22 (1975) 463-472

Aiherosclerosis,

463

0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

RAT

HEART

JER-SHUNG

LIPOPROTEIN

TWU, ARLENE

LIPASE

S. GARFINKEL

AND

MICHAEL

C. SCHOTZ

Research, Veterans Administration Wadsworth Hospital Center, Los Angeles, Calif. Department of’Medicine, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.)

90073,

and

(Received January 13th, 1975) (Accepted March 17th, 1975)

SUMMARY

using

Rat heart lipoprotein lipase was highly heparin-Sepharose 4B. When extracts

purified by affinity of acetone powder

chromatography were applied to

columns, lipase activity was firmly bound to the gel matrix and was later eluted with 1.5 A4 NaCl. At this stage the eluted enzyme was purified 1500-fold. Disc gel electrophoresis yielded a single protein band corresponding with the enzyme activity. The apparent molecular weight was 60,000. The purified enzyme was highly unstable; however, its activity could be partially stabilized by glycerol or ethylene glycol. In studying the purified enzyme we observed: (i) a cofactor in serum was required for full enzymatic activity; ApoLp-Glu could be substituted for this cofactor; (ii) ApoLpSer was inhibitory to lipase activity; (iii) NaCl and protamine sulfate also markedly inhibited the lipase activity; (iv) heparin stimulated the enzymatic activity.

Key words : Affinity chromatography - Heart - Heparin - Lipoprotein Iipase

INTRODUCTIO&

The enzyme lipoprotein lipase appears to be a necessary factor in the transport of glyceride fatty acid from the circulation into tissue l. Although initially observed in post-heparin plasma, this enzyme was first characterized in extracts of heart tissuea. Attempts to purify lipoprotein lipase by conventional methods have been largely unsuccessful, presumably because of the inherent instability of preparations of this enzyme. However, with the recent development of affinity chromatography, specificalAbbreviations: apoLp-Ala = apolipoprotein-alanine, apoLp-Glu = apolipoprotein-glutamic, apoLp-Ser = apolipoprotein-serine, LPL = lipoprotein lipase, VLDL = very low density lipoprotein. This work was supported in part by grants from the United States Public Health Service AM 4706, American Heart Association 72-673, and American Heart Association, Greater Los Angeles Affiliate 492-lG2.

464

JER-SHUNG

ly, chromatography

with Sepharose

TWU,

to which heparin

A. S. GARFINKEL,

is covalently

M. C. SCHOTZ

linked,

a number

of investigators have purified lipoprotein lipase(s) from a variety of sources. These sources include bovine milks, chicken4, rats, and pig adipose tissue6, and rat postheparin plasmaT. From one major source of lipoprotein lipase, heart tissue, no definitive study of the purified enzyme has been reported. In the course of our work on metabolic regulation

of rat heart lipoprotein

lipases we succeeded

In this report we describe some characteristics of the purification MATERIALS

AND

in purifying

of the purified

this lipase 1500-fold.

heart enzyme and details

procedure. METHODS

Tri[l-i4C]oleoyl

glycerol

(132 mCi/mmole)

and

tri[9,10-sH]oleoyl

glycerol

(6.3 Ci/mmole) were purchased from Applied Science Laboratories, Inc., and DHOM Products, Ltd., respectively. Silicic acid thin-layer chromatography indicated that these triglycerides were 99 % radiochemically pure. Heparin (160 USP units/mg) and protamine sulfate were obtained from Calbiochem. The following proteins were purchased from Sigma Chemical Co. : trypsin, glyceraldehyde phosphate dehydrogenase, aldolase, ovalbumin, glutamate dehydrogenase and bovine albumin. The peptides (apoLp-Glu, apoLp-Ser, and apoLp-Ala), the gift of Drs. F. Aladjem and Y. Sambray, University of Southern California School of Medicine, had been isolated from human VLDL by the method of Herbert et ~1.9. These peptides were as homogeneous as described in that report. Heparin-Sepharose was prepared as described by Iveriusra. All chemicals were reagent grade. The lipase assay measured the release from triolein of either i4C-or sH-labelled fatty acidil. The assay system contained the following components in a total volume of 1.0 ml: 10 pmoles unlabelled triolein, 0.17 PCi tri[l-t%]oleoyl glycerol, or 83 PCi tri[9,10-3H]oleoyl glycerol; 0.53 mg egg lecithin; 20 mg bovine 0.4 ml fasted dog serum; and various amounts of enzyme solutionre.

