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Colorimetric method for the assay of heparin content in immobilized heparin preparations
ANALYTICAL
BIOCHEMISTRY
Calorimetric
109,
466-473
(1980)
Method for the Assay of Heparin Immobilized Heparin Preparations
P. K. SMITH, A. K. MALLIA,AND Biochemical
Research
G.T.
July
in
HERMANSON'
Division, Pierce Chemical Cornpuny, Rockfbrd. Illinois 61105 Received
Content
P.O.
Bo.r 117.
18, 1980
The properties of the metachromatic dye toluidine blue have been utilized to determine calorimetrically the amount of heparin covalently coupled to Sepharose. The method involves monitoring the dye depletion in the supernatant at 631 nm as Toluidine blue is adsorbed onto the heparin polymer upon the beaded matrix. The procedure represents a simple assay technique which allows the direct quantitation of heparin in immobilized heparin preparations.
Heparin is a linear polysaccharide composed of repeating units of uranic acid, containing an o-sulfate group at the c-2 position, and D-glucosamine, usually N-sulfated with an additional o-sulfate group at c-6 (1). Due to its unique structure and surface charge distribution, heparin is able to interact strongly with several different proteins in a specific manner. The significance of heparin’s biological activities, especially its interaction with blood-clotting factors and human serum lipoproteins, has made this glycosaminoglycan the focus of many investigations (2-4). Recently, with the advent of affinity chromatographic techniques, heparin immobilized on insoluble beaded polymers has become very nearly an “allpurpose” ligand for the purification of a wide range of proteins and biomolecules (5-10). For this reason the heparin content in immobilized heparin preparations has become an important parameter to investigators using this affinity support. A number of methods are known for the determination of heparin in solution, and the methodology has been recently reviewed ’ The author addressed.
to whom
correspondence
0003-2697/80/180466-08$02.00/O Copyright D 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
should
be
466
(11). However, application of these methods to directly determine heparin covalently coupled to an insoluble support such as agarose is limited or nonapplicable. Techniques which have been developed for the determination of immobilized heparin have been for the most part indirect methods based on the described means of determining soluble heparin. Using this approach, the concentration of heparin in the reaction mixture prior to coupling is compared to the free heparin content left in the supernatant and washings after coupling. The difference is taken as the amount bound. Probably the most widely used such procedure (3,4,9,10,12- 15) involves the uranic acid carbazole reaction as described by Bitter and Muir (17) and later modified by Galambos (18). Although this method is accurate enough for determining dissolved heparin, the procedure is tedious and inconvenient in that concentrated sulfuric acid is required. Other investigators have used the dye-binding interactions of otoluidine blue (5,8,11,19-21) to fluorimetritally quantitate unbound glycosaminoglycan before and after coupling. Additional possibilities along this line involve the dyes acridine orange and alcian blue (22,23).
HEPARIN
CONTENT
IN IMMOBILIZED
However, all of these methods have one thing in common: they are designed to measure heparin in solution and not in its immobilized form on an insoluble beaded matrix. These may in fact yield spurious results if the washing procedure after coupling is not performed well, because free glycosaminoglycans have been shown to bind immobilized glycosaminoglycans (13). Attempts to directly measure heparin coupled to a support such as Sepharose have also surfaced in the literature. Several investigators (16,24,28) have used the sulfate analysis of Antonopoulos (25) or Terho and Hartiala (26) to estimate the amount of heparin coupled after subtracting the indigenous sulfate content of the matrix. These methods are very time consuming and the results may be suspect due to the high blank which can result from the Sepharose itself and the uncertain structure of the heparin polymer (1). In addition. the method may involve the known human carcinogen benzidine. Andersson et al. (27) determined bound heparin by amino acid analysis for glucosamine after hydrolysis in 2 M HCl at 110°C for 24 h. This method would seem to be the most accurate to date, but the limited availability of amino acid analyzers makes it inapplicable to most laboratories. Another recent technique (28) involved a potentiometric titration of the immobilized heparin. In this, the amount of base needed to achieve equivalence as obtained from the resulting titration curves was compared with standard heparin solutions to determine the concentration of heparin on the matrix. However, this procedure also requires specialized equipment ( 11). The importance of finding a simple and precise method of measuring the heparin content in heparin-/I-agarose preparations was amplified in our laboratory, since we are regularly involved with preparing large batches of that particular affinity matrix. Monitoring the free heparin concentration before and after coupling was difficult due to the large wash volumes which resulted.
