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Determination of tissue heparin
ARCHIVES OF BIOCEEMIBTBY AND BIOPHYBICS 70,
169-177 (1967)
Determination of Tissue HeparS Leon Freeman, Richard Poshma,
Lily Gordon and Walter Marx
of Biochemistry and Nutrition, School of Medicine, of Southern California, Los Angeles, California
From the Department
University
Received October 8, 1956
Most of the procedures published on the extraction of heparin from animal tissues are based on the work of Charles and Scott (l-3) who developed a method for preparative rather than analytical purposes, involving the extraction and purification of the anticoagulant from relatively large amounts of tissue, relatively rich in heparin. For a project dealing with the heparin content of rat and rabbit tissues (4), a technique was developed for the measurement of the anticoagulant in small samples and in tissues which are of low heparin content. The present communication describes this procedure. PROCEDURE
Extraction and Deproteinizatim The tissues are frozen as soon as possible and stored in the frozen state. A sample of l-5 g. (depending on the size and expected heparin content of the organ) is thoroughly homogenized with an equal weight of cold water. The homogenate is immediately poured into 5 vol. of acetone; after standing for a few minutes, the suspension is centrifuged and the clear supernatant fluid discarded. The sediment is suspended in 5-10 ml. of 0.5 M NH&l buffer, pH 8.5, and the mixture is heated in a boiling water bath for 15 min. The sample is then transferred to dialysis tubing closed at one end and provided with a short piece of glass tubing at the other end; the filled bag is suspended in a jar containing 0.5 1. of the buffer described above. One milliliter of a 500Joglycerol solution containing 100 mg. 1 This investigation was supported, in part, by research grant H 1596 from the National Heart Institute, of the National Institutes of Health, U. S. Public Health Service, and by grants from Darwin Laboratories and Lederle Laboratories Division, American Cyanamid Co. 169
170
FREEMAN,
POSTHUMA,
GORDON
AND
MARX
trypsinz is added, and the tube attached to the open end of the bag is stoppered. The jar is then incubated at 37°C. for 48 hr. Then a second milliliter of enzyme solution is added to the contents of the bag, the jar is filled with fresh buffer, and the incubation is continued for an additional 48 hr. The sample is then dialyzed against running tap water overnight. The contents of the bag are transferred to a 50-ml. centrifuge tube, NaCl is added to a concentration of lye, and the solution is heated in a boiling water bath for 15 min. to inactivate and coagulate the enzyme. The mixture is cooled to room temperature and centrifuged at 2500 r.p.m. for 5 min., and the supernatant fluid is decanted into a 250-ml. centrifuge tube; the sediment is discarded. To the former, 5 vol. of acetone are added; after standing for 30 min., the mixture is centrifuged at 2500 r.p.m. for 5 min., and the clear fluid phase is discarded. The sediment is dissolved in a small volume of 1% NaCl and transferred to a 50-ml. centrifuge tube; 5 vol. of methanol are added, and, after standing for 30 min., the mixture is centrifuged at 2509 r.p.m. for 5 min. The clear supernatant fluid is discarded, and the tube is inverted and drained for several minutes. The precipitate is transferred to a tared vial or tube of about 20 ml. capacity, with a volume of water not exceeding 5 ml.; the solution is then lyophilized and the residue is weighed.
Determination
of Anticoagulant
Activity
The samples are dissolved in isotonic saline at a concentration of l-5 units/ml. The activity is determined by the procedure described in the U. S. Pharmacopeia XIV (U.S.P.), with the following modification: The unknown and standard samples are diluted to concentrations of anticoagulant which will permit the formation of a full clot within a period of 30-60 min. In this manner clotting times are compared rather than the extent of clotting at the end of 1 hr. A fresh standard is prepared daily from a U.S.P. Reference Standard Heparin Sodium or from a purified preparation of heparin sodium, U.S.P.3 Potency is expressed in U.S.P. units. EXPERIMENTAL
Three commercial proteinase preparations, i.e., trypsin, pepsin,* and papain6 were compared, under the conditions described above regarding their digestive action and the yield of heparin extracted. In each case, a buffer was selected with a pH corresponding to the pH optimum of the particular enzyme under investigation; in the experiments with papain, Naz$ and KCN were added to the digestion medium as activators. The * Purified Trypsin Concentrate supplied by Southern California Gland Co.; 1 mg. of this preparation liberates 2 mg. of tyrosine equivalent from hemoglobin under conditions as described by Northrop (“Crystalline Enzymes,” p. 155. Columbia University Press, New York, 1939). 1 Supplied by Southern California Gland Co. 4 Pepsin 1:15,000, 100 mg./ml., supplied by Cudahy Packing Co. 6 Papain, Merck and Co., 50 mg./ml.
