer and stored in the refrigerator. Two drops of octanol were added per 100 ml to suppress foaming. METHODS
The determination of elastolytic activity with the three substrates and nonlabeled elastin was performed under identical experimental conditions. Unless otherwise indicated the buffer used throughout was 0.2 M Tris-HCl,
DETERMINATION
OF ELASTOLYTIC TABLE
EXTINCTION
Chromophore RB-SCN Orcein Congo red
COEFFICIENTS
1 OF CHROMOPHORES
Chromophoreb (wdmg of elastin)
Wavelength (nm) 550 590
112 262 562
500
423
ACTIVITY
Binding capacity of chromophore’ 1.00 0.11
75.9 8.2 5.0
0.06
a Extinction coefficients were determined in 10 mm cells on solutions of the chromophores dissolved in buffer. * Micrograms of chromophore per milligram of elastin was calculated as the ratio of slope of solubilized substrate (Fig. 1) and the extinction coefficient times 10,000. c The binding capacity of RB-SCN was unity.
pH 8.8, containing 0.01% sodium azide. In general, buffer and elastase, totaling 2 ml vol, were separately pipetted into lo-ml conical flasks. Then 1 ml (20 mg) of substrate was pipetted into each flask from a rapidly stirring suspension in buffer. The flasks were loosely stoppered and incubated in a shaking water bath at 37°C for 20 min. The flasks were set in ice, adjusted to 10 ml with cold buffer, and filtered on Whatman No. 41 paper into test tubes. Absorbance and/or fluorescent intensity of the filtrates was determined against a blank that contained all reagents except elastase and was taken through the incubation and filtration procedure. The filtrates of elastin-rhodamine were read in lo-mm cuvettes at 550-nm wavelength in a Coleman Model 111 spectrophotometer. Fluorescent intensity was determined in 12-mm cuvettes in a Turner Model 111 fluorometer using filters No. 546 and No. 590 in the excitation and emission sources, respectively. Usually, a l/IO dilution of the filtrates was necessary TABLE RELEASE
OF LABEL
2
BY VARIOUS
TREATMENTS
Treatment” Substrate
0.1 N NaOH
Elastin-rhodamine Elastin-orcein Elastin-congo red
40.5 85.7
10.7b
0.1 N I-ICI
Buffer
Albumin
3.5 7.7 22.5
4.5 16.7 31.4
1.2 92.9 95.7
u Substrates were treated with boiling acid, base, and buffer and with albumin at 37°C. washed and lyophilized. Total absorbance was determined on completely solubilized quantities. b All values are percentages of label removed by treatments in comparison with nontreated substrates.
424
PAUL F. HUEBNER
FIG. 2. Relationship between absorbance (A) or fluorescence (B) and elastase concentration using elastin-rhodamine (R) read at 550 nm; elastin-orcein (0) read at 590 nm; and elastin-Congo red (C) read at 500 nm. Fluorescent intensity (-----) of elastin-rhodamine.
in order to obtain fluorescent readings within the range of the fluorometer. The absorbances of filtrates from assays with elastin-orcein, elastinCongo red, and elastin were determined at 590,500, and 275 nm, respectively. The latter was corrected for the absorbance of elastase included in the filtrates. The quantity of substrate digested was calculated by dividing the absorbance of the filtrate by the slope of the curve of total absorbance of solubilized substrate. Total absorbance of the substrates was determined after completely solubilizing known quantities (2-10 mg) by incubating at 37°C with elastase (150 pg) for about 2 hr. The incubates were brought to 10 ml with buffer, and absorbance or fluorescent intensity was determined. Substrate suspensions (10 mg/ml) were tested for release of the label by heating for 30 min on a boiling-water bath in acid (0.1 M HCl), in base (0.1 M NaOH), in buffer, and by incubation at 37°C with bovine serum albumin (5%, w/v, in buffer). After the various treatments the substrates were washed with water on No. 41 filter paper until the filtrates were colorless and lyophilized. Total absorbance of treated substrates was again determined and compared to values obtained for nontreated substrates. The protein assay of filtered incubates was determined by the microKjeldahl nitrogen method. Protein content was estimated using a factor of 16.34% nitrogen in nonlabeled elastin (15). Prior to nitrogen determination, the filtrates were dialyzed against three changes of 1000 vol of cold distilled water. The effect of particle size on the rate of elastolytic activity was determined using elastin-rhodamine sieved to particle size lOO- 150, 150-200, and 200-400 mesh and particle size passing the 400-mesh screen. The particles were suspended in buffer, and 1 ml (20 mg) of each suspension was incubated for 20 min with 2 ml of buffer containing 20 pg of elastase.