467
HEPARIN
and the methods available for direct heparin determinations on the matrix seemed too time consuming or inaccurate. We were looking for a quick calorimetric assay which would allow us to easily quality-control each batch. Our research led us to the metachromatic dye o-toluidine blue which has long been used as a calorimetric indicator of heparin concentration in solution (21), and more recently as a qualitative indicator for monitoring heparin coupling to agarose (29). In this paper, we describe the use of this dye to quantitatively determine the amount of heparin bound to an insoluble support. namely, Sepharose 4B. MATERIALS
AND METHODS
Heparin was obtained from Riker Laboratories, Inc., Northridge, California, with an activity of 40.000 USP units/ml. Sepharose 4B was from Pharmacia Fine Chemicals, Piscataway, New Jersey. Cyanogen bromide was obtained from Pierce Chemical Company, Rockford, Illinois. Toluidine blue (C.I. Basic Blue 17: C.I. No. 52040) was a product of Allied Chemical. All other chemicals were reagent grade or better. Absorbance spectra were recorded on a Perkin-Elmer Hitachi 200 spectrophotometer. Centrifugations were performed using an International Centrifuge Model C-50.
Immobilized heparin was prepared essentially according to the method of Andersson (27) with some modifications. Heparin (8 x 10” USP units or 6.7 g assuming 120 unitsimg (31)) was dissolved in 1500 ml of ice-cold deionized water and the pH was adjusted to 7.8. To the stirred heparin solution was added 2.5 g of CNBr. When the CNBr had completely dissolved, 400 ml of washed Sepharose 4B was added, and the solution was kept at pH 11 for 25 min by the addition of 6 M NaOH. The suspension was then allowed to stir overnight at room temperature. After 24 h the gel was filtered
468
SMITH,
MALLIA,
and washed on a fritted glass filter funnel with 10 liters of deionized water. Excess reactive groups were blocked by reacting with 1 M ethanolamine at pH 9.0 for 3 h. Heparin-II-Sepharose 4B was finally washed extensively with each of the following: (a) water, 4 liters; (b) 0.1 M sodium acetate, pH 4.7, 2 liters; (c) 0.5 M sodium bicarbonate, 2 liters; and (d) water, 10 liters, The immobilized heparin preparation was stored in 0.02% sodium azide at 4°C until used. Calorimetric Determinution Bo:tnd to Agarose
of Hepcrrin
The reaction between toluidine blue and heparin was performed essentially as described by Macintosh (21) but with certain modifications which allow us to determine the heparin content of immobilized heparin directly. All operations were conducted at room temperature.
(1) Standard heparin solution 50 ~1 heparin ( 16.6 mg) diluted to 100 ml with 0.2% NaCl. This results in a 0.0166% heparin standard. (2) 0.005% Toluidine blue 25 mg toluidine blue dissolved in 500 ml 0.01 N HCl containing 0.2% NaCl. (3) 0.2% NaCl (4) hexane Procedure. Two and one-half milliliters of the 0.005% Toluidine blue solution was pipetted into each of 13 test tubes. In tubes 1-7, varying amounts of the standard heparin solution were added which would result in a standard curve representing a concentration range of approximately lo70 Fg of heparin. In tubes 8-12, amounts of a I:4 diluted slurry of heparin-/I-Sepharose 4B in water were added which would constitute a range of 0.025 to 0.15 ml settled gel. Tube 13 was a blank which contained either a Sepharose 4B slurry or the same volume of 0.2% NaCl. Each tube was then diluted with 0.2% NaCl to a total volume
AND
HERMANSON
of 5 ml and agitated by a Vortex mixer for 30 s. Hexane (5 ml) was then added to each tube and the tubes were shaken vigorously for another 30 s to separate the heparindye complex formed in tubes 1-7. Since the heparin-dye complex in tubes 8-12 forms upon the gel surface only, hexane is not actually required. It was included to insure uniformity of treatment. After centrifugation (1000 rpm, 3 min) of tubes 8- 13 to remove suspended gel from the solution, the aqueous layers of all the tubes were sampled and diluted 1: 10 with absolute ethanol. The absorbance at 631 nm was then read for each within 30 min. RESULTS
AND DISCUSSION
Upon binding to a polyanionic substrate, metachromatic dyes undergo a color change which is accompanied by a shift in the absorbance spectrum to lower wavelengths. It is now commonly believed that this phenomenon is due to the tendency of dye molecules to reversibly polymerize in close proximity to such a substrate (3). The polymers arrange themselves in such a fashion so as to exhibit an absorption spectrum slightly different from that of the monomers (30). The metachromatic behavior of Toluidine blue as it binds native heparin in solution is illustrated in Fig. 1. As shown, the absorbance maximum of a dilute toluidine blue solution in water occurs at approximately 631 nm. However, in a solution containing heparin this band is displaced toward the 540 to 560-nm range depending on the concentration of both dye and heparin. A simple calorimetric method for the determination of heparin based on this decrease in absorbance at 63 1 nm is not immediately possible, because of metachromatic band overlap. Macintosh (21) devised a method of overcoming this problem through removal of the heparindye complex from solution by adsorption at the interface of an organic solvent (light petroleum). Thus removed, the decrease
HEPARIN
CONTENT
IN IMMOBILIZED
HEPARIN
469
0.6
0.1
600 WAVELENGTH
Inm)
FIG. I. Metachromatic spectra of toluidine blue binding to heparin. Standard toluidine blue solution (0.005% in 0.01 N HCI containing 0.2% NaCl) was diluted I:10 with 0.2% NaCI. To the dye solution was added a 0.0166% heparin solution in IO-PI aliquots and the spectrum was scanned after each addition.