DETERMINATION
OF TISSUE
171
HEPARIN
TABLE I Digestive Action of Proteolytic Enzymes on Beef Lung Homogenate EIlZyllW
Papain Trypsin/papain Pepsin Trypsin/pepsin Trypsin
Total solids~ig&t& No. of experiments Mean
6 1 5 8 13
%
%
33 50 64 63 91
2246 48-77 42-89 W-94
5 Samples of 5 g. beef lung were treated as described in text, and the total solids remaining were determined. TABLE II Yield of Heparin Activity Extracted from Beef Lung Homogenate by Means of Proteolytic Digestion E~yllXS
No. of experiments
HepAn&hi$ty units/g.”
Papain Trypsin/papain Pepsin Trypsin/pepsin Trypsin
1 1 2 2 2
20 27 14, 12 19, 21 50, 46
a Samples of 5 g. beef lung were treated as described in text, and the anticoagulant activities of the products were determined. b Of wet weight tissue.
enzymes were used alone, or in combination. In the latter case, the digestion was carried out in two successive steps. The efficiency of the proteolysis was evaluated by a determination of the solid residue remaining, and of the amount of anticoagulant activity extracted, measured as described above. The results of a number of experiments using beef lung are summarized in Tables I and II. It is obvious that incubation with trypsin alone gave a more complete digestion, and a significantly higher heparin yield than any of the other enzymes or enzyme combinations tested. At the same time, the results with trypsin alone were more consistent. The amount of heparin activity extracted after tryptic followed by peptic digestion was only about one fourth of that obtained with trypsin alone; on the other hand, after incubation with the two enzymes, a significantly greater amount of precipitate was visible, and the quantity of nondialyzable material remaining was about six times as great as that
172
FREEMAN,
POSTHUMA,
GORDON
AND
MARX
TABLE III Yield of Heparin Activity Extracted from Supernatant Fluid and Sediment Formed during Proteolytic Digestion of Beef Lung Homogenate No. of experiments
Mean total solids remaining
Total heparin activit extra&da Mean and range of va f ues Supernat. 8. sedimentb Total
WT.
Trypsin/pepsin
4
units
5
100
units
124
(2zq Trypsin
units
530 125 (82-155)
(5J23) 0
125
0 Samples of 5 g. beef lung were treated as described in text. b The sediment formed during peptic digestion was redigested with trypsin and the resulting extract assayed for heparin.
found following hydrolysis with trypsin alone. We suspected, therefore, that under conditions of peptic digestion (pH = about 2), a part of the heparin was precipitated in combination with protein, perhaps with the enzyme itself, and thus made unavailable for subsequent extraction. In order to test this hypothesis, a number of experiments was made in which the material sedimented during peptic proteolysis was redigested with trypsin, and the heparin content of the resulting digest was measured. The results, summarized in Table III, indicate, in agreement with our hypothesis, that, when pepsin was used, only about one-third of the heparin present was extracted, while about two-thirds remained in the insoluble sediment. Further digestion of the latter with trypsin liberated the anticoagulant. Table III also shows that, although heparin was precipitated under conditions of peptic digestion, its anticoagulant activity was not destroyed. Redigestion of the minor sediment resulting from tryptic proteolysis did not yield additional heparin activity. After it became clear that trypsin was the enzyme of choice, attempts were made to establish the amount of trypsin and the digestion period that would permit the highest recoveries. The results, shown in Table IV, indicate that the greatest heparin yield was obtained using two l-ml. portions of the trypsin solution described, and an incubation period of twice 48 hr. Recovery Experiments
In order to investigate whether any of the steps used in our extraction procedure would inactivate heparin, recovery experiments were made in which tissue samples were divided into two portions, and to one of these, a measured amount of heparin sodium U.S.P. was added. The
DETERMINATION
OF
TISSUE
173
HEPARIN
TABLE IV Yield of Heparin Activity Extracted from Beef Lung Homogenate after Tryptic Digestion (Effects of variations in amount of trypsin and period of digestion) No. of experiments
2 6 2 2
Amount of trypsin solution ml.