DETERMINATION
MESH
100-150
OF ELASTOLYTIC
ACTIVITY
zoo-400
400
150-200
425
FIG. 3. Relationship between the particle size of elastin-rhodamine and the rate of elastolysis. Incubation flasks contained 20 mg of elastin-rhodamine of particle size lOO- 1.50mesh (A); HO-200 mesh (B); 200-400 mesh (C); and particles passing the 400-mesh screen (D).
In kinetic experiments, 1 to 40 mg of 200-400-mesh elastin-rhodamine were incubated for 20 min with a constant amount of elastase (3, 10, or 20 pg) in 3 ml of buffer. Units for the rate of elastolysis, v, are milligrams of substrate solubilized per milliliter of incubation volume per minute of incubation time. In time-course experiments, 20 mg of substrate was incubated with and without shaking for 5 to 80 min in 3 ml of buffer containing 15 pg of elastase. Elastolysis was expressed as milligrams of substrate solubilized. The effect of temperature on elastolysis was determined by incubating 20 pg of elastase with 200-400-mesh elastin-rhodamine at 0 to 62°C for 20 min. The elastolytic activity at various pH’s of the Tris- HCl buffer was determined using 200-400-mesh elastin-rhodamine. In these determinations, the substrate was suspended in buffer of the pH under investigation, and absorbance read against a blank of the same pH. Elastolytic activity was expressed as milligrams of substrate solubilized per milligram of elastase. RESULTS AND DISCUSSION
The total absorbance of the three substrates and nondyed elastin after complete solubilization by elastase is shown in Fig. 1. The solubilized elastin-rhodamine exhibited approximately four times more light absorbed than either the solubilized elastin-orcein or the elastin-Congo red. The total absorbance of substrates would vary slightly between preparations. However, the absorbance of elastin-rhodamine was always highest among the three labeled substrates. Extinction coefficients indicated that solutions (l%, w/v, in buffer) of RB-SCN, orcein, and Congo red absorb light in the ratio of 1:2:5 (Table 1). Although orcein and Congo red have extinction coefficients greater than RB-SCN, the binding capacity of RB-SCN is 9 and 15 times greater than orcein and Congo red, respectively. Consequently, the solubilized products of elastin-rhodamine are four and
426
PAUL F. HUEBNER
20 40 TEMPERATURE
60 “c
6
8
10
12
PH
FIG. 4. Effects of temperature (A) and pH (B) on the rate of elastolysis of elastin-rhodamine (200-400 mesh, 20 mg/incubation flask).
three times more absorbant than elastin-orcein and elastin-Congo red, respectively. The release of label by hot chemical treatments and by incubation with albumin (Table 2) indicated that RB-SCN was least susceptible to removal from elastin. Resistance to removal of RB-SCN was attributed to the covalent linkage between the free amino groups of elastin and the -SCN moiety of the dye (1). Orcein and Congo red were readily removed by all treatments. Albumin removed most of the ionic bonded dyes, apparently by decreasing the electrostatic charge. This effect by soluble protein in eluting such ionic bonded dyes has been documented for both orcein (18) and Congo red (2). The combined effects of high binding capacity and resistance to elution contribute to the high sensitivity of the elastin-rhodamine substrate. Figure 2B shows that 0.1 pg/ml of elastase was easily detected by fluorescence using elastin-rhodamine as substrate. The absorbance responses with elastin-orcein and elastin-Congo red at this low level of enzyme concentration were barely detectable. Increased elastase concentration (Fig. 2A) yielded linear elastase activity with all labeled substrates, elastin-rhodamine having the highest response. Nitrogen determinations on the dialyzed filtrates showed that similar quantities of all substrate were solubilized. The sensitivity then, among the three labeled substrates, was directly related to absorbance of the label and the amount of label bound to elastin. Variations in the particle size of the elastin-rhodamine substrate significantly affected the rate of elastolysis during the 20-min incubation period (Fig. 3). The rate doubled as the particle size decreased. This
DETERMINATION
OF ELASTOLYTIC
C”r”e No. I
.Ob
Elk% 20
ACTIVITY
427
Kinetic Parameters Vmax Km (mghl /mm) mg/ml
.034 ,020 .oos
.70 .50 .70
FIG. 5. Relationship of substrate concentration and the rate of elastolysis using elastin-rhodamine (200-400 mesh) with three levels of elastase (3, i0, or 20 &incubation flask). The inset indicates kinetic parameters derived from each curve. V,,, was determined as the intercept of the ordinate with the flat portion of the curve. K, was determined as the intercept of the abscissa from M V,,, plotted on each curve.