in dye in the aqueous layer could then be directly determined and correlated to the heparin concentration. Immobilized heparin has unique properties of its own which were unknown when Macintosh developed this technique. Immobilization of molecules has allowed the interaction of selected ligands with a solution in relatively normal fashion even though they themselves are not truly in solution. Thus, heparin coupled to a beaded polymer like Sepharose can still associate with Toluidine blue and elicit the characteristic metachromasia upon the gel surface while at the same time remain easily removable from solution by centrifugation.
Quantitation of the amount of heparin bound to such insoluble supports should then be possible by simply monitoring the dye depletion in the supernatant and comparing it to known heparin standards. In Fig. 2, a typical standard curve is shown for various known concentrations of native heparin in solution. The linear decrease in absorbance at 631 nm with increasing heparin concentrations was a typical phenomenon observed if the heparindye complex (and therefore the metachromatic band) was first removed by shaking with hexane. It was found that this curve is reproducibly linear over the native heparin concentration range of lo-70 pg
470
SMITH,
which corresponds to a dye approximately IO-50%. If immobilized heparin was analogous manner, but without for the precipitation of the
MALLIA,
depletion
AND
of
treated in an the necessity heparin-dye
HERMANSON
complex upon a water-hexane interface, then similar results were obtained. Figure 3 illustrates for two separate determinations the corresponding decrease in dye concentration in the supernatant as increasing
0.55
0.50
0.40
0.35 I
I 20.0
I
I 40.0 NATIVE
I HEPARIN.
I 60.0
I
I 80.0
og
FIG. 2. Standard curve showing the decrease in absorbance in a Toluidine blue solution at 63 I nm with increasing concentrations of native heparin (see Materials and Methods). The heparin-dye complex which formed was removed from solution by adsorption at the interface of a hexane layer prior to reading the absorbance of the dye layer. All absorbance readings represent a I:10 dilution of the aqueous layer with absolute ethanol.
HEPARIN
CONTENT
IN IMMOBILIZED
amounts of heparin-I(-Sepharose 4B were added to the dye solution. The spectral characteristics of this dye depletion upon the addition of immobilized heparin are shown in Fig. 4. Noteworthy is the fact that as opposed to the alternate case of native
471
HEPARIN
heparin additions (Fig. l), there is no metachromasia apparent in the solution when aliquots of heparin-/I-Sepharose are added. This shows that the metachromatic effect is formed solely upon the gel surface and can be removed prior to reading the
0.55
0.50
: F; (0 0.45 s E b B Q
0.40
0.35
I 25
I 50 Heparin-I/-Sepharo&
I 75
I 1Cil 48. ill settled
I 125
1 150
gel
FIG. 3. The results of duplicate determinations of the amount of heparin in heparin-+Sepharose 4B. The dye depletion in the supernatant t 1: 10 diluted with ethanol) was monitored at 63 1 nm after various amounts of immobilized heparin were added. The heparin-dye complex formed upon the agarose beads was removed from solution by centrifugation prior to measuring the absorbance.
