New colored and fluorescent amine substrates for activated fibrin stabilizing factor (Factor XIIIa) and for transglutaminase

ANALYTICAL

BIOCHEMISTRY

131,419-425

(1983)

New Colored and Fluorescent Amine Substrates for Activated Stabilizing Factor (Factor XIII,) and for Transglutaminase

Fibrin

L. LORAND,K. N. PARAMESWARAN, P.T. VELASCO, L. K.-H. Hsu, AND G. E. SIEFRING, JR. Department of Biochemistry, Molecular. and Cell Biology, Northwestern University, Evanston, Illinois 60201 Received October 5. 1982 Two fluorescent (FITC and 6-chloro-2-methoxyacridine) and an intensely colored (dabsyl) derivative of cadaverine were synthesized. following earlier work from this laboratory with dansylcadaverine, in order to enlarge the scope of possibilities for labeling some y-glutamine sites in proteins. Enzyme affinities of the amine substrates for human Factor XIII, and for guinea pig liver transglutaminase were measured. The utility of dabsylcadaverine was further demonstrated by activity staining of these enzymes. following electrophoresis in agarose, and by measuring the Factor XIII zymogen of human plasma calorimetrically.

The synthesis of novel amines played an important role in research with enzymes of the endo-y-glutamine:t-lysine transferase class. The fluorescent amine: dansylcadaverine or N-(5-aminopentyl)-5-dimethylaminolnaphthalenesulfonamide (l), for example, proved to be an excellent substrate for all known transamidases of this type (such as fibrinoligase, i.e., thrombin and Ca*+-activated fibrin stabilizing factor or Factor XIII, and transglutaminase). Among numerous other uses, it has often been employed for the enzyme-directed site specific modification of yglutamine residues in proteins (2,3). Nevertheless, there could be circumstances in which it might be preferable to use a substrate containing a fluorophore other than the dansyl group or else a chromophore with a high extinction coefficient. In this paper, we report the synthesis of two fluorescent (I and II) and an intensely colored (III) monosubstituted derivative of diaminopentane (i.e., cadaverine): (I) N-(5-aminopentyl)-N’-(S-fluoresceinyl)thiourea or N-(fluoresceinylthiocarbamoyl)- 1,5-diaminopentane or FITCcadaverine, (II) 9-(5-aminopentylamino)6 - chloro - 2 - methoxyacridine, and (III) N- (5 - aminopentyl) - 4 - (p- (dimethylamino)419

phenylazo)benzene sulfonamide or dabsylcadaverine. Enzyme affinities and general utilities of these new compounds were evaluated. MATERIALS

AND

METHODS

Purifications of the fibrin stabilizing factor zymogen (i.e., Factor XIII) from outdated human plasma (4) and of guinea pig liver transglutaminase (5) were carried out by published procedures. Human cr-thrombin was a gift of Dr. J. W. Fenton II of the New York State Department of Health, Albany, New York. Dimethylation of Hammarsten casein was performed by treatment with formaldehyde and sodium borohydride (6). Salts, buffers, and reagents were of the highest purity available. Thin-layer chromatographic analysis was carried out on sheets of EM silica gel F-254 (Eastman-Kodak) using a variety of solvent systems: A-chloroform/isopropanol/glacial acetic acid 17/ l/ 1 (v/v/v); B-anhydrous ether/absolute ethanol 14/4 (v/v); C-anhydrous ether/absolute ethanol/concentrated ammonium hydroxide 14/4/2 (v/v/v); Dchloroform/isopropanol/glacial acetic acid 17/ 0003-2697183 $3.00 Copyright All

rights

0

I983

of reproductv~n

by

Academic in any

Press. form

Inc. reserved.

420

LORAND

3/l (v/v/v); E-n-butanol/glacial acetic acid/ water 4/ l/ 1 (v/v/v); F-n-butanol/glacial acetic acidfwater/pyridine 30/6/24/20 (v/v/v/ v); and G-anhydrous ether/absolute ethanol/ 58% NH40H 70/20/10 (v/v/v). Enzyme affinities were evaluated by competitive kinetics against the incorporation of [ 14C]putrescine into iV,N’-dimethylcasein, as described by Lorand et al. (7), and were measured in relation to dansylcadaverine as reference compound. RESULTS

A. Synthesis of New Compounds (I) The trijluoroacetate ine.

Ho ‘THO

\

of FITC-cadaver-

COOH

/

o/

HN-i-NH(CH2J5NH2.