relationship was due to the increase in reactive surface area made available for elastolysis. The effects of temperature and pH on the rate of elastolysis of elastin-rhodamine are shown in Fig. 4. The rate increased with temperature from 0 to 52°C then decreased at 57°C due to denaturing of the enzyme. The rate also increased with pH reaching a maximum between 9.5 and 11. Kinetic determinations were performed using elastin-rhodamine as the substrate. Rate of elastolysis was expressed as milligrams of substrate solubilized per milliliter of incubation volume per minute of incubation time. The rate was plotted against substrate concentration, S. The results of tests containing three levels of enzyme over a 40-fold concentration of substrate are shown in Fig. 5. The three curves indicated maximum rate of elastolysis when S exceeded 5 mg/ml and nearly constant K, values (ca. 0.70 mg/ml). The latter was approximately l/10 the K, value reported for elastin-Congo red (14). Maximum velocity (V,,,) was nearly linear with respect to enzyme concentration. Time-course experiments indicated linear elastolysis up to 20 min of incubation and a twofold increase in the rate of elastolysis when the incubates were shaken (Fig. 6). Presumably, shaking aids the reaction by
428
DETERMINATION
OF ELASTOLYTIC
ACTIVITY
FIG. 6. Relationship of elastolysis and time of reaction. Elastin-rhodamine (200-400 mesh) was incubated for various time periods with 15 pg of elastase with shaking (curve 1 at 80 oscillations/min) and without shaking (curve 2).
keeping the substrate particles homogenously dispersed throughout the elastase solution. The experimentation with the three labeled substrates has led us to advance the conclusion that elastin-rhodamine was the most effective substrate for the determination of elastolytic activity. The properties of low K,, high absorbance, and well-defined particle size have provided fairly conclusive evidence for its efficiency under the described conditions of pH 8.8 and 37°C incubation temperature. If the conditions were changed to optimum (pH 9.5 and 52°C) then the rate of elastolysis would double during the 20-min incubation period. The elastin-rhodamine substrate has been of value in other respects. Nanogram quantities of elastase are rapidly detected by incubation in the wells of a gel composed of agar and solubilized elastin-rhodamine. Additionally, the proteolytic activity of nonspecific enzymes of plant origin, such as crystalline papain, may be assayed when incubated with elastin-rhodamine at pH 6.2 in 0.1 M citrate-HCl buffer. REFERENCES 1. Rinderknecht, H., Geokas, M. C., Silverman, D., Lillard, Y., and Haverback, B. J. (1%8) Clin. Chim. Acra 19, 327-339. 2. Banga, I., and Ardelt, W. (1%7) Biochim. Biophys. Acta 146, 284-286. 3. Undenfriend, S. (1962) in Fluorescent Assay in Biology and Medicine (Kaplan, N. O., and Scheraga, H. A., ed.), p. 100, Academic Press, New York. 4. Lewis, U. J., Williams, D. E., and Brink, N. G. (1956)J. Biol. Chem. 222, 705-720. 5. Baumstark, J. S., Bardawil, W. A., Sbarra, A. J., and Hayes, N. (1%3) Biochim. Biophys.
Acta
77, 676-679.
6. Wintersberger, E., Cox, D. J., and Neurath, H. (1962) Biochemistry 1, 1069-1077. 7. Hummel, B. C. W. (1959) Canad. J. Biochem. Physiol. 37, 1393-1399. 8. Folk, J. E., and Schirmer, E. W. (1963) J. Biol. Chem. 238, 3884-3894.
DETERMINATION
OF ELASTOLYTIC
ACTIVITY
429
9. Folk, J. E., Piez, K. A., Carroll, W. R., and Gladner, J. (1960) J. Biol. Chem. 235, 2272-2277. 10. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1%2) Nature (London ) 195, 281-283. 11. Sachar, L. A., Winter, K. K., Sicher, N., and Frankel, S. (1955) Proc. Sot. Exp. Bio/. Med.
90, 323-326.
12. Chemical Memorandum No. 8, Technical Development Laboratories, Laboratory Division, National Communicable Disease Center, Savannah, Ga. 13. Lowry, 0. H., Gilligan, D. R., and Katersky, E. M. (1941) J. Biol. Chem. 139, 795-804. 14. Hall, D. A. (1966)Biochem. .Z. 101, 29-36. 15. Pierce, J. A., and Hocott, J. A. (1959)J. Clin. Invest. 39, 8-14. 16. Mallory, D. A., and Travis, J. (1975) Biochemistry 14, 722-730. 17. Feinstein, G., Hofstein, R., Koifmann, J., and Sokolovsky, M. (1974) Eur. J. Biochem. 43, 569. 18. Rinderknecht, H., Geokas, M. C., Silverman, P., and Haverback, B. J. (1968) C/in. Chim. Acta 21, 197-203.