2 2 2 2
x x x x
1.0 0.5 1.0 0.5
Heparin activity extracted0 MeaIl Individual values unwg.b unit&b
Period of digestion days
4 4 2 2
30, 24, 22, 22,
30 25, 25, 26, 28, 30 24 24
30 26 23 23
a Samples of 5 g. beef lung were treated as described in text. b Of wet weight tissue. TABLE V E&action of Heparin Activity from Beef Lung Homogenate; Recovery of Added Heparin Exphi
Enzymes used
1
Trypsin/pepsinb
2
Trypsin
:d::f
units 0 50 0 25
Heparin activity recovereda Total Added units units
160 217 155 136
57 25
a Samples of 5 g. beef lung were treated as described in text. b The sediment obtained after pepsin digestion was redigested with trypsin, and the extracts were combined before assaying for heparin.
heparin content of both samples was then determined as described. Table V shows that the anticoagulant activity was recovered completely, within the limits of error of the method. hfeproducibiility
of Method
Eight 5-g. samples of the same beef liver homogenate were processed separately, and the resulting fractions were assayed for heparin using the method given. The following values were found: 12.4; 12.6; 13.0; 13.2; 13.6; 13.6; 14.4; 15.5 units/g. Furthermore, four different tissue samples were processed according to the procedure presented; the activities of the final extracts were then determined by two individuals working independently in different laboratories. The results summarized in Table VI give an indication of the reproducibility of the assay method.
174
FREEMAN,
POSTHUMA,
GORDON
TABLE Heparin
AND
MARX
VI
Assay; Comparison Working
of Results Obtained by Two Individuals Independently in Different Laboratories Heparin activity
SE%lUpl~
Laboratory
A
utaits/g.”
1 2 3 4 D Samples of 5 g. each of rat intestine were treated as described in text. b Of wet weight tissue.
3.0 7.5 8.0 41.1
found” Laboratory
B
units/g.b 3.0 7.5 6.5 42.6
(I), rat lung (9, S), and beef lung (4)
Protamine Reversal of Anticoagulant
Activity
Samples of rabbit, rat, and beef tissues treated as described were assayed for heparin in the presence and absence of protamine. Addition of protamine6 completely inhibited the anticoagulant activity of the extracts, in agreement with observations by Chargaff and Olson (5) and others. Paper Electropkoresis and Metachromasia Samples of rat, rabbit, and beef tissues were processed as mentioned, and the extracts obtained were subjected to paper electrophoresis. A horizontal apparatus (closed-chamber type), Whatman paper No. 1, 30 X 3 cm., and 0.05 M acetate buffer, pH 4.5 were used. A volume of 0.01 ml. of a solution containing about 5 units of heparin was pipetted onto each paper strip, and 300 v. and 2 ma. were applied for 4 hr.; then the paper was stained with Azure A. For comparison, a sample of heparin sodium U.S.P. was examined under identical conditions. All tissue preparations showed essentially the same patterns, with the major staining component represented by a single somewhat elongated spot, exhibiting the red-purple color typical of the dye-heparin complex and a mobility identical with that observed for heparin sodium U.S.P. No other metachromatic region was seen, except a very small spot at the origin. It is of interest that, prior to electrophoretic separation, the tissue extracts did not exhibit any metachromatic activity. Even the addition of purified heparin to a mixture of tissue extract and Azure A failed to show metachromasia. Furthermore, when a tissue preparation was added 6 Protamine sulfate, Van Camp Sea Food Co.
DETERMINATION
OF TISSuE
HEPARIN
175
to a mixture of purified heparin and Azure A, the red-purple color typical of the heparindye complex disappeared, and only the blue color of the free Azure A remained. Apparently the tissue extracts contain material which interferes with the metachromatic reaction; electrophoresis separates this fraction from heparin. Interference with metachromasia by certain tissue components was noted by other investigators (6). In spite of this interference with the metachromatic reaction, no influence on anticoagulant activity was noted. The compound responsible for this phenomenon has not been characterized. DISCUSSION
Heparin occurs in tissues tightly bound to protein, and, in order to measure its anticoagulant activity, it is necessary to cleave this complex. For this purpose, Charles and Scott, and other workers proposed to use autolysis, followed by tryptic digestion of the material extracted from the autolyzate (l-3). However, autolysis does not liberate all heparin present, and, therefore, extraction is not complete under these conditions. Furthermore, it is not practical, since Jaques et al. demonstrated heparinase activity in several mammalian tissues (7, S), and it is obvious that inactivation of this enzyme is essential for a quantitative recovery of the anticoagulant. In the present work this is accomplished by treatment of the sample with acetone, and, then, with heat (lOO’C.), immediately after homogenization. During these steps, not only heparinase, but also the enzymes responsible for autolysis are inactivated. Therefore the autolysis step had to be abandoned. However, the denaturation of the proteins and the removal of some of the lipides markedly facilitate the following proteolytic digestion. The latter step is carried out under conditions of simultaneous dialysis. This permits a continuous removal of dialyzable digestion products, and, therefore, inhibition of the proteolysis by an accumulation of split products is avoided, and the equilibrium is shifted toward complete hydrolysis. Further advantages of this technique include (a) an improved maintenance of the pH of the digestion medium-a consequence of the latter being in equilibrium with an excessof buffer in the outside compartment; and (5) a separation from the sample of dialyzable compounds which influence the clotting reaction-material which would otherwise interfere with the assay of heparin. As a result of the dialysis, a relatively small amount of heparin is present in a relatively large volume. For purposes of concentration, the anti-