CFjCOOH

Fluorescein isothiocyanate ( 160 mg, 0.4 1 mmol) was added to a partial solution of tBoc-cadaverine hydrochloride (100 mg, 0.42 mmol) in 18 ml of dry tetrahydrofuran and 0.12 ml (0.86 mmol) of triethylamine. The reaction mixture was stirred at room temperature for 48 h and evaporated under reduced pressure. The residue was mixed with 40 ml of water and 10 ml of cold 0.5 N hydrochloric acid and was extracted with ethyl acetate (3 X 50 ml). The organic extract was washed with saturated sodium chloride (2 X 50 ml), dried, filtered, and the filtrate was concentrated to a 6-ml volume under reduced pressure. After adding 20 ml of petroleum ether to the concentrate and cooling, the yellow precipitate was filtered off to give 185 mg (76%), m.p. 214-2 16°C. TLC’ showed one ’ Abbreviations used: TLC, thin-layer chromatography; t-Boc, tertiary butyloxycarbonyl; dabsyl, 4-p(dimethylamino)phenylazobenzenesulfonyl; dansyl, 5-dimethylamino-l -naphthalenesulfonyl; FITC, fluoresceinylthiocarbamoyl.

ET

AL.

fluorescent spot with Rf’s of 0.54 in system D, 0.84 in E, and 0.88 in F. This precursor was deblocked for 0.5 h at room temperature with 50% (v/v) trifluoroacetic acid in dichloromethane and the mixture was evaporated under reduced pressure at room temperature. To the residue was added 30 ml of anhydrous ether and cooled. The bright orange precipitate was filtered off, washed with anhydrous ether and redissolved in anhydrous methanol (20 ml) at room temperature. Heating was avoided alter preliminary tests indicated slight decomposition of the product, as evidenced by TLC. The methanolic solution was filtered to remove a small amount of insoluble material and the filtrate was concentrated to 2 ml under reduced pressure at room temperature. Anhydrous ether (25 ml) was added to the above concentrate, cooled, and the crystallized product was filtered off, washed with anhydrous ether and dried under vacua to give 70 mg (59%) m.p. 180-183°C (dec.). TLC showed one fluorescent spot coincident with the ninhydrin positive spot and with Rf’s of 0.08 in system D, 0.49 in E, and 0.67 in F which also revealed a trace of a fluorescent tail. In 50 mM Tris-HCl, pH 7.5, the absorption spectrum showed X,,, = 493 nm (t = 75,560); at 238 nm, E = 53,300. Fluorescence maxima were: X,,, = 490 nm, &, = 520 nm. Anal. Calcd for CZ8HZ6F3N307S: C, 55.53; H, 4.33; N, 6.94. Found: C, 55.01; H, 4.58; N, 7.04. (II) The Dihydrochloride methoxyacridine cadaverine.

of 6-chloro-2-

HN(CH215NH2.2HCI I

pJyJ”;“ Cl

To a solution of 239 mg (1 mmol) of t-Boccadaverine hydrochloride in 10 ml of absolute ethanol were added 0.14 ml (1 mmol) of triethylamine and 278 mg (1 mmol) of 6,9-dichloro-2-methoxyacridine, followed by 7 mI

AMINE

SUBSTRATES

FOR

of dry tetrahydrofuran. After gentle heating of the mixture on a steam bath for 4 h, 1.5 g of phenol was added. The mixture was heated for a further 3.5 h while the reaction was followed by the disappearance of the ninhydrin positive spot due to t-Boc-cadaver-me on TLC. A further 50 mg portion of 6,9-dichloro-2methoxyacridine was added and the mixture was heated for another 6 h. After allowing to stand at room temperature for 18 h, the mixture was evaporated to give a brown solid residue. The latter was deblocked to remove the t-Boc group with 2.2 ml of 6 N hydrochloric acid at room temperature for 0.5 h and mixed with water (70 ml) and chloroform ( 100 ml). The chloroform layer was extracted once more with 0.1 N hydrochloric acid (75 ml). The combined aqueous acidic extract was made basic (pH 11) with cold 4 N sodium hydroxide and extracted with chloroform (5 X 50 ml). After the TLC examination of the chloroform extract revealed impurities, it was reextracted with 0.5 N hydrochloric acid (3 X 30 ml), the extract was basified and extracted again with (III)

