luorometric ubstrate for Sulfatase and Lipase George G . Guilbault and James Hieserman Department of Chemistry, Louisiana State Unioersity in New Orleans, Lakefroat Campus, New Orleans, La. 70122
New fluorometric substrates are described for the assay of the enzyme sulfatase and lipase. p-Naphthol sulfate and 4-methyl umbelliferone sulfate are useful for the assay of sulfatases, and N-methyl indoxyl myristate is an excellent substrate for the analysis of lipase. As little as unit per ml of lipase or 1 0 - 3 unit per ml of sulfatase can be determined by a direct reaction rate method in 2-3 minutes with a precision and accuracy of about 1.5%.
IN RECENT PUBLICATIONS (1-3), the use of fluorometric substrates for the sensitive assay of enzymes has been described. New substrates for lipase described include fluorescein dibutyrate (4, 5) and 4-methyl umbelliferone heptanoate, octanoate, and nonanoate (6). Interference from pseudo cholinesterase made the latter substrates of only limited interest. Ravinand Seligman (7) have proposed the use of the esterase resistant 2-naphthyl myristate as a colorimetric substrate for the reliable assay of lipase in diagnosis of human pancreatitis. Since the myristate ester of naphthol was a specific, though less sensitive, reagent €or lipase, it was reasoned that the myrjstate and other higher molecular weight esters of N-methyl indoxyl (highly fluorescent molecules, esters of which had been recently proposed by Guilbault et al. (8) for cholinesterase) should be both sensitive and specific substrates for lipase. Hence, five new substrates were prepared as substrates for lipase: the hexanoate, heptanoate, octanoate, nonanoate, and myristate esters of N-methyl indoxyl. These substrates were compared to other esters for lipase (4-6, 7) from aspects of stability in solution, non-enzymic hydrolysis, rate of enzymic hydrolysis, specificity, and sensitivity. N-methyl indoxyl myristate was found to be the best substrate for lipase. Many colorimetric substrates have been described for sulfatase : nitrocatechol sulfate (P), p-nitrophenyl sulfate (101, and p-acetylphenyl sulfate (11). Leaback (12) and Sherman and Stanfield (13) have proposed the use of 4-methyl umbelliferone sulfate as a fluorometric substrate for the assay of sulfatase. We have prepared five fluorometric substrates for sulfatase: the sulfate esters of indoxyl, @-naphthol, 4-methyl umbellif(1) G. G. Guilbault, ANAL.CHEM., 38, 527R (1966). (2) Zbid., 40, 45913 (1968). (3) 6. G. Guilbault, “Enzymatic Methods of Analysis,” Pergamon
Press, London, New York, 1969. (4) D. Kramer and G. G. Guilbault, ANAL.CHEM., 35, 588 (1963). (5) 0. G. Guilbault and D. Kramer, ibid., 36, 409 (1964). (6) G. G. Guilbault, M. H. Sadar, and D. Arcenaux, Anal. Lett., 1, 551 (1968). (7) H. A. Ravin and A. M. Seligman, Arch. Biochem. Biophys., 42, 337 (1953). (8) . . G. G. Guilbault, M. H. Sadar, R. Glazer, and C. Skou, Anal. Lett., 1, 365 (1968). (9) A. B. ROY.Biochem. J., 62, 41 (1956). (10) Y . A. Leon, R. D. Bdbrook, and E. D. Comer, Biochem. J . , 75, 612 (1960). (11) K. S. Dodgson and B. Spencer, ibid., 55, 315 (1953). (12) D. H. Leaback, ibid., 78, 22 (1961). (13) W. R. Sherman and E. F. Stanfield, ibid., 102,905 (1967). 2006
erone, fluorescein, and resorufin, and have compared these with each other and with other colorimetric substrates for sulfatase. Of these 0-naphthyl sulfate and 4-methyl umbelliferone sulfate appear to be optimum for assay of various types of sulfatase. As little as lod4unit per ml of lipase or unit per ml of sulfatase can be determined by a direct reaction rate method in 2-3 minutes with a precision and accuracy of about 1.5 %. EXPERIIVIENTAL