176
FREEMAN, POSTHUMA, GORDON AND MARX
coagulant is precipitated with acetone, the best precipitant of heparin among the water-miscible organic solvents, as far as we know. However tissue extracts may contain anticoagulants of a lipide nature (9, 10) which are not acetone-soluble and which would interfere with the assay of heparin. In order to remove these compounds, the sample is reprecipitated with methanol from a more concentrated solution. The weight of the lyophilized residue is usually quite constant for any given tissue. It is obtained as a check on the digestion and dialysis; e.g., if for any reason the digestion should be incomplete, the residue would be unusually heavy; if a leak should develop in the dialysis tubing, the weight of the residue would be unusually low. Some investigators have proposed to assay heparin by means of its metachromatic reaction (11, 12). However, other tissue components survive the procedure described; some of these substances interfere with the metachromatic reaction as shown in the Experimental section; others are known to react with toluidine blue or Azure A, such as, e.g., chondroitin sulfate. Other workers have pointed out that the metachromatic reaction is not specific for heparin (13, 14). It is obvious, therefore, that a quantitative determination of the heparin present in the final samples cannot be based on the metachromatic reaction. An assay involving the anticoagulant activity, on the other hand, is more specific for heparin, and, under the conditions described, the results are considered more significant. Therefore, the heparin content of the final extracts was measured according to the method of the U. S. Pharmacopeia modified as mentioned above. The procedure outlined is considered specific for heparin, since the measurement is based on anticoagulant activity, and as no other known, naturally occurring anticoagulant can survive the treatment involved, as far as we know. Additional characteristic criteria such as neutralization of anticoagulant activity by protamine, electrophoretic behavior, and metachromatic activity were considered further evidence for the heparin nature of the anticoagulant present in the final extracts. The method has been applied to a large number of tissue samples derived from different animal species,and varying in heparin content from about 1 to 45 units/g. [Table VI; (4)]. SUMMARY
A method is described for the determination of heparin in relatively small tissue samples, including those relatively low in heparin. The pro-
DETERMINATION
OF TISSUE
HEPARIN
177
cedure includes, among other steps, initial denaturation of heparinase, proteolysis under conditions of simultaneous dialysis, and removal of lipides. The heparin content of the preparation obtained is measured by means of its anticoagulant activity according to a modification of the technique of the U. S. Pharmacopoeia XIV. The method outlined is considered to be specific for heparin. The heparin nature of the anticoagulant present in the final extract was confirmed by several criteria such as protamine reversal of anticoagulant activity and electrophoretic and metachromatic behavior. ADDENDUM
After this paper had gone to press, the final step of the procedure (the methanol precipitation whose purpose was to remove lipide-soluble anticoagulants) was modified as follows: The sediment resulting from the precipitation with acetone is triturated with about 30 ml. of petroleum ether and the mixture gently warmed and agitated; it is then centrifuged, and the supernatant fluid discarded; the remaining solvent is completely removed by a gentle air stream at 60-70’ C, and the sediment is dissolved in water, lyophiiized and assayed as described. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
CHARLES, A. F., AND SCOTT, D. A., J. Biol. Chem. 102, 425 (1933). CHARLES, A. F., AND SCOTT, D. A., J. Biol. Chem. 102, 437 (1933). KUIZENGA, M. H., AND SPAULDING, L. B., J. Biol. Chem. 148, 641 (1943). MARX, W., MARX, L., RUGGERI, L., RUC~ER, P., AND FREEMAN, L., Proc. Sot. Exptl. Biol. Med. 94, 217 (1957). CHARGAFF, E., AND OLSON, K. B., J. Biol. Chem. 122, 153 (1938). KELLY, J. W., Arch. Biochem. Biophys. 66, 130 (1955). JAQUES, L. B., AND KEERI-SZANTO, E., Can. J. Med. Sci. 39, 353 (1952). CEO, M. H., AND JAQUES, L. B., Federation Proc. 14, 327 (1955). CHARGAFF, E., J. BioE. Chem. 121, 175 (1937). TOCANTINS, L. M., AND MCBRIDE, T. J., Proc. Sot. Exptl. Biol. Med. 99, 110
(1948). 11. COPLEY, A. L., AND SCHNEDORF, J. G., Am. J. Physiol. 133, 562 (1941). 12. JAQUES, L. B., MONKHOUSE, F. C., AND STEWART, M., J. Physiol. (London) 109, 41 (1949). 13. JAQUES, L. B., Rev. hbmatol. 7, 74 (1952). 14. WALTON, K. W., Brit. J. Pharmacol. 9, 1 (1954).