serum albumin; The final buffer

concentration was 0.10 M Tris-HCl, pH 8.0, containing 0.15 A4 NaCI. Incubations were carried out for 1 hr at 37°C. The released labelled free fatty acids were extracted into 0.1 N KOH and determined in a Packard Liquid Scintillation Spectrophotometerll. For enzyme purification male Sprague-Dawley rats fed ad lib. and weighing between 150 and 250 g were sacrificed by decapitation. Hearts were excised, trimmed of fat, minced, and acetone powders prepared is. All powders were used within 10 days of preparation. One gram of dry acetone powder was routinely taken for enzyme purification. The delipidated powder was homogenized at 4°C in 35 ml of 5 mM sodium barbital buffer, pH 7.2 (the term barbital buffer refers to this particular buffer) using a Ten Broeck homogenizer. The resulting suspension was centrifuged at 30,000 x g for 20 min at 4°C to remove the insoluble material. The clear supernatant (30 ml) was applied to a heparin-Sepharose column (1 x 10 cm) at 4°C equilibrated with barbital buffer. Following passage of the sample, the column was

RAT HEART

LIPOPROTEIN

465

LIPASE

washed with 10 ml of barbital

buffer. All fractions

collected

bined. Further elution was carried out with 30 ml of barbital followed by 30 ml of barbital buffer containing 1.5 A4 NaCl.

to this point were combuffer in 0.75 M NaCl

For polyacrylamide gel electrophoresis the purified enzyme was desalted by dialysis and concentrated by vacuum dialysis or lyophylization. Disc gel electrophoresis was performed in 7.5 “/, acrylamide gel, pH 9.5, at 4°C for 1 to 2hr14. Protein bands were visualized by Coomassie blue staining. The minimum

molecular

weight of the purified

enzyme was determined

by the

method of Weber and Osbornr5 using trypsin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, ovalbumin, and bovine serum albumin as standards. The gels were run for 4 hr at room temperature. Localization of enzymic activity on polyacrylamide gel after electrophoresis at 4°C was done by slicing the gel into 0.5 cm divisions, grinding the gel slices with a glass rod and incubating with substrate to determine lipase activity. Parallel gels were run simultaneously for protein staining. Protein was determined by the method of Lowry et aZ.16,using bovine serum albumin as standard. RESULTS

The result of a typical purification

carried

out with 1 g of acetone

powder

are

summarized in Table 1. Over 60 % of the applied protein and about 50% of total lipolytic activity did not bind to the heparin-Sepharose gel. Reapplication of this unadsorbed protein onto another heparin-Sepharose column did not show further binding of lipoprotein lipase activity to the gel. A solution of 0.75 M NaCl released from the gel matrix almost all of the bound protein, but essentially none of the tightly bound lipoprotein lipase activity. However, on increasing the NaCl concentration to TABLE 1 PURIFICATION

Step

OF RAT

HEART

LIPOPROTEIN

Volume

Protein

(ml)

(mg)

Extracta

30.0

Unadsorbedb protein 0.75 M NaCl 1.5 M NaW

38.5 28.0 6.7

LIPASE

Total activity (mpmoles FFAjhr)

Specific activity (mpmoles FFAlmg proteinlhr)

Purification

Yield

(fold)

f%)

300

6003

20.1

1.0

100

206 94 0.108

2844 0 3333

13.8 0 30,861

0.7 0 1540

47 0 55

a Sodium barbital (0.005 M, pH 7.2) extract of 1 g rat heart acetone powder. b Combined eluates of extract applied to the column and washing buffer in the absence of NaCI. c Pooled fractions from tubes 3 to 5 of Fig. 1.