The hemifumarate

TRANSGLUTAMINASES

421

chloroform as above. After a work-up, followed by evaporation of the chloroform, the oily residue was redissolved in a mixture of isopropanol (2 ml) and anhydrous ether (25 ml). Anhydrous hydrogen chloride in tetrahydrofuran was added until the pH of the solution was 2.0. After cooling, the precipitated product was collected, washed with anhydrous ether, dried, and recrystallized from a mixture of anhydrous methanol, isopropanol and anhydrous ether to give 230 mg (55%) of bright yellow solid with a m.p. 258-260°C. TLC showed one fluorescent spot coincident with the ninhydrin positive spot with R,‘s of 0.72 in system G, 0.07 in A, and a trace of ninhydrin negative fluorescent spot in front. The absorption spectrum in 50 InM Tris-HCl, pH 7.5, showed the following maxima and shoulders (Sh): 265(Sh), 278 (t = 38,800) 325(Sh), 340 (c = 3,860) 400(Sh). 420 (t = S,SOO), and 440 (E = 7,710) nm. In the same solvent the fluorescence maxima were: X,,, = 420 and X,, = 490 nm. Anal. Calcd for Cr9H2&13N30: C, 54.75; H, 5.80; N, 10.08. Found: C, 54.58; H, 5.73: N, 10.04.

of dabsylcadaverine.

First, dabsyl chloride or 4-p-(dimethylamino)phenylazobenzenesulfonyl chloride was prepared by adding 30 ml of phosphorous oxychloride to 16.35 g (50 mmol) of the sodium salt of methyl orange. The mixture was warmed to 50-60°C with drying tube protection for 3 h, cooled to room temperature and poured into 400 ml of water, containing crushed ice, and using small amounts of cold water to complete transfer of the reaction product. The mixture was then neutralized with a saturated solution of sodium bicarbonate with stirring and cooling. The precipitated dabsyl chloride was quickly extracted with dichloromethane (5 X 10 ml). The extract was washed with water (2 X 200 ml) and

dried over anhydrous sodium sulfate. The dried extract was filtered and the filtrate was concentrated to a 50-ml volume. Upon addition of petroleum ether (70 ml; b.p. 3060°C) and cooling, the precipitated dark red crystals were filtered off, washed with petroleum ether, and dried to give 10.81 g (66%), m.p. 194-196°C (dec.); Lit. J. K. Lin and J. Y. Chang(S), m.p. 186-188°C. TLCshowed a single spot with R, = 0.84 in solvent system A and 0.89 in solvent system B. Dabsyl chloride (1.62 g, 5 mmol) was dissolved in 600 ml of dry benzene and added, dropwise, over a period of 5 h to a stirred solution of 3 ml (25 mmol) of 1,5-diamino-

422

LORAND

pentane in 50 ml of benzene. The reaction mixture was stirred for 18 h and was extracted with 300 ml of 0.1 N hydrochloric acid. The benzene layer was reextracted once more with hydrochloric acid ( 100 ml) and the combined aqueous acidic layer was extracted with ether ( 100 ml). The bis-dabsylcadaverine byproduct thus remained in the organic extract. The aqueous layer (pH 2.0) was made basic with sodium carbonate to pH 11 .O. It was then extracted with dichloromethane (4 X 200 ml). The combined organic extract was washed first with a 1: 1 mixture of saturated sodium bicarbonate and saturated sodium carbonate solution (2 X 200 ml) and then with saturated sodium chloride solution. The separation of dabsylcadaverine from unreacted cadaverine was monitored by TLC which showed an R, of 0.35 for the former and an Rf of 0.06 for the latter in system C. The extract was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness under reduced pressure at 80°C to give bright red crystals of the presumed free base form of dabsylcadaverine. The above product was redissolved in a mixture of hot isopropanol and anhydrous methanol (50 ml of each). To the clear solution was added a hot solution of 580 mg (5 mmol) of fumaric acid in 15 ml of anhydrous methanol which resulted in an orange-colored precipitate almost immediately. The crystallization mixture was cooled to 4°C for 18 h and the crystals were filtered off,