472
SMITH,
MALLIA,
AND
I
HERMANSON I
I
I
0.7
0.6
0.5
WAVELENGTH
(nm)
FIG. 4. The spectral characteristics ofToluidine blue depletion in the supematant upon the addition of immobilized heparin. The heparin-dye complex. and therefore the metachromatic band, were removed by centrifugation and the supernatant was diluted I:10 with absolute ethanol prior to scanning the spectrum. (1) 25 1.11heparin-I(-Sepharose. (2) 125 ~1 heparin-+Sepharose.
absorbance at 631 nm by centrifugation at 1000 t-pm. Like the soluble heparin determinations, the curve formed by immobilized heparin was also found to be linear over the range of lo-50% dye depletion in the supernatant (or lo-70 pg heparin present). Direct extrapolation of these absorbance readings onto the standard heparin curve gave a heparin content upon the agarose beads of 0.58 ? 0.02-mg/ml settled gel.’ Performing the assay using only washed Sepharose 4B resulted in no metachromasia 2 Mean
f standard
deviation
of the mean.
upon the gel surface. However, one limitation was noticed: when using relatively large volumes of gel (e.g., 0.5 ml), an increase in absorbance at 63 1 nm occurred. This was probably due to the fact that, although the total volumes of two representative tubes (e.g., one containing gel and the other consisting of only a 0.2% NaCl blank) may be identical, the one containing gel has a lower effect& solution volume. This is because the Sepharose polymer itself is not truly in solution and. therefore, takes up some of the space which would normally be available for the dye molecules. Thus, a “forced”
HEPARIN
CONTENT
IN IMMOBILIZED
concentration of Toluidine blue results in all tubes which contain gel. However, this inherent error may be reduced to a minimum by using only very low gel volumes (~0.15ml settled gel). The spectral similarity between Toluidine blue binding to free and immobilized heparin demonstrates the applicability of this method for the assay of heparin content in immobilized heparin preparations. The linear characteristics of dye depletion with increasing heparin concentration (in both native and immobilized forms) further proves the validity of this technique. In addition, we have observed that this method can be used for the quantitation of the amount of dextran sulfate in immobilized dextran sulfate, and may well prove to be a useful technique for the determination of other immobilized glycosaminoglycans which illicit metachromatic behavior with o-toluidine blue.
8. 9.
10. II.
12. 13. 14.
15. 16. 17. 18. 19. 20.
ACKNOWLEDGMENTS The authors wish to gratefully acknowledge Dr. Robert Vigna for his critical reviews of the manuscript, Mrs. Carole Preston for her patient secretarial help, and Mrs. Cheryl Roettger for drawing the figures.
21. 22. 23.
24.
REFERENCES I. Park. J. W.. and Chakrabarti. B. (1977) Biochrm. Biophys. Res. Commun. 78, 604-608. 2. Chung, M. C., and Ellerton, N. F. (1976) Biopolymers 15, 1409- 1423. 3. Lagunoff, D. (1974) Biochemisfry 13, 3982-3986. 4. Srinivasan, S. R.. Radhakrishnamurthy. B.. and Berenson, G. S. (1975) Arch. Bioc~twm. Biophys. 170, 334-340. 5. Gertler. M. M., Chien, M., and Yue. R. H. (1979) Thromhos. Hwmostus. 41, 567-575. 6. Davison, B. L., Leighton, T.. and Rabinowitz. J. C. ( 1979) J. Biol. Chem. 254, 9220-9226. 7. Heimark, R. L., and Davie. E. W. (1979) Biochrmisrry 18, 5743-5750.
25. 26. 27. 28.
29. 30. 31.