169-177 (1967)
Determination of Tissue HeparS Leon Freeman, Richard Poshma,
Lily Gordon and Walter Marx
of Biochemistry and Nutrition, School of Medicine, of Southern California, Los Angeles, California
From the Department
University
Received October 8, 1956
Most of the procedures published on the extraction of heparin from animal tissues are based on the work of Charles and Scott (l-3) who developed a method for preparative rather than analytical purposes, involving the extraction and purification of the anticoagulant from relatively large amounts of tissue, relatively rich in heparin. For a project dealing with the heparin content of rat and rabbit tissues (4), a technique was developed for the measurement of the anticoagulant in small samples and in tissues which are of low heparin content. The present communication describes this procedure. PROCEDURE
Extraction and Deproteinizatim The tissues are frozen as soon as possible and stored in the frozen state. A sample of l-5 g. (depending on the size and expected heparin content of the organ) is thoroughly homogenized with an equal weight of cold water. The homogenate is immediately poured into 5 vol. of acetone; after standing for a few minutes, the suspension is centrifuged and the clear supernatant fluid discarded. The sediment is suspended in 5-10 ml. of 0.5 M NH&l buffer, pH 8.5, and the mixture is heated in a boiling water bath for 15 min. The sample is then transferred to dialysis tubing closed at one end and provided with a short piece of glass tubing at the other end; the filled bag is suspended in a jar containing 0.5 1. of the buffer described above. One milliliter of a 500Joglycerol solution containing 100 mg. 1 This investigation was supported, in part, by research grant H 1596 from the National Heart Institute, of the National Institutes of Health, U. S. Public Health Service, and by grants from Darwin Laboratories and Lederle Laboratories Division, American Cyanamid Co. 169
170
FREEMAN,
POSTHUMA,
GORDON
AND
MARX
trypsinz is added, and the tube attached to the open end of the bag is stoppered. The jar is then incubated at 37°C. for 48 hr. Then a second milliliter of enzyme solution is added to the contents of the bag, the jar is filled with fresh buffer, and the incubation is continued for an additional 48 hr. The sample is then dialyzed against running tap water overnight. The contents of the bag are transferred to a 50-ml. centrifuge tube, NaCl is added to a concentration of lye, and the solution is heated in a boiling water bath for 15 min. to inactivate and coagulate the enzyme. The mixture is cooled to room temperature and centrifuged at 2500 r.p.m. for 5 min., and the supernatant fluid is decanted into a 250-ml. centrifuge tube; the sediment is discarded. To the former, 5 vol. of acetone are added; after standing for 30 min., the mixture is centrifuged at 2500 r.p.m. for 5 min., and the clear fluid phase is discarded. The sediment is dissolved in a small volume of 1% NaCl and transferred to a 50-ml. centrifuge tube; 5 vol. of methanol are added, and, after standing for 30 min., the mixture is centrifuged at 2509 r.p.m. for 5 min. The clear supernatant fluid is discarded, and the tube is inverted and drained for several minutes. The precipitate is transferred to a tared vial or tube of about 20 ml. capacity, with a volume of water not exceeding 5 ml.; the solution is then lyophilized and the residue is weighed.
Determination
of Anticoagulant
Activity
The samples are dissolved in isotonic saline at a concentration of l-5 units/ml. The activity is determined by the procedure described in the U. S. Pharmacopeia XIV (U.S.P.), with the following modification: The unknown and standard samples are diluted to concentrations of anticoagulant which will permit the formation of a full clot within a period of 30-60 min. In this manner clotting times are compared rather than the extent of clotting at the end of 1 hr. A fresh standard is prepared daily from a U.S.P. Reference Standard Heparin Sodium or from a purified preparation of heparin sodium, U.S.P.3 Potency is expressed in U.S.P. units. EXPERIMENTAL
Three commercial proteinase preparations, i.e., trypsin, pepsin,* and papain6 were compared, under the conditions described above regarding their digestive action and the yield of heparin extracted. In each case, a buffer was selected with a pH corresponding to the pH optimum of the particular enzyme under investigation; in the experiments with papain, Naz$ and KCN were added to the digestion medium as activators. The * Purified Trypsin Concentrate supplied by Southern California Gland Co.; 1 mg. of this preparation liberates 2 mg. of tyrosine equivalent from hemoglobin under conditions as described by Northrop (“Crystalline Enzymes,” p. 155. Columbia University Press, New York, 1939). 1 Supplied by Southern California Gland Co. 4 Pepsin 1:15,000, 100 mg./ml., supplied by Cudahy Packing Co. 6 Papain, Merck and Co., 50 mg./ml.