ET AL.

washed with cold anhydrous methanol and dried first by suction and then at 100°C for 30 min to give 1.54 g (69%) of bright orange crystals, m.p. 232-234°C (dec.). TLC gave a single ninhydrin positive spot with an R, = 0.35 in solvent system C. In 50 mM TrisHCl, pH 7.4, the absorption spectrum showed x max= 470 nm and 6 = 23,900; in 0.1 N HCl x mm = 502 nm and c = 46,250. Anal. Calcd for C21H29N504S: C, 56.35; H, 6.53; N, 15.65. Found: C, 56.04; H, 6.51; N, 15.60. B. Enzyme Studies 1. Competitive inhibition. Assays with either purified human fibrinoligase (i.e., activated fibrin stabilizing factor or Factor XIII,) or with guinea pig liver transglutaminase were used to show that the three newly synthesized amines could, indeed, serve as substrates for endo-y-glutamine:c-lysine transferases of this type. Kinetic analysis of inhibition against the incorporation of [ i4C]putrescine into N, N’-dimethylcasein (7) revealed that all of them were good competitive inhibitors. Apparent K; values are presented in Table 1 in comparison to dansylcadaverine as reference compound. 2. Activity staining. Inasmuch as dansylcadaverine fluorescence has previously been exploited for the electrophoretic identification of enzyme activity in agarose as well as in polyacrylamide gels (9), it was of interest to find out if the high color intensity due to

TABLE 1 APPARENT K, VALUES MEASURED FOR THE INHIBITION OF [“‘CIPVTRESCINE INCORPORATION INTO N,N’-DIMETHYLCASEIN BY MONOSUBSTITUTED CADAVERINES, AT pH 7.5, USING EITHER GUINEA PIG LIVER TRANSGLUTAMINASE (TGase) OR HUMAN FIBRINOLIGASE (FACTOR XIII.) AS ENZYMES”

Compound Reference I II III

Substituent group Dansyl FITC 6-Chloro-2-methoxyacridine Dabsyl

o For details of methodology, see Lorand et al. (7). b Not determined.

With TGase 4 4 10 NDb

With Factor XIII, 9 18 27 18

AMINE

SUBSTRATES

FOR TRANSGLUTAMINASES

the methylorange substituent in compound III (i.e., dabsylcadaverine) would allow for a direct staining of Factor XIII, and of transglutaminase activities without the necessity of resorting to uv illumination. Following electrophoresis (2 V cm-‘, 4°C 2 h) on a 1% agarose-0.3% N,N’-dimethylcasein matrix, the gel was treated at 37°C for 1 h with a solution containing 0.2 mM dabsylcadaverine, 20 mM CaC& ,2 mM dithiothreitol, and 14 NIH units/ ml of thrombin in a buffer of 0.05 M TrisHCl, pH 7.5. Thrombin was included only to promote enzyme generation from the Factor XIII zymogen. The gel was fixed in 45% (v/ v) ethanol-lo% (v/v) acetic acid for 30 mitt, pressed, and dried in warm air exactly as described previously for dansylcadaverine (9). Destaining was completed by further washing

+

1

2

FIG. 1. Color staining of enzymes with dabsylcadaverine, following electrophoresis. For experimental details, see text. In track 1, human Factor XIII (4 pg protein in 10 ~1 solution) and in track 2. guinea pig liver transglutaminase (10 pg protein in 10 ~1 solution) were applied.