HEPARIN
473
Goldstein, J., and Safer, B. (1979) in Methods in Enzymology (Moldave. K., ed.). Vol. 60. pp. 165-181. Academic Press. New York. Molinari. A. M.. Medici, N.. Moncharmont. B.. and Puca. G. A. (1977) Proc. Nut. Acad. Sc,i. USA 74, 4886-4890. Brennessel. B. A.. Buhrer, D. P.. and Gottlieb. A. A. (1978) Anal. Biochem. 87, 411-417. Jaques, L. B. (1977) in Methods in Biochemichal Analysis (Glick. D.. ed.), Vol. 24, pp. 203-312. Wiley, New York. Iverius. P.-H. (1971) Biochrm. J. 124, 677-683. Ototani. N.. and Yosirawa. Z. (1978) .I. Biwhem. 84. 1005- 1008. Mulder, E., Foekens, J. A.. Peters. M. J.. and Van Der Molen, H. J. (1979) FEBS Lclr. 97, 260-264. Sica. V.. and Bresciani. F. (1979) Biochcv~is~r~ 18, 2369-2378. Sternbach. H.. Engelhardt, R.. and Lezius, A. G. ( 1975) Gtr. 1. Biwhrm. 60, S I -ST. Bitter, T.. and Muir, H. M. (1962) Anut. Bioc.hcm. 4, 330-334. Galambos. J. T. (1967) Awl. Biochr~m. 19. 119- 132. Gentry, P. W.. and Alexander. B. (1973) Bioc hc~m. Biophy.s. REJ.. Commun. 50, 500-509. Jaques. L. B.. and Bell, H. J. (1959) in Methods in Biochemical Analysis (Glick, D.. ed.). Vol. 7. pp. 253-309, Wiley, New York. Macintosh, F. C. (1941) Biochcm. /. 35, 776-782. Gold, E. W. (1979) Anirt. Bitrc~tzrm. 99, 183- 188. Diakun, G. P.. Edwards. H. E.. Allen. J. C., Phillips. G. 0.. Cundall. R. B. (1979) A&. Biwhrm. 94, 378-382. Miller-Andersson. M.. Borg, H., and Andersson. L.-O. (1974) Thromhos. Rcs. 5. 439-452. Antonopoulos. C. A. (1962) A(.I~I Chcm. J’cwml. 16, 1521-1522. Terho. T. T., and Hartiala. K. (1971) Auo/. Biwhc~m. 41, 471-476. Andersson, L.-O.. Borg. H.. and Miller-Andersson. M. ( 1975) Thromtwr. Rc.F. 7, 451-459. Varshavskaya. M. Ya.. Klibanov. A. L., Goldmacher, V. S.. and Torchilin. V. P. (1979) Anal. Bicwhem. 95, 449-4.5 I. Audhya. T. K., and Walter, R. (1979) J. Rirpl. Chem. 254, 291-298. Michaelis. L.. and Granick. S. (1945) J. Amer. Chcm. Ser. 67, 1212-1219. The Merck Index (1976). 9th Ed.. Merck & Co.. Inc.. Rahway, New Jersey.
BIOCHEMISTRY
Calorimetric
109,
466-473
(1980)
Method for the Assay of Heparin Immobilized Heparin Preparations
P. K. SMITH, A. K. MALLIA,AND Biochemical
Research
G.T.
July
in
HERMANSON'
Division, Pierce Chemical Cornpuny, Rockfbrd. Illinois 61105 Received
Content
P.O.
Bo.r 117.
18, 1980
The properties of the metachromatic dye toluidine blue have been utilized to determine calorimetrically the amount of heparin covalently coupled to Sepharose. The method involves monitoring the dye depletion in the supernatant at 631 nm as Toluidine blue is adsorbed onto the heparin polymer upon the beaded matrix. The procedure represents a simple assay technique which allows the direct quantitation of heparin in immobilized heparin preparations.
Heparin is a linear polysaccharide composed of repeating units of uranic acid, containing an o-sulfate group at the c-2 position, and D-glucosamine, usually N-sulfated with an additional o-sulfate group at c-6 (1). Due to its unique structure and surface charge distribution, heparin is able to interact strongly with several different proteins in a specific manner. The significance of heparin’s biological activities, especially its interaction with blood-clotting factors and human serum lipoproteins, has made this glycosaminoglycan the focus of many investigations (2-4). Recently, with the advent of affinity chromatographic techniques, heparin immobilized on insoluble beaded polymers has become very nearly an “allpurpose” ligand for the purification of a wide range of proteins and biomolecules (5-10). For this reason the heparin content in immobilized heparin preparations has become an important parameter to investigators using this affinity support. A number of methods are known for the determination of heparin in solution, and the methodology has been recently reviewed ’ The author addressed.
to whom
correspondence
0003-2697/80/180466-08$02.00/O Copyright D 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
should
be
466
(11). However, application of these methods to directly determine heparin covalently coupled to an insoluble support such as agarose is limited or nonapplicable. Techniques which have been developed for the determination of immobilized heparin have been for the most part indirect methods based on the described means of determining soluble heparin. Using this approach, the concentration of heparin in the reaction mixture prior to coupling is compared to the free heparin content left in the supernatant and washings after coupling. The difference is taken as the amount bound. Probably the most widely used such procedure (3,4,9,10,12- 15) involves the uranic acid carbazole reaction as described by Bitter and Muir (17) and later modified by Galambos (18). Although this method is accurate enough for determining dissolved heparin, the procedure is tedious and inconvenient in that concentrated sulfuric acid is required. Other investigators have used the dye-binding interactions of otoluidine blue (5,8,11,19-21) to fluorimetritally quantitate unbound glycosaminoglycan before and after coupling. Additional possibilities along this line involve the dyes acridine orange and alcian blue (22,23).