Enzymes. LIPASE. Stock 1 mg/ml solutions were prepared from porcine pancreas [Calbiochem Co., activity 2.0 Wilson units per mg-one unit represents 0.05 meq of titratable fatty acid formed from the action of enzyme powder on 1.0 ml of neutral olive oil in 30 minutes at 37 ‘C,as assayed by the procedure of Lazo-Wasem (1411. Triply distilled water was used for all solutions. SULFATASE.Stock 5 mg/ml solutions were prepared from limpets (Type 111, Sigma, 5 unit/mg-one unit represents 1 pmole of p-nitrocatechol sulfate cleaved per hour at pH 5.0 and 37 “C) and from Helix Pomatia (Type H-1, Sigma, 15 unit/mg). All dilutions were made with triply distilled water. Substrates. The sulfates of 4-methyl umbelliferone and 0-naphthol were prepared by a procedure similar to that of Sherman and Stanfield (13). The products were identified using IR, NMR, and mass spectrometry. Elemental analysis provided further proof of isolation of the desired compounds. Results agreed within experimental error. Mass spectral analysis was accomplished with a Bendix Timeof-Flight mass spectrometer equipped with a solid sample injection system. A low ionization voltage (10 eV) was used to avoid sample decomposition and the parent ion was used for identification. Stock solutions of these sulfate esters were prepared in methyl cellosolve at a concentration of 10-lM. N-methyl indoxyl acetate, propionate, and butyrate esters were prepared as described in a previous paper (8). The heptanoate ester of 4-methyl umbelliferone was obtained from T. J. Jacks and the Southern Regional Research Labs (15). Fluorescein dibutyrate was obtained from Eastman Organics. The hexanoate, heptanoate, octanoate, nonanoate, and myristate esters of N-methyl indoxyl were prepared from N-methyl indoxyl acetate and the acyl chloride in a procedure similar to that used for N-methyl indoxyl butyrate (8). The N-methyl fatty acid esters are oils and cannot be purified by recrystallization and not easily by vacuum distillation. They were best purified by absorption chromatography using a neutral aluminum oxide column and elution with pet. ether. The oils are light sensitive and should be protected from light before use. The fatty acid esters were identified by elemental analysis and IR spectra. The IR identification was based on the shift of the C-0 stretching frequency of the ester. For the acetate ester (starting material), the stretching frequency is about 8.13 microns. In the fatty acid esters, this stretching frequency is shifted to about 8.60 microns with disappearance of the absorption at 8.13 microns. The remainder of the (14) E. A. Lazo-Wasem, J. Pharm. Sci., 50, 999 (1961).
ANALYTICAL CHEMISTRY, VOL. 41, NO. 14, DECEMBER 1969
Table I. Comparison of Various Substrates for Lipase Lowest Fluorescence detectable Substrate wavelengths Blank" VIllZXb concn (pgiml) Km (mM) 0.080 1.7 x 4.0 7.0 =t0 . 5 X Fluorescein dibutyrate ,A, = 490mp , A, = 520 mp 0.01 9.6 x 10-5 0.035 7.3 i 0.5 X 4-Methyl umbelliferone ,A, = 330 mp hem = 450 mp heptanoate 0.014 3.6 x 10-4 0.033 1.4 =t0 . 3 X N-Methyl indoxyl acetate A, = 430 mp ,A, = 500 m M 0.050 1 . 2 A 0 . 3 x 10-5 Same 0.0 2.2 x 10-4 N-Methyl indoxyl hexanoate 0 050 2.1 i.0.3 x 10-5 Same 0.0 1.7 x 10-4 N-Methyl indoxyl heptanoate 3.0 i 0.3 X lod6 N-Methyl indoxyl octanoate Same 0.0 1.65 x 10-4 0.025 0.050 2.2 k 0 . 3 x 10-5 Same 0.0 1.6 x 10-4 N-Methyl indoxyl nonanoate 0.10 2 . 2 =I 0 . 3 x 10-5 1.2 x 10-4 Same 0.0 N-Methyl indoxyl myristate a Rate of nonenzymic hydrolysis expressed in A fluorescence units per minute. Maximum rate in moles per liter of substrate hydrolyzed per mg of porcine pancreas lipase per minute. Blank rate subtracted. Rate calculated by dividing the observed rate in A fluorescence per minute by the total fluorescence per M of each product of hydrolysis, pH = 7.5. I
Table 11. Comparison of Various Substrates for Sulfatase Range Substrate Fluorescence Sulfatase of concn. (sulfate K salt) wavelengths (type) VmaXa (unit/ml) M K , (mM) Indoxyl A, = 380mp I11 2 x 10-6 0.040 -1.40 1.4 k 0.2 X ,A, = 470 mp H-1 7.5 x 10-6 0.06 -1.50 1 . 4 zk 0 . 2 X 0.0015-0.10 1.4 i.0.2 x 10-4 4-Methyl umbelliferone A,, = 338mp I11 2 . 7 x 10-6 0.0015-1.5 1.2 zk 0 . 2 x 10-5 ,A, = 451 mp H- 1 2.2 x 10-6 0.015 -3.0 5.4 i 0 . 3 x 10-5 fi-Naphthyl A, = 325 mp H-1 5.6 x 10-5 ,A, = 410mp Blank rate subtracted. Rate calculated by dividing a Maximum rate in moles per liter of substrate split per mg of sulfatase per minute. the observed rate in A fluorescence per minute by the total fluorescence per M of each product of hydrolysis, pH = 5.4.