DETERMINATION
OF TISSUE
171
HEPARIN
TABLE I Digestive Action of Proteolytic Enzymes on Beef Lung Homogenate EIlZyllW
Papain Trypsin/papain Pepsin Trypsin/pepsin Trypsin
Total solids~ig&t& No. of experiments Mean
6 1 5 8 13
%
%
33 50 64 63 91
2246 48-77 42-89 W-94
5 Samples of 5 g. beef lung were treated as described in text, and the total solids remaining were determined. TABLE II Yield of Heparin Activity Extracted from Beef Lung Homogenate by Means of Proteolytic Digestion E~yllXS
No. of experiments
HepAn&hi$ty units/g.”
Papain Trypsin/papain Pepsin Trypsin/pepsin Trypsin
1 1 2 2 2
20 27 14, 12 19, 21 50, 46
a Samples of 5 g. beef lung were treated as described in text, and the anticoagulant activities of the products were determined. b Of wet weight tissue.
enzymes were used alone, or in combination. In the latter case, the digestion was carried out in two successive steps. The efficiency of the proteolysis was evaluated by a determination of the solid residue remaining, and of the amount of anticoagulant activity extracted, measured as described above. The results of a number of experiments using beef lung are summarized in Tables I and II. It is obvious that incubation with trypsin alone gave a more complete digestion, and a significantly higher heparin yield than any of the other enzymes or enzyme combinations tested. At the same time, the results with trypsin alone were more consistent. The amount of heparin activity extracted after tryptic followed by peptic digestion was only about one fourth of that obtained with trypsin alone; on the other hand, after incubation with the two enzymes, a significantly greater amount of precipitate was visible, and the quantity of nondialyzable material remaining was about six times as great as that
172
FREEMAN,
POSTHUMA,
GORDON
AND
MARX
TABLE III Yield of Heparin Activity Extracted from Supernatant Fluid and Sediment Formed during Proteolytic Digestion of Beef Lung Homogenate No. of experiments
Mean total solids remaining
Total heparin activit extra&da Mean and range of va f ues Supernat. 8. sedimentb Total
WT.
Trypsin/pepsin
4
units
5
100
units
124
(2zq Trypsin
units
530 125 (82-155)
(5J23) 0
125
0 Samples of 5 g. beef lung were treated as described in text. b The sediment formed during peptic digestion was redigested with trypsin and the resulting extract assayed for heparin.
found following hydrolysis with trypsin alone. We suspected, therefore, that under conditions of peptic digestion (pH = about 2), a part of the heparin was precipitated in combination with protein, perhaps with the enzyme itself, and thus made unavailable for subsequent extraction. In order to test this hypothesis, a number of experiments was made in which the material sedimented during peptic proteolysis was redigested with trypsin, and the heparin content of the resulting digest was measured. The results, summarized in Table III, indicate, in agreement with our hypothesis, that, when pepsin was used, only about one-third of the heparin present was extracted, while about two-thirds remained in the insoluble sediment. Further digestion of the latter with trypsin liberated the anticoagulant. Table III also shows that, although heparin was precipitated under conditions of peptic digestion, its anticoagulant activity was not destroyed. Redigestion of the minor sediment resulting from tryptic proteolysis did not yield additional heparin activity. After it became clear that trypsin was the enzyme of choice, attempts were made to establish the amount of trypsin and the digestion period that would permit the highest recoveries. The results, shown in Table IV, indicate that the greatest heparin yield was obtained using two l-ml. portions of the trypsin solution described, and an incubation period of twice 48 hr. Recovery Experiments
In order to investigate whether any of the steps used in our extraction procedure would inactivate heparin, recovery experiments were made in which tissue samples were divided into two portions, and to one of these, a measured amount of heparin sodium U.S.P. was added. The
DETERMINATION
OF
TISSUE
173
HEPARIN
TABLE IV Yield of Heparin Activity Extracted from Beef Lung Homogenate after Tryptic Digestion (Effects of variations in amount of trypsin and period of digestion) No. of experiments
2 6 2 2
Amount of trypsin solution ml.