HEPARIN
CONTENT
IN IMMOBILIZED
However, all of these methods have one thing in common: they are designed to measure heparin in solution and not in its immobilized form on an insoluble beaded matrix. These may in fact yield spurious results if the washing procedure after coupling is not performed well, because free glycosaminoglycans have been shown to bind immobilized glycosaminoglycans (13). Attempts to directly measure heparin coupled to a support such as Sepharose have also surfaced in the literature. Several investigators (16,24,28) have used the sulfate analysis of Antonopoulos (25) or Terho and Hartiala (26) to estimate the amount of heparin coupled after subtracting the indigenous sulfate content of the matrix. These methods are very time consuming and the results may be suspect due to the high blank which can result from the Sepharose itself and the uncertain structure of the heparin polymer (1). In addition. the method may involve the known human carcinogen benzidine. Andersson et al. (27) determined bound heparin by amino acid analysis for glucosamine after hydrolysis in 2 M HCl at 110°C for 24 h. This method would seem to be the most accurate to date, but the limited availability of amino acid analyzers makes it inapplicable to most laboratories. Another recent technique (28) involved a potentiometric titration of the immobilized heparin. In this, the amount of base needed to achieve equivalence as obtained from the resulting titration curves was compared with standard heparin solutions to determine the concentration of heparin on the matrix. However, this procedure also requires specialized equipment ( 11). The importance of finding a simple and precise method of measuring the heparin content in heparin-/I-agarose preparations was amplified in our laboratory, since we are regularly involved with preparing large batches of that particular affinity matrix. Monitoring the free heparin concentration before and after coupling was difficult due to the large wash volumes which resulted.
467
HEPARIN
and the methods available for direct heparin determinations on the matrix seemed too time consuming or inaccurate. We were looking for a quick calorimetric assay which would allow us to easily quality-control each batch. Our research led us to the metachromatic dye o-toluidine blue which has long been used as a calorimetric indicator of heparin concentration in solution (21), and more recently as a qualitative indicator for monitoring heparin coupling to agarose (29). In this paper, we describe the use of this dye to quantitatively determine the amount of heparin bound to an insoluble support. namely, Sepharose 4B. MATERIALS
AND METHODS
Heparin was obtained from Riker Laboratories, Inc., Northridge, California, with an activity of 40.000 USP units/ml. Sepharose 4B was from Pharmacia Fine Chemicals, Piscataway, New Jersey. Cyanogen bromide was obtained from Pierce Chemical Company, Rockford, Illinois. Toluidine blue (C.I. Basic Blue 17: C.I. No. 52040) was a product of Allied Chemical. All other chemicals were reagent grade or better. Absorbance spectra were recorded on a Perkin-Elmer Hitachi 200 spectrophotometer. Centrifugations were performed using an International Centrifuge Model C-50.
Immobilized heparin was prepared essentially according to the method of Andersson (27) with some modifications. Heparin (8 x 10” USP units or 6.7 g assuming 120 unitsimg (31)) was dissolved in 1500 ml of ice-cold deionized water and the pH was adjusted to 7.8. To the stirred heparin solution was added 2.5 g of CNBr. When the CNBr had completely dissolved, 400 ml of washed Sepharose 4B was added, and the solution was kept at pH 11 for 25 min by the addition of 6 M NaOH. The suspension was then allowed to stir overnight at room temperature. After 24 h the gel was filtered
468
SMITH,
MALLIA,
and washed on a fritted glass filter funnel with 10 liters of deionized water. Excess reactive groups were blocked by reacting with 1 M ethanolamine at pH 9.0 for 3 h. Heparin-II-Sepharose 4B was finally washed extensively with each of the following: (a) water, 4 liters; (b) 0.1 M sodium acetate, pH 4.7, 2 liters; (c) 0.5 M sodium bicarbonate, 2 liters; and (d) water, 10 liters, The immobilized heparin preparation was stored in 0.02% sodium azide at 4°C until used. Calorimetric Determinution Bo:tnd to Agarose
of Hepcrrin
The reaction between toluidine blue and heparin was performed essentially as described by Macintosh (21) but with certain modifications which allow us to determine the heparin content of immobilized heparin directly. All operations were conducted at room temperature.