466

JER-SHUNG

1.5M NaCl

TWU,

A. S. GARFINKEL,

M. C. SCHOTZ

-W

lb Tube #

Fig. 1. Purification of rat heart lipoprotein lipase by heparin-Sepharose affinity chromatography. An extract (30 ml) ,prepared from 1 g of acetone powder, as described in the text, was applied to a heparinSepharose 4B column (1 x 10 cm) equilibrated with 0.005 M sodium barbital buffer, pH 7.2. Unadsorbed protein was collected and combined with the next 10 ml of washing buffer. After elution with 30 ml buffer containing 0.75 M NaCl (flow rate 30 ml/hr), the column was washed with 30 ml buffer containing 1.5 M NaCl and the eluate collected in 2 ml fractions. Aliquots of all fractions were assayed immediately after elution and results are summarized in Table 1. Enzyme activity is expressed as mpmoles fatty acid released per ml of enzyme solution per hr.

STACKING

‘-

0

GEL

loo

200

CPM

Fig. 2. Polyacrylamide gel electrophoresis of rat heart lipoprotein lipase. Samples in 1.5 M NaCl were first desalted by dialysis for 2 hr, than concentrated by vacuum dialysis. A major protein band is seen in the diagram of the separation gel in the center of the figure with enzyme activity corresponding to this band on the right. Polyacrylamide gel electrophoresis of rat heart LPL in the presence of sodium dodecyl sulfate, Coomassie blue stained, is pictured on the left. Details of the conditions of electrophoresis and assay of gel slices for enzyme activity are given in the text.

RAT HEART LIPOPROTEIN LIPASE

i&---_+0

2

Incubation

24

3 time

467

(hr)

Fig. 3. Stability of purified heart lipoprotein lipase in 1.5 M NaCl at 4 “C in the absence and presence of 1 M ethylene glycol. TABLE 2 STABILITY

OF PURIFIED

LIPOPROTEIN

Additions

LIPASE

Relative

LPL activity&

(%I

None Bovine serum albumin (1 mg/ml) Glycerol (20 %) Ethylene glycol (1 M)

16 27 56 25

a Purified LPL activity in the presence of 1.2 MNaCl stored for 7 days at -20°C. is expressed as a percentage of that present at time of storage.

The enzyme activity

1.5 M, a homogeneous lipoprotein lipase activity appeared in the eluate accompanied by a measurable protein band (Fig. 1). The total lipoprotein lipase activity recovered in this step represented about 50% of that applied to the column. This highly purified enzyme represented a 1500-fold purification of the original extract. The results of this purification were consistently reproducible. On polyacrylamide gel electrophoresis at pH 9.5 the purified enzyme from the 1.5 M NaCl eluate revealed a major protein band (Rf 0.75) (Fig. 2). Assay of the gel slices yielded a lipolytic activity peak at the site corresponding to this major protein band (Fig. 2) and at the stacking gel. Polyacrylamide gel electrophoresis (10 %) carried out in the presence of sodium dodecyl sulfate (Fig. 2) indicated a major protein band (70-80x) plus two faint bands below the major one. The stability of the purified enzyme eluted in 1.5 A4 NaCl was investigated in the presence of several chemical agents. As shown in Fig. 3, ethylene glycol (1 M) partially prevented the rapid disappearance of enzyme activity. The sample with 1 M ethylene glycol retained 50% of the original activity after 24 hr of storage at 4 “C, whereas the control without ethylene glycol retained only 16% of the original lipo-

JER-SHUNG

468

TWU,

A. S. GARFINKEL,

M. C. SCHOTZ

300,

250-

360

1

.

L

f 240-

/

i? k. .

5 120E

10

20

30 40 Time (min)

50

/

i

- Serum 0

/

60

FxYZ7Z74

Enzyme ( ml 1

Fig. 4. Linearity of hydrolysis of triglyceride by purified lipoprotein lipase in the presence and absence of serum with time of incubation and amount of enzyme. Equal aliquots of purified enzyme preparation were assayed in the presence (0) and absence (0) of serum for varying periods of time up to 60 min (figure on the left); and varying amounts of enzyme were assayed after a 60 min incubation (figure on the right). Enzyme activity is expressed as m~~moles fatty acid released during incubation at 37°C.