spectra is essentially identical. Further identification was provided by NMR which easily indicates the presence of any acetate ester in the fatty acid ester. None was observed in any of the fatty esters. The melting point of N-methyl indoxyl acetate is 58 OC, that of N-methyl indoxyl myristate 53-54 OC. The other esters are oils which melt below 30 "C, and decompose upon heating. Elemental analysis Qn all compounds agree with the structures proposed within experimental error. Stock solutions of all esters (lO-*M) were prepared in methyl cellosolve. The substrates : N-methyl indoxyl acetate and myristate, 4-methyl umbelliferone sulfate, and 0naphthyl sulfate are now available commercially from Isolabs, Inc., Elkhart, Ind. Apparatus. All fluorescence measurements were made with an Aminco Bowman spectrophotofluorometer (SPF) grating instrument with a Xenon lamp and a Beckman linear recorder. A constant temperature of 25 "C was maintained with a thermo-electric cooler. The excitation and emission wavelengths used are given in Tables I and 11. Procedure. COMPARISONS OF SUBSTRATES. Test solutions of the various substrates were prepared by diluting stock solutions with buffer. The blank rate was determined by recording the change in fluorescence with time, AF/min, over a period of 5 minutes. Then 0.1 ml of a stock solution of porcine pancrease or sulfatase was added and the rate of enzymic cleavage determined by dividing the observed rate, AFlmin, or AAbs/min, by the fluorescence of hydrolyzed product per concentration of original substrate in M or the molar absorptivity. The lowest detectable concentration of lipase reported is that concentration required to give an enzymic rate twice that of the blank rate (Le., a AF/min of 0.10 if the blank is 0.05). The molar fluorescence of Nmethyl indoxyl and o-naphthol is 1 X 106M-', for 4-methyl
umbelliferone it is 1 x 106M-1 on the SPF used under the conditions of assay specified. DETERMINATION OF LIPASE. Three milliliters of 0.1M phosphate buffer, pH 7.5, and 0.1 ml of a 10-ZM solution of N-methyl indoxyl myristate are placed in a fluorescence cell in the SPF, and the instrument is adjusted to read zero. At zero time 0.1 ml of the lipase to be assayed is added, and the change in the fluorescence with time, AF/min, is automatically recorded. A calibration plot of AF/min 6s. enzyme concentration is made and used for all enzyme analyses. DETERMINATION OF SULFATASE. Three milliliters of 0.1M acetate buffer, pH 5.4, and 0.1 ml of a 10-IM solution of pnaphthyl or 4-methyl umbelliferyl sulfate are used in a procedure identical to that used above. One-tenth ml of sulfatase is assayed and the enzyme concentration is calculated from a calibration plot as above. RESULTS AND DISCUSSION
Substrates for Lipase. The results obtained in a comparison study of various substrates for lipase are listed in Table I and Figure 1. From aspects of stability, the 4methyl umbelliferone heptanoate and all the N-methyl indoxyl esters are extremely stable in solution, whereas the fluorescein dibutyrate hydrolyzes in solution at a fair rate. The N-methyl indoxyl esters may be stored as stock solutions in methyl cellosolve for weeks with no apparent hydrolysis. Moreover, none of the esters had an appreciable rate of nonenzymatic hydrolysis at pH 7.5. Of all the substrates tested, fluorescein dibutyrate had the best K, value, but 4.0 wg/ml of porcine pancreas was required to give an enzymic rate twice that of the blank rate. The
ANALYTICAL CHEMISTRY, VOL. 41, NO. 14, DECEMBER 1969
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250
5.0
Oct 200
4.0
nF/min
AFWn 3.0
IO-^ 2a
100
1.0
50
0.5
4.c
3 .O
1.0
pg/ml
Figure 1. Plot of rate of lipase catalyzed hydrolysis of 5 N-methyl indoxyl esters, AF/min, GS. enzyme concentration in m/ml A - - A Heptanoate ester X X Hexanoate ester 0 o Octanoate ester o--.--o Nonanoate ester 0--O Myristate esker
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highest rates of enzymic hydrolysis were observed with the N-methyl indoxyl esters, the rate decreasing in the order acetate > hexanoate > heptanoate > octanoate > nonanoate > myristate. The most sensitive ester is N-methyl indoxyl octanoate. As little as 2.5 X pg/ml can be detected using this ester compared to 0.1 pg/ml with the myristate
Table 111. Effect of Various Enzymes on Hydrolysis of N-Methyl Indoxyl Esters AFlmin.