(1) Standard heparin solution 50 ~1 heparin ( 16.6 mg) diluted to 100 ml with 0.2% NaCl. This results in a 0.0166% heparin standard. (2) 0.005% Toluidine blue 25 mg toluidine blue dissolved in 500 ml 0.01 N HCl containing 0.2% NaCl. (3) 0.2% NaCl (4) hexane Procedure. Two and one-half milliliters of the 0.005% Toluidine blue solution was pipetted into each of 13 test tubes. In tubes 1-7, varying amounts of the standard heparin solution were added which would result in a standard curve representing a concentration range of approximately lo70 Fg of heparin. In tubes 8-12, amounts of a I:4 diluted slurry of heparin-/I-Sepharose 4B in water were added which would constitute a range of 0.025 to 0.15 ml settled gel. Tube 13 was a blank which contained either a Sepharose 4B slurry or the same volume of 0.2% NaCl. Each tube was then diluted with 0.2% NaCl to a total volume
AND
HERMANSON
of 5 ml and agitated by a Vortex mixer for 30 s. Hexane (5 ml) was then added to each tube and the tubes were shaken vigorously for another 30 s to separate the heparindye complex formed in tubes 1-7. Since the heparin-dye complex in tubes 8-12 forms upon the gel surface only, hexane is not actually required. It was included to insure uniformity of treatment. After centrifugation (1000 rpm, 3 min) of tubes 8- 13 to remove suspended gel from the solution, the aqueous layers of all the tubes were sampled and diluted 1: 10 with absolute ethanol. The absorbance at 631 nm was then read for each within 30 min. RESULTS
AND DISCUSSION
Upon binding to a polyanionic substrate, metachromatic dyes undergo a color change which is accompanied by a shift in the absorbance spectrum to lower wavelengths. It is now commonly believed that this phenomenon is due to the tendency of dye molecules to reversibly polymerize in close proximity to such a substrate (3). The polymers arrange themselves in such a fashion so as to exhibit an absorption spectrum slightly different from that of the monomers (30). The metachromatic behavior of Toluidine blue as it binds native heparin in solution is illustrated in Fig. 1. As shown, the absorbance maximum of a dilute toluidine blue solution in water occurs at approximately 631 nm. However, in a solution containing heparin this band is displaced toward the 540 to 560-nm range depending on the concentration of both dye and heparin. A simple calorimetric method for the determination of heparin based on this decrease in absorbance at 63 1 nm is not immediately possible, because of metachromatic band overlap. Macintosh (21) devised a method of overcoming this problem through removal of the heparindye complex from solution by adsorption at the interface of an organic solvent (light petroleum). Thus removed, the decrease
HEPARIN
CONTENT
IN IMMOBILIZED
HEPARIN
469
0.6
0.1
600 WAVELENGTH
Inm)
FIG. I. Metachromatic spectra of toluidine blue binding to heparin. Standard toluidine blue solution (0.005% in 0.01 N HCI containing 0.2% NaCl) was diluted I:10 with 0.2% NaCI. To the dye solution was added a 0.0166% heparin solution in IO-PI aliquots and the spectrum was scanned after each addition.
in dye in the aqueous layer could then be directly determined and correlated to the heparin concentration. Immobilized heparin has unique properties of its own which were unknown when Macintosh developed this technique. Immobilization of molecules has allowed the interaction of selected ligands with a solution in relatively normal fashion even though they themselves are not truly in solution. Thus, heparin coupled to a beaded polymer like Sepharose can still associate with Toluidine blue and elicit the characteristic metachromasia upon the gel surface while at the same time remain easily removable from solution by centrifugation.
Quantitation of the amount of heparin bound to such insoluble supports should then be possible by simply monitoring the dye depletion in the supernatant and comparing it to known heparin standards. In Fig. 2, a typical standard curve is shown for various known concentrations of native heparin in solution. The linear decrease in absorbance at 631 nm with increasing heparin concentrations was a typical phenomenon observed if the heparindye complex (and therefore the metachromatic band) was first removed by shaking with hexane. It was found that this curve is reproducibly linear over the native heparin concentration range of lo-70 pg
470
SMITH,
which corresponds to a dye approximately IO-50%. If immobilized heparin was analogous manner, but without for the precipitation of the
MALLIA,
depletion
AND
of
treated in an the necessity heparin-dye
HERMANSON
complex upon a water-hexane interface, then similar results were obtained. Figure 3 illustrates for two separate determinations the corresponding decrease in dye concentration in the supernatant as increasing
0.55
0.50
0.40
0.35 I
I 20.0
I
I 40.0 NATIVE
I HEPARIN.