2 2 2 2
x x x x
1.0 0.5 1.0 0.5
Heparin activity extracted0 MeaIl Individual values unwg.b unit&b
Period of digestion days
4 4 2 2
30, 24, 22, 22,
30 25, 25, 26, 28, 30 24 24
30 26 23 23
a Samples of 5 g. beef lung were treated as described in text. b Of wet weight tissue. TABLE V E&action of Heparin Activity from Beef Lung Homogenate; Recovery of Added Heparin Exphi
Enzymes used
1
Trypsin/pepsinb
2
Trypsin
:d::f
units 0 50 0 25
Heparin activity recovereda Total Added units units
160 217 155 136
57 25
a Samples of 5 g. beef lung were treated as described in text. b The sediment obtained after pepsin digestion was redigested with trypsin, and the extracts were combined before assaying for heparin.
heparin content of both samples was then determined as described. Table V shows that the anticoagulant activity was recovered completely, within the limits of error of the method. hfeproducibiility
of Method
Eight 5-g. samples of the same beef liver homogenate were processed separately, and the resulting fractions were assayed for heparin using the method given. The following values were found: 12.4; 12.6; 13.0; 13.2; 13.6; 13.6; 14.4; 15.5 units/g. Furthermore, four different tissue samples were processed according to the procedure presented; the activities of the final extracts were then determined by two individuals working independently in different laboratories. The results summarized in Table VI give an indication of the reproducibility of the assay method.
174
FREEMAN,
POSTHUMA,
GORDON
TABLE Heparin
AND
MARX
VI
Assay; Comparison Working
of Results Obtained by Two Individuals Independently in Different Laboratories Heparin activity
SE%lUpl~
Laboratory
A
utaits/g.”
1 2 3 4 D Samples of 5 g. each of rat intestine were treated as described in text. b Of wet weight tissue.
3.0 7.5 8.0 41.1
found” Laboratory
B
units/g.b 3.0 7.5 6.5 42.6
(I), rat lung (9, S), and beef lung (4)
Protamine Reversal of Anticoagulant
Activity
Samples of rabbit, rat, and beef tissues treated as described were assayed for heparin in the presence and absence of protamine. Addition of protamine6 completely inhibited the anticoagulant activity of the extracts, in agreement with observations by Chargaff and Olson (5) and others. Paper Electropkoresis and Metachromasia Samples of rat, rabbit, and beef tissues were processed as mentioned, and the extracts obtained were subjected to paper electrophoresis. A horizontal apparatus (closed-chamber type), Whatman paper No. 1, 30 X 3 cm., and 0.05 M acetate buffer, pH 4.5 were used. A volume of 0.01 ml. of a solution containing about 5 units of heparin was pipetted onto each paper strip, and 300 v. and 2 ma. were applied for 4 hr.; then the paper was stained with Azure A. For comparison, a sample of heparin sodium U.S.P. was examined under identical conditions. All tissue preparations showed essentially the same patterns, with the major staining component represented by a single somewhat elongated spot, exhibiting the red-purple color typical of the dye-heparin complex and a mobility identical with that observed for heparin sodium U.S.P. No other metachromatic region was seen, except a very small spot at the origin. It is of interest that, prior to electrophoretic separation, the tissue extracts did not exhibit any metachromatic activity. Even the addition of purified heparin to a mixture of tissue extract and Azure A failed to show metachromasia. Furthermore, when a tissue preparation was added 6 Protamine sulfate, Van Camp Sea Food Co.
DETERMINATION
OF TISSuE
HEPARIN
175
to a mixture of purified heparin and Azure A, the red-purple color typical of the heparindye complex disappeared, and only the blue color of the free Azure A remained. Apparently the tissue extracts contain material which interferes with the metachromatic reaction; electrophoresis separates this fraction from heparin. Interference with metachromasia by certain tissue components was noted by other investigators (6). In spite of this interference with the metachromatic reaction, no influence on anticoagulant activity was noted. The compound responsible for this phenomenon has not been characterized. DISCUSSION
Heparin occurs in tissues tightly bound to protein, and, in order to measure its anticoagulant activity, it is necessary to cleave this complex. For this purpose, Charles and Scott, and other workers proposed to use autolysis, followed by tryptic digestion of the material extracted from the autolyzate (l-3). However, autolysis does not liberate all heparin present, and, therefore, extraction is not complete under these conditions. Furthermore, it is not practical, since Jaques et al. demonstrated heparinase activity in several mammalian tissues (7, S), and it is obvious that inactivation of this enzyme is essential for a quantitative recovery of the anticoagulant. In the present work this is accomplished by treatment of the sample with acetone, and, then, with heat (lOO’C.), immediately after homogenization. During these steps, not only heparinase, but also the enzymes responsible for autolysis are inactivated. Therefore the autolysis step had to be abandoned. However, the denaturation of the proteins and the removal of some of the lipides markedly facilitate the following proteolytic digestion. The latter step is carried out under conditions of simultaneous dialysis. This permits a continuous removal of dialyzable digestion products, and, therefore, inhibition of the proteolysis by an accumulation of split products is avoided, and the equilibrium is shifted toward complete hydrolysis. Further advantages of this technique include (a) an improved maintenance of the pH of the digestion medium-a consequence of the latter being in equilibrium with an excessof buffer in the outside compartment; and (5) a separation from the sample of dialyzable compounds which influence the clotting reaction-material which would otherwise interfere with the assay of heparin. As a result of the dialysis, a relatively small amount of heparin is present in a relatively large volume. For purposes of concentration, the anti-