423

with ethanol-acetic acid, with the addition of a few drops of dilute HCl to enhance the red color of enzyme bands. As seen in Fig. 1, the appearance of the colored electrophoretogram was the same as the fluorescent pattern reported for dansylcadaverine. 3. Calorimetric assay for Factor XIII in human plasma. It was thought that some clin-

ical laboratories might find it advantageous if, in addition to the fluorescent technique based on dansylcadaverine for measuring Factor XIII in human plasma samples (lo), a colorimetric method of comparable sensitivity was also available. The protocol with compound III (i.e., dabsylcadaverine) was patterned on the earlier use of dansylcadaverine. To 0.4 ml of titrated human plasma 0.1 ml of 55% glycerol was added and the mixture was warmed at 56°C for 4 min. It was placed on ice for the purpose of rapid cooling to 22°C (room temperature), and 0.35 ml of buffer (0.05 M Tris-HCl, pH 7.8, containing 10% glycerol), 0.1 ml of 0.2 M fresh glutathione, 20 ~1 of thrombin (1000 NIH units/ml of 0.75 M NaCl) were added. Following 20 min of incubation, 0.54 ml of buffer, 40 ~1 of dabsylcadaverine (compound III dissolved in 0.1 N HCl), 0.4 ml of 2% N,N’-dimethylcasein and 20 ~1 of 0.16 M CaC& were admixed. The glutathione, CaC12, and casein derivative were dissolved in the glycerol containing Tris buffer. Incorporation of dabsylcadaverine was allowed to proceed at 22°C and, at the desired time (see Fig. 2) the reaction was terminated by adding 2 ml of ice-cold 10% trichloroacetic acid, followed by centrifugation. Free dabsylcadaverine was removed by repeated washing of the precipitate with 10 ml of ethanol/ acetone (50/50) containing 0.1 N HCl, each time followed by centrifugation, until the supematant was no longer colored. The precipitate was then washed with acetone and dried (50-60°C). The dabsylcadaverine-labeled protein was taken up in 0.75 ml of trypsin (0.22 mg/ml) in 0.1 M ammonium bicarbonate (pH 8) and was digested at 37°C for 18 h, when 0.75 ml of 9 M urea in 0.3 N HCl was added. Absorbancy of the solution was

424

LORAND

MIN

FIG. 2. Progression curve for the incorporation of the highly colored dabsylcadaverine into N,N’-dimethylcasein, as catalyzed by the Factor XIII. generated in normal human plasma. For experimental details, see text.

read at 502 nm against a standard curve of dabsylcadaverine (compound III; 0- 10 PM) solution made up similarly in an equal volume of 0.1 M ammonium bicarbonate and 9 M urea in 0.3 N HCl. Nonenzymatic controls were prepared by substituting 20 ~1 of 0.05 M EDTA (in 10% glycerol containing 0.05 M Tris-HCl, pH 7.8) for CaC12 throughout the procedure. The time curve for a human plasma specimen with normal fibrin stabilizing factor (Factor XIII) content is presented in Fig. 2. It can be seen that the incorporation of dabsylcadaverine proceeded in a linear fashion for the duration of the experiment and, even though a negligible volume (0.4 ml) of plasma was employed as source of the Factor XIII zymogen, a sufficient amount of dabsylcadaverine-labeled, red-colored protein was produced to yield an optical density of about 0.2 AU in 15 min of reaction. DISCUSSION

In addition to the crosslinking of proteins, endo-y-glutamine:+lysine transferases show an activity for incorporating amines into certain y-glutamine sites of a number of protein substrates. These enzymes are widely distributed in nature as both extra- and intracellular constituents, and several have already been isolated. Human fibrinoligase (activated fibrin stabilizing factor or coagulation Factor

ET AL.