I 60.0
I
I 80.0
og
FIG. 2. Standard curve showing the decrease in absorbance in a Toluidine blue solution at 63 I nm with increasing concentrations of native heparin (see Materials and Methods). The heparin-dye complex which formed was removed from solution by adsorption at the interface of a hexane layer prior to reading the absorbance of the dye layer. All absorbance readings represent a I:10 dilution of the aqueous layer with absolute ethanol.
HEPARIN
CONTENT
IN IMMOBILIZED
amounts of heparin-I(-Sepharose 4B were added to the dye solution. The spectral characteristics of this dye depletion upon the addition of immobilized heparin are shown in Fig. 4. Noteworthy is the fact that as opposed to the alternate case of native
471
HEPARIN
heparin additions (Fig. l), there is no metachromasia apparent in the solution when aliquots of heparin-/I-Sepharose are added. This shows that the metachromatic effect is formed solely upon the gel surface and can be removed prior to reading the
0.55
0.50
: F; (0 0.45 s E b B Q
0.40
0.35
I 25
I 50 Heparin-I/-Sepharo&
I 75
I 1Cil 48. ill settled
I 125
1 150
gel
FIG. 3. The results of duplicate determinations of the amount of heparin in heparin-+Sepharose 4B. The dye depletion in the supernatant t 1: 10 diluted with ethanol) was monitored at 63 1 nm after various amounts of immobilized heparin were added. The heparin-dye complex formed upon the agarose beads was removed from solution by centrifugation prior to measuring the absorbance.
472
SMITH,
MALLIA,
AND
I
HERMANSON I
I
I
0.7
0.6
0.5
WAVELENGTH
(nm)
FIG. 4. The spectral characteristics ofToluidine blue depletion in the supematant upon the addition of immobilized heparin. The heparin-dye complex. and therefore the metachromatic band, were removed by centrifugation and the supernatant was diluted I:10 with absolute ethanol prior to scanning the spectrum. (1) 25 1.11heparin-I(-Sepharose. (2) 125 ~1 heparin-+Sepharose.
absorbance at 631 nm by centrifugation at 1000 t-pm. Like the soluble heparin determinations, the curve formed by immobilized heparin was also found to be linear over the range of lo-50% dye depletion in the supernatant (or lo-70 pg heparin present). Direct extrapolation of these absorbance readings onto the standard heparin curve gave a heparin content upon the agarose beads of 0.58 ? 0.02-mg/ml settled gel.’ Performing the assay using only washed Sepharose 4B resulted in no metachromasia 2 Mean
f standard
deviation
of the mean.
upon the gel surface. However, one limitation was noticed: when using relatively large volumes of gel (e.g., 0.5 ml), an increase in absorbance at 63 1 nm occurred. This was probably due to the fact that, although the total volumes of two representative tubes (e.g., one containing gel and the other consisting of only a 0.2% NaCl blank) may be identical, the one containing gel has a lower effect& solution volume. This is because the Sepharose polymer itself is not truly in solution and. therefore, takes up some of the space which would normally be available for the dye molecules. Thus, a “forced”
HEPARIN
CONTENT
IN IMMOBILIZED
concentration of Toluidine blue results in all tubes which contain gel. However, this inherent error may be reduced to a minimum by using only very low gel volumes (~0.15ml settled gel). The spectral similarity between Toluidine blue binding to free and immobilized heparin demonstrates the applicability of this method for the assay of heparin content in immobilized heparin preparations. The linear characteristics of dye depletion with increasing heparin concentration (in both native and immobilized forms) further proves the validity of this technique. In addition, we have observed that this method can be used for the quantitation of the amount of dextran sulfate in immobilized dextran sulfate, and may well prove to be a useful technique for the determination of other immobilized glycosaminoglycans which illicit metachromatic behavior with o-toluidine blue.
8. 9.
10. II.
12. 13. 14.
15. 16. 17. 18. 19. 20.
ACKNOWLEDGMENTS The authors wish to gratefully acknowledge Dr. Robert Vigna for his critical reviews of the manuscript, Mrs. Carole Preston for her patient secretarial help, and Mrs. Cheryl Roettger for drawing the figures.
21. 22. 23.
24.
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25. 26. 27. 28.
29. 30. 31.
HEPARIN
473
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