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coagulant is precipitated with acetone, the best precipitant of heparin among the water-miscible organic solvents, as far as we know. However tissue extracts may contain anticoagulants of a lipide nature (9, 10) which are not acetone-soluble and which would interfere with the assay of heparin. In order to remove these compounds, the sample is reprecipitated with methanol from a more concentrated solution. The weight of the lyophilized residue is usually quite constant for any given tissue. It is obtained as a check on the digestion and dialysis; e.g., if for any reason the digestion should be incomplete, the residue would be unusually heavy; if a leak should develop in the dialysis tubing, the weight of the residue would be unusually low. Some investigators have proposed to assay heparin by means of its metachromatic reaction (11, 12). However, other tissue components survive the procedure described; some of these substances interfere with the metachromatic reaction as shown in the Experimental section; others are known to react with toluidine blue or Azure A, such as, e.g., chondroitin sulfate. Other workers have pointed out that the metachromatic reaction is not specific for heparin (13, 14). It is obvious, therefore, that a quantitative determination of the heparin present in the final samples cannot be based on the metachromatic reaction. An assay involving the anticoagulant activity, on the other hand, is more specific for heparin, and, under the conditions described, the results are considered more significant. Therefore, the heparin content of the final extracts was measured according to the method of the U. S. Pharmacopeia modified as mentioned above. The procedure outlined is considered specific for heparin, since the measurement is based on anticoagulant activity, and as no other known, naturally occurring anticoagulant can survive the treatment involved, as far as we know. Additional characteristic criteria such as neutralization of anticoagulant activity by protamine, electrophoretic behavior, and metachromatic activity were considered further evidence for the heparin nature of the anticoagulant present in the final extracts. The method has been applied to a large number of tissue samples derived from different animal species,and varying in heparin content from about 1 to 45 units/g. [Table VI; (4)]. SUMMARY
A method is described for the determination of heparin in relatively small tissue samples, including those relatively low in heparin. The pro-
DETERMINATION
OF TISSUE
HEPARIN
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cedure includes, among other steps, initial denaturation of heparinase, proteolysis under conditions of simultaneous dialysis, and removal of lipides. The heparin content of the preparation obtained is measured by means of its anticoagulant activity according to a modification of the technique of the U. S. Pharmacopoeia XIV. The method outlined is considered to be specific for heparin. The heparin nature of the anticoagulant present in the final extract was confirmed by several criteria such as protamine reversal of anticoagulant activity and electrophoretic and metachromatic behavior. ADDENDUM
After this paper had gone to press, the final step of the procedure (the methanol precipitation whose purpose was to remove lipide-soluble anticoagulants) was modified as follows: The sediment resulting from the precipitation with acetone is triturated with about 30 ml. of petroleum ether and the mixture gently warmed and agitated; it is then centrifuged, and the supernatant fluid discarded; the remaining solvent is completely removed by a gentle air stream at 60-70’ C, and the sediment is dissolved in water, lyophiiized and assayed as described. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
CHARLES, A. F., AND SCOTT, D. A., J. Biol. Chem. 102, 425 (1933). CHARLES, A. F., AND SCOTT, D. A., J. Biol. Chem. 102, 437 (1933). KUIZENGA, M. H., AND SPAULDING, L. B., J. Biol. Chem. 148, 641 (1943). MARX, W., MARX, L., RUGGERI, L., RUC~ER, P., AND FREEMAN, L., Proc. Sot. Exptl. Biol. Med. 94, 217 (1957). CHARGAFF, E., AND OLSON, K. B., J. Biol. Chem. 122, 153 (1938). KELLY, J. W., Arch. Biochem. Biophys. 66, 130 (1955). JAQUES, L. B., AND KEERI-SZANTO, E., Can. J. Med. Sci. 39, 353 (1952). CEO, M. H., AND JAQUES, L. B., Federation Proc. 14, 327 (1955). CHARGAFF, E., J. BioE. Chem. 121, 175 (1937). TOCANTINS, L. M., AND MCBRIDE, T. J., Proc. Sot. Exptl. Biol. Med. 99, 110
(1948). 11. COPLEY, A. L., AND SCHNEDORF, J. G., Am. J. Physiol. 133, 562 (1941). 12. JAQUES, L. B., MONKHOUSE, F. C., AND STEWART, M., J. Physiol. (London) 109, 41 (1949). 13. JAQUES, L. B., Rev. hbmatol. 7, 74 (1952). 14. WALTON, K. W., Brit. J. Pharmacol. 9, 1 (1954).