1 0

-‘ h 0

0.2

200

0.4 0.6 NaCl (M)

400

ciao

0.8

800

l,Oco

1.0

20

40 Heparin

60 (pg /ml)

00

100

Qcm

Protamine sulfate (gg I ml 1

Fig. 5. Effects of NaCI, heparin, and protamine sulfate on purified heart lipoprotein lipase. N&lEnzyme activity was determined without prior incubation at the NaCl concentrations given on the abscissa and related to that determined in the presence of 0.15 MNaCI. Heparin andprotumine sulfate - Enzyme activity is related to that determined in the absence of heparin or protamine sulfate.

RAT HEART

LIPOPROTEIN

469

LIPASE

1O-r... hoa-

\

r .t c 2

.

06_ \

i

.

; 04_ .z r 2 ;L 0.2..

0

5

IO

15

ApoLp-Glu

I

0

AilpOoLp%a

(:ilrnl)

20 (pg

25

30

0

80

/ml)

80

Fig. 6. Effect of peptides, isolated from VLDL, on purified heart lipoprotein lipase. Increasing levels of apoLP-Glu were substituted for dog serum in the enzyme assay or the enzyme assay system contained 0.1 ml dog serum and increasing amounts of apoLp-Ser or apoLp-Ala. Enzyme activity is related to that determined in the absence of added peptides.

protein lipase activity. When the purified enzyme in 1.2 M NaCl was stored for a week at -2O”C, addition of glycerol (20 %) preserved 60% of the initial enzymic activity (Table 2). Under these conditions ethylene glycol (1 M) and bovine serum albumin (1 mg/ml) were less effective as preservative agents than was glycerol. The rate of catalysis of triglyceride substrate by the purified enzyme wasexamined in the presence and absence of dog serum (Fig. 4). With serum present the purified enzyme catalyzed the release of fatty acid at a linear rate for at least 60 min. In the absence of serum some residual enzyme activity was measurable, but the rate of catalysis quickly reached a plateau during the incubation period. It can be seen also in Fig. 4 that during a 60 min incubation the release of fatty acid was proportional to amount of enzyme added. The effects of sodium chloride, heparin, and protamine sulfate are summarized in Fig. 5. The activity of the purified enzyme is very sensitive to NaCl in the assay system. Enzyme activity was inhibited more than 70 % in the presence of 0.3 M NaCl. All enzyme activity was determined without prior incubation at the NaCl concentration being investigated. Enzyme activity was consistently stimulated about 60% on

470

JER-SHUNG

TWU,

A. S. GARFINKEL,

M. C. SCHOTZ

--GLYCERALDEHYDE PKSPHATE DEHYDROCENASE

II

0.2

0.4 0.6 Mobility

0.8

1.0

Fig. 7. Determination of the minimum molecular weight of rat heart lipoprotein lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 6 marker proteins used were bovine serum albumin, glutamate dehydrogenase, ovalbumin, aldolase, glyceraldehyde phosphate dehydrogenase and trypsin. The arrow indicates the mobility of heart lipoprotein lipase.

addition of 5 to 100 pug of heparin. Higher concentrations (1 mg/ml) of heparin did not result in inhibition. The highly purified heart lipoprotein lipase was inhibited by protamine sulfate. At a concentration of 200 pg protamine sulfate/ml assay mixture 50 % of enzyme activity was lost. Increasing the protamine sulfate concentration did not cause further significant inhibition. As shown in Fig. 6, the purified heart lipoprotein lipase requires a peptide, apoLp-Glu, as a cofactor for full catalytic activity. In the presence of 30 pg apolpGlu/ml, a 30-fold stimulation of lipoprotein lipase activity was observed. In the absence of serum but with increasing concentrations of apoLp-Glu, a hyperbolic curve was obtained. When serum (0.1 ml) was present in the assay mixture, apolpSer exhibited an inhibitory effect on lipoprotein lipase activity (Fig. 6). ApoLp-Ala showed only a slight inhibitory

effect on enzyme

activity

under these conditions.