XIII,) and guinea pig liver transglutaminase have been studied rather extensively. They display different specificities for the glutamine residues of a given protein (2). The enzymes also show characteristic specificities for small molecular weight amine substrates and, in general, primary amines which carry large apolar substituents on an alkyl side chain of a length comparable to that of the lysine residue in proteins are the best. This was first demonstrated with dansylcadaverine or N-(5-aminopentyl)-5-dimethylamino-lnaphthalenesulfonamide which was synthesized in this laboratory for the specific purpose of the enzyme-directed labeling of reactive yglutamine sites in proteins with a dansyl fluorophore ( 1) and which has since gained wide acceptance as a substrate. In an effort to enlarge the scope of such labeling possibilities, we have undertaken the synthesis of two new fluorescent (i.e., compounds I and II) and an intensely colored derivative of cadaverine. FITC-cadaverine or N-(fluoresceinyl-thiocarbonyl)1,5-diaminopentane (I) is characterized by h,,, = 490 nm, &,, = 520 nm, and 6-chloro-2-methoxyacridine cadaverine or 9-(5-aminopentylamino)6-chloro-2-methoxyacridine (II) by h,,, = 420 = 490 nm. N-(5-Aminopentyl)4nm, A,, (p - ( dimethylamino ) phenylazo ) benzenesulfonamide, or dabsylcadaverine (III) is a highly colored methyl orange derivative with X,,, = 502 and t = 46,250 in acid media. As seen from the results presented in Table 1, all of these compounds displayed good affinities for the enzymes which were comparable to that of dansylcadaverine. While it was shown previously that the yellow-colored, 2,4dinitrobenzene derivatives of cadaverine can act as substrates for transglutaminase [see compounds in series C in Table II of the article by Lorand et al. (7)], none could match the color intensity of dabsylcadaverine. As presented in this paper, this methyl orange derivative could be readily employed for the direct straining of activated human fibrin stabilizing factor (coagulation Factor XIII,) and of transglutaminase in electro-

AMINE

SUBSTRATES

FOR TRANSGLUTAMINASES

phoretic gels (Fig. 1). Furthermore, the dabsylcadaverine substrate allowed the colorimetric determination of the Factor XIII zymogen in human plasma where previously only fluorescence (lo- 12) or isotope methodologies ( 13) were available. ACKNOWLEDGMENTS Thanks are due to Dr. M. Michalska and Dr. P. Stenberg and to Mrs. A. Quraishi for assistance. This work was aided by a U. S. Public Health Service Research Career Award (5KO6 HL-03512) and by a grant from the National Institutes of Health (HL-16346). REFERENCES 1. Lorand, L., Rule, N. G., Ong, H. H., Furlanetto, R., Jacobsen, A., Downey, J., Oner, N., and BrunerLorand, J. (1968) Biochemistry 7, 1214-1223. 2. Lorand, L., Chenoweth, D., and Gray, A. ( 1972) Ann. N. Y. Acad. Sci. 202, 155-17 1. 3. Fretto, L. J., Ferguson, E. W., Steinman, H. M., and McKee, P. A. (1978) J. Biol. Chem. 253, 21842195.

425

4. Curtis, C. G., and Lorand, L. (1976) in Methods in Enzymology (Lorand, L., ed.), Vol. 45, pp. 17719 1, Academic Press, New York. 5. Connellan, J. M., Chung, S. I., Whetzel, N. K., Bradley, L. M., and Folk, J. E. (1971) J. Biol. Chew. 246, 1093-1098.

6. Lin, Y., Means, G. E.. and Feeney, R. E. ( 1969) J. Biol. Chem. 244, 189-793. 7. Lorand, L., Parameswaran, K. N., Stenbcrg, P., Tong, Y. S., Velasco. P. T.. Jiinsson. N. A.. Mikiver. L.. and Moses, P. (1979) Biochem&y 18, 1756-1;65. 8. Lin, J. K., and Chang, J. Y. (1975) Anal. Chem. 47, 1634-1638. 9. Lorand, L., Siefring, G. E., Jr., Tong, Y. S., BrunerLorand, J., and Gray, A. J., Jr. (1979) Annl. Biochem. 93, 453-458. 10. Lorand, L., Urayama, T., de Kiewiet, J. W. C., and Nossel, H. L. (1969) J. Clin. Invest. 48, 10541064. 11. Lorand, L.. Urayama. T., Atencio, A. C., and Hsia, D. Y.-Y. (1970) Amer. J. Hum. Genel. 22,89-95. 12. Henriksson, P., Hedner, U., Nilsson, I. M., Boehm, J., Robertson, B., and Lorand, L. (I 974) Pediarric Rex 8, 789-79 1. 13. Lorand, L.. Campbell-Wilkes, L. K., and Cooperstein, L. (1972) Anal. B&hem. 50, 623-63 I.