Enzymea Lipase, porcine pancreas (Calbiochem) Cholinesterase, horse serum (Sigma) Cholinesterase,bovine erythrocyte (Sigma) Cholinesterase, bee (these labs) Cholinesterase.chicken liver (these labs) Cholinesterase,eel
Acetate Heptanoate Myristate 1.8 1.o 0.8 4 2
0.6
0
1 .o
0
0
1.03
0
0
5.2
1.8
0
1.2
0
0
Chymotrypsin (Sigma) 0 0 a Concentration of all enzymes 0.031 mg/ml.
0
I
(Sigma)
Figure 2. Plot of rate of sulfatase (type H-1) catalyzed hydrolysis of 3 sulfate esters, AF/min, us. enzyme concentration in Pg/ml 0--0 4-Methyl umbelliferone sulfate A - - A @-Naphthylsulfate 0--0 Indoxyl sulfate
-
ester or 5 X pg/ml using the nonanoate ester. However, more linear, reproducible results were obtained with the latter 2 esters (Figure 1). The nonlinearity observed with the lower esters (hexanoate, heptanoate, and octanoate) is probably due to the action of esterase impurities in the lipase on the esters yielding an erratic response. Using optimum substrate concentrations and pE1, from 0.050 to 5.0 pg/ml of porcine pancrease lipase could be assayed with N-methyl indoxyl nonanoate and 0.2 to 5.0 pg/ml with N-methyl indoxyl myristate with an accuracy of about 1.5% and a precision of about 1.2%. This represents an increase in 3 orders of magnitude over conventional titrimetric and manometric methods for lipase (2-6). With all of the N-methyl indoxyl esters a pH of 7.5, phosphate buffer, 0.1M) appeared to yield the lowest non-enzymic rate and the highest rate of enzymic cleavage. A substrate concentration of 3.3 X 10-4M(0.1 ml of stock 10-2M diluted to 3 ml) was found to be optimum for all esters, except N-methyl indoxyl acetate (3 X 10-aM). High rates of non-enzymic hydrolysis were observed with phosphate buffer solutions in which precautions against growth of micro-organisms were not taken. This nonenzymic hydrolysis is probably due to enzymes in the microorganisms. Lipases from certain plants (Le., peanut) are inhibited by phosphate ion (15),however; in a measurement of the activity of such enzymes use of tris rather than phosphate buffer is necessary. (15) T. J. Jacks andH. W. Kircher, Anal. Biochem., 21,279 (1967).