At

a concentration of 80 ,ug/ml for each peptide, 50 % and 18 % inhibition of the original lipoprotein lipase activity was found for apoLp-Ser and apoLp-Ala, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of samples of the purified enzyme yielded one major protein band (Fig. 2). This major protein band accounted for over 70-80% of the total protein stained by Coomassie blue. Its electrophoretic mobility indicated a molecular weight of 60,000 (Fig. 7). DISCIJSSIOPi

Rat heart lipoprotein lipase was highly purified (1500 x) by affinity chromatography with heparin-Sepharose 4B as the insoluble gel matrix. The high affinity of this lipase for heparin results in a firm binding between enzyme and matrix. Increasing the ionic strength to 0.75 M NaCl released the proteins non-specifically bound to the matrix. Higher ionic strength (1.5 A4 NaCl) liberated the lipoprotein lipase activity. We observed that 45 % of lipase activity applied to the column was not retarded by the heparin-Sepharose gel. The possiblity that this was due to overloading the col-

RAT HEART

LIPOPROTEIN

471

LIPASE

umn was ruled out by the failure of this fraction

to bind to a new heparin-Sepharose

gel.

purified lipoprotein lipase from adipose tissue and skim Other investigatorss*s milk using heparin-Sepharose 4B column chromatography with initial extracts containing 0.5 M NaCl. These columns were eluted with a linear NaCl gradient from 0.5 to 1.5 M. It was reported that by this procedure almost all lipase activity in the initial extract was bound to the column (85 y0 for skim milk and 100 o/, for swine adipose tissue) and subsequently the lipase could be released by increasing ionic strength. In contrast

we found

that with the crude extract

of heart tissue, adding

0.5 M NaCl

did not result in complete binding of lipase to the gel matrix. The discrepancy between these findings may be due to species or tissue differences. Since no significant amount of contaminating protein (Fig. 2) was found in the final 1.5 M NaCl eluate and since the tightly bound

lipase was not liberated

by 0.75 M NaCI, we employed

a disconti-

nuous salt gradient. Employing heparin-Sepharose columns Dolphin and Rubinstein17 have recently reported preliminary data concerning the interaction of a partially purified rat heart lipoprotein lipase with VLDL and chylomicrons from rat serum. The enzyme activity was fully protected in some way when it was absorbed on the gel matrix, but once it was released into 1.5 M NaCl it then exhibited highly unstable characteristics. Although ethylene glycol (1 M) slowed the decay of the enzyme activity (Fig. 3), glycerol (20%) was the most effective of the three stabilizing agents studied and seems best for enzyme storage (Table 2). The inhibition of the enzyme by protamine sulfate (Fig. 5) suggests that the purified heart lipase may have a low p1 and therefore form a complex with highly basic protamine sulfate rendering the active site of lipase less accessible to substrate. In the absence of serum or apoLp-Glu, the enzyme still exhibited some lipolytic activityis. The results of the kinetic studies shown in Fig. 4 suggest that some cofactor present in the serum is required for full enzymatic activity or serum in some manner stabilizes enzyme activity. ApoLp-Glu, isolated from human VLDL, can substitute for serum (Fig. 6). Further studies on the interaction between apoLp-Glu and lipase are needed to resolve the role of this peptide in activating lipase. Another peptide, ApoLp-Ser, isolated from VLDL, exhibited a marked inhibitorye ffect on lipase activity when serum was included in the assay mixture (Fig. 6). It has been reported that the peptide, apoLp-Ala, is a major inhibitor of lipoprotein lipase from other sourcessJ9,sO. Therefore, it is of interest that the purified heart LPL is not significantly inhibited by this peptide but is inhibited by apoLp-Ser. These effects suggest that these peptides may be involved in the regulation of the lipolytic process. The minimum molecular weight of rat heart lipase as determined by disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed an approximate value of 60,000 (Fig. 7). This value coincides with that reported for skim milk, 64,0003, and swine adipose tissue, 62,0006. Our purified lipase gave only one protein band on disc-gel electrophoresis. However, more than one protein was visualized on the sodium dodecyl sulfate disc gel. The two faint bands which migrated below the major band indicate that the enzyme preparation requires further purification. All the properties mentioned above fulfill the classic criteria for defining the

472

JER-SHUNG TWU, A. S. GARFINKEL, M. C. SCHOTZ

isolated rat heart lipase as a lipoprotein lipase. Among a number of problems still remaining to be examined are the nature of the two small proteins, and the characteristics of the lipase activity which does not bind to heparin-Sepharose gel. ACKNOWLEDGEMENTS

The authors

wish to thank Mrs. Judith Nikazy for excellent technical

assistance.