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The effect of various enzymes on the hydrolysis of N-methyl indoxyl-acetate, -heptanoate and -myristate under the optimum conditions is given in Table 111. All enzymes used were of the highest available purity. In addition to lipase, other esterases (horse serum, bovine erythrocyte, bee, chicken liver, and eel cholinesterases) catalyze the hydrolysis of N-methyl indoxyl acetate, making it a poor substrate for the specific assay of porcine pancreas lipase. The heptanoate ester is more selective, but, like @-naphtholmyristate (7), the myristate ester of N-methyl indoxyl is completely specific for lipase, and is not hydrolyzed by other esterases. Thus this substrate appears to be an excellent one for the assay of lipase. Substrates for Sulfatase. Because fluorescence methods have been shown to be from 3 to 4 orders of magnitude more sensitive than colorimetric techniques for the assay of enzymes (1-3), it was reasoned that the use of fluorogenic substrates would provide the ultimate in sensitivity for assay of sulfatases. Since fluorescein, resorufin, indoxyl, @-naphthol,and umbelliferone are the most fluorescent molecules known, we attempted the preparation of the sulfate esters of these substances. These substrates were compared with respect to stability, Michaelis constant (Km)for the enzyme-substrate complex, spontaneous hydrolysis, and rate of enzymic cleavage. The results of this study are summarized in Table I1 and Figure 2. All five substrates were very stable, both when stored as a stock solution in methyl cellosolve as well as in solution at pH 5.4. Of all the fluorogenic substrates tested, 4-methyl umbelliferone sulfate had the best Km value, indoxyl sulfate the poorest. The Km obtained for 4-methyl umbelliferone sulfate, 1.4 X IO-4, is lower than that obtained by Sherman and Stanfield (13) with the same enzyme. The reason for the difference is not evident. The highest rate of enzymic hydrolysis was observed with @-naphthylsulfate, V,,, decreasing in the order @-naphthyl sulfate > 4 MUS > indoxyl sulfate > nitrocatechol sulfate > p-nitrophenyl sulfate. The most sensitive substrate is 4 MUS, as little as unit per ml of sulfatase being detectable, compared to 10-2 unit per ml using @-naphthylsulfate and 0.04 unit per ml using indoxyl sulfate. The better sensitivity offered by 4 MUS over @-naphthylsulfate was due to the greater fluorescence of 4 MU. The observed rate of change in fluorescence with time, AFjmin, is greater with 4 MUS than @-naphthyl sulfate even though the V,,, (moles
per liter per mg of enzyme per minute) is higher with the latter substrate. More linear, reproducible results are obtained using @-naphthylsulfate (accuracy and precision about 1 %) than 4 MUS [accuracy and precision about 2-3 % (Figure 2)]. The optimum pH for both limpet and Helix Pomatia sulfatase was found to be 5.4 in 0.1M acetate buffer in agreement with that found by others (9-13). The optimum substrate concentrations were 1 X 10-4Mfor 4 MUS (Type H-1 enzyme), 1 X 10-3M for 4 MUS (Type I11 enzyme), 1 X 10-8Mfor @-naphthylsulfate (Types H-1 and III), 1 X 10-aM for indoxyl sulfate (Type H-l), and 1 X 10-2M for indoxyl sulfate (Type 111). Using optimum substrate concentrations and pH, from 0.0015-0.10 unit per ml of Type I11 sulfatase and from 0.00151.5 unit per ml of Type H-1 sulfatase can be assayed using 4-methyl umbelliferyl sulfate with a precision of 2% and an accuracy of 2.5 % by an initial reaction rate method in 2 to 3 minutes. From 0.015-3.0 unit/ml of Type I11 or H-1 sulfatase can be assayed with @-naphthylsulfate with a precision of about 0.9 % and an accuracy of about 1.2 %. The sensitivity of the assay can be increased by measuring the fluorescence of 4-methyl umbelliferone at a pH of 10 (13) or by a preincubation step as described by Sherman and Stanfield (13)to a theoretical limit of 2 X 10-6 unit detectable with a 100-min incubation. However, it is judged that the current limit of lova unit per ml determinable directly is sufficient for most analysis. Finally, the effect of other enzymes on the hydrolysis of the sulfate esters was tested under the optimum conditions. No other enzymes, butycholinesterases (horse, cow, human, insect), acetylcholinesterases (beef, eel), lipase (pancreas), acid or alkaline phosphatases, oxidases, etc., had any effect on any of the esters. Thus 4-methyl umbelliferone sulfate and @-naphthyl sulfate appear to be excellent substrates for the assay of sulfatases. ACKNOWLEDGMENT
The authors thank M. H. Sadar and D. Hackney who assisted in some of the experiments. RECEIVED for review April 23, 1969. Accepted September 19, 1969. The financial assistance of the Office of Saline Water, Grant No. 1401-0001-1337,is gratefully acknowledged.
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