REFERENCES 1 ROBINSON,D. S., The function of the plasma triglycerides in fatty acid transport. AND E. H. STOTZ (Eds.),

2

3 4 5 6 7 8 9 10

11 12 13 14 15 16 17

18 19 20

In: M. FLORKIN

1970, pp. 51-116. KORN, E. D., Clearing factor, a heparin-activated lipoprotein lipase, Part 1 (Isolation and characterization of the enzyme from normal rat heart), J. BioL Chem., 215 (1955) 1. EGELRUD, T. AND OLIVECRONA,T., The purification of a lipoprotein lipase from bovine skim milk, J. Biol. Chem., 247 (1972) 6212. EGELRUD,T., Reversible binding of lipoprotein lipase from hen adipose tissue to insolublized heparin, Biochim. Biophys. A&a, 296 (1973) 124. GRETEN,H. AND WALTER, B., Purification of rat adipose tissue lipoprotein lipase, FEBS Lett., 35 (1973) 36. BENSADOUN,A., EHNHOLM,C., STEINBERG,D. AND BROWN, W. V., Purification and characterization of lipoprotein lipase from pig adipose tissue, J. Biol. Chem., 249 (1974) 2220. JANSEN,H., VAN ZUYLEN-VAN WEGGEN, A., AND HULSMANN,W. C., Lipoprotein lipase from heart and liver - an immunological study, Biochem. Biophys. Res. Commun., 55 (1973) 30. SCHOTZ, M. C. AND GARFINKEL,A. S., Effect of nutrition on species of lipoprotein lipase, Biochim. Biophys. Acta, 270 (1972) 472. HERBERT,P. N., SHULMAN,R. S., LEVY, R. 1. AND FREDRICKSON,D. S., Fractionation of the Capoproteins from human plasma very low density lipoproteins, J. Biol. Chem., 248 (1973) 4941. IVERIUS, P. H., Coupling of glycosamino glycans to agarose beads (Sepharose 4B), Biochem. J., 124 (1971) 677. SCHOTZ, M. C., GARFINKEL,A. S., HUEBOTTER,R. J. AND STEWART,J. E., A rapid assay for lipoprotein lipase, J. Lipid Res., 11 (1970) 68. SCHOTZ, M. C. AND GARFINKEL,A. S., A simple lipase assay using trichloroacetic acid, J. Lipid Res., 13 (1972) 824. GARFINKEL,A. S. AND SCHOTZ, M. C., Separation of molecular species of lipoprotein lipase from adipose tissue, J. Lipid Res., 13 (1972) 63. CLARKE, J. T., Simplified “disc” (polyacrylamide gel) electrophoresis, Ann. N. Y. Acad. Sci., 121 (1964) 428. WEBER, K. AND OSBORN, M., The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J. Biol. Chem., 244 (1969) 4406. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L. AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. DOLPHIN, P. J. AND RUBINSTEIN,D., The metabolism of very low density lipoprotein and chylomicron by purified lipoprotein lipase from rat heart and adipose tissue, Biochem. Biophys. Res. Commun., 57 (1974) 808. EGELRUD,T. AND OLIVECRONA,T., Purified bovine milk (lipoprotein) lipase - activity against lipid substrates in the absence of exogenous serum factors, Biochim. Biophys. Actu, 306 (1973) 115. BROWN, W. V. AND BAGINSKY,M. L., Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein, Biochem. Biophys. Res. Commun., 46 (1972) 375. HAVEL, R. J., FIELDING,C. J., OLIVECRONA,T., SHORE,V. G., FIELDING,P. E. AND EGELRUD,T., Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoprotein lipase from different sources, Biochemistry, 12 (1973) 1828. Comprehensive

Biochemistry,

Vol. 18, Elsevier, Amsterdam,

Rat heart lipoprotein lipase.

Rat heart lipoprotein lipase was highly purified by affinity chromatography using heparin-Sepharose 4B. When extracts of acetone powder were applied t...
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