Iozzo Renato V. Proteoglycan Protocols

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Preparation for Amino Acid Sequence Analysis 75

8. Tubes can either be dried in a Savant Speedvac or frozen for storage. If peptides are being

sent to a core facility, there is no need to ship them cold if they have been dried. Transfer-

ring peptides from one container to another may result in complete loss.

At the end of the run, or series of runs, rinse the injection loop with deionized water

in both the load and inject positions to remove salt.

The relative size of peaks will be largely related to the amino acids that are present

in the peptides. The largest peaks will likely have tryptophan as one of the constitu-

ent amino acids. The next largest peaks will likely contain tyrosine. Smaller peaks

may be from contaminants or will contain no aromatic amino acids. Peaks that contain

single peptides will be symetrical or may tail slightly. If the HPLC separation has been

preceded by gel filtration, it is possible to estimate purity by determining how many

fractions a given peak is in. Typically, a single peptide should not elute from the

Fig. 1. The use of lag time to ensure that peaks are collected into a single tube. To ensure that

peaks from reversed-phase HPLC analyses are collected into single tubes, it is necessary to know

the time between a peptide arriving in the flow cell (which translates into an absorbance change)

and the peptide exiting the tube downstream from the flow cell. In a typical scenario, the tube

after the flow cell will be 20–30 cm long and will have an internal diameter of 0.1 mm, resulting

in a volume of 20 µL. If the flow cell has a volume of 5 µL, then this means that at 250 µL / min, the

lag time between the peak entering the flow cell and it appearing at the outlet will be 6 s. In practice,

the point that is most important is when the apex of the peak is in the flow cell. For this, a volume of

half the flow cell + the outlet tube will give a lag of 5.4 sub at 250 µL/min and 6.75 sub at 200 µL/

min. It is also possible to determine this empirically by injecting a colored dye into the system. Note

that for maximum peptide purity, it is important to collect the peak, but not the leading and trailing

edges of the peak.

76 Neame

size-exclusion volume in a volume greater than 1 ml for a 0.5 ml sample size (two or

three 0.5-ml fractions).

An estimate of the likely amount of a peptide can be obtained from the UV trace.

With the UV monitor set at 220 nm and using a 2.1-mm-internal-diameter column, a

“typical” peak of 15 pmol of a 12-amino acid peptide should have a peak height of

0.01–0.02 AU.

4. Notes

1. It is possible to digest samples on blots or in gels. This is described in more detail in another

volume of this series (3,4). An earlier version of the blot digestion procedure has also been

published (11). These methods work well for globular proteins, but in the case of

proteoglycans it can be difficult to obtain a sample density that provides an adequate signal-

to-noise ratio. Proteoglycans often do not stain well with Coomassie blue or Ponceau S, so

it can be difficult to identify the location of the substrate. It is unusual to

be able to load a sufficient quantity of proteoglycan onto an SDS-PAGE gel to obtain the

necessary amount and concentration of material. As little as 25 pmol of material can be

digested (2.5 µg of the small proteoglycan, decorin), although 2–4 times this much is rec-

ommended. For reasonable results, this should be in a total gel weight of 50 mg. A blank

area of gel should be used as a control.

2. Chymotrypsin tends to cut proteins in too many places to be useful; it typically cuts at the

C-terminal of tyrosine, tryptophan, and phenylalanine, but will also cut at leucine. Other

enzymes (endoprotease Glu-C, for example) can be a little unpredictable. Endoprotease Glu-C

(V8 protease) will generally cut on the C-terminal of glutamate, but can also cut at aspartate.

3. Guanidine HCl (4 M) as a chaotrope for size-exclusion chromatography has the advantage

that it gives good yields and prevents peptides precipitating. However, it is corrosive, and

crystals of salt can cause scoring of moving parts in pumps. To avoid damage to hardware,

chromatography systems should be washed thoroughly after use. Guanidine also precludes

use of direct SDS-PAGE analysis, which would make it difficult to analyze GAG-contain-

ing peptides using Western blotting. Urea is an alternative chaotrope, but it should be free

of cyanates, which will carbamylate amino groups (the N-terminal and lysine side chains).

Urea can be cleaned by passing it down an ion-exchange column. All buffers should be

filtered through a 0.2 µm filter. This is particularly important for the high-concentration

chaotropes.

If high-salt buffers are used, syringe-type pumps, such as those found on the Pharmacia

FPLC, are easier to use than the piston-based pumps found on most HPLC systems. Some

HPLCs have a pump design that enables the rear of the piston to be irrigated with water; this

eliminates the problem of salt buildup on the rear of the piston and subsequent scoring of the

piston and damage to the pump seals.

4. Some HPLC users prefer not to inject guanidine onto their columns. It is good courtesy to

use your own HPLC column, but in fact the guanidine will not damage anything as long as

it is at a pH below 7 and it is not left in contact with moving parts.

5. The fractions containing glycosaminoglycan can be concentrated on a Speedvac, desalted,

and applied directly to the sequencer. While this may give more than one sequence, if a

cDNA-derived protein sequence is available, it should be possible to deconvolute the amino

acids at each cycle to determine where in the protein the glycosaminoglycan is attached.

On occasion, it is possible to separate glycosaminoglycan-substituted peptides by

reversed-phase HPLC, although the peaks are extremely broad (6,10).

Preparation for Amino Acid Sequence Analysis 77

References

1. Edman, P. (1956) Mechanism of the phenylisothiocyanate degradation of proteins. Nature

177, 667–668.

2. Williams, K. R., Samander, S. M., Stone, K. L., Saylor, M., and Rush, J. (1996) Matrix-assisted

laser desorption ionization mass spectrometry as a complement to internal protein sequenc-

ing, in The Protein Protocols Handbook (Walker, J. M., ed.), Humana Press, Totowa, NJ, pp.

541–555

3. Fernandez, J., and Mische, S. M. (1996) Enzymatic digestion of membrane-bound pro-

teins for peptide mapping and internal sequence analysis, in The Protein Protocols Hand-

book (Walker, J. M., ed.), Humana Press, Totowa, NJ, pp. 405–414.

4. Stone, K. L. and Williams, K. R. (1996) Enzymatic digestion of proteins in solution and in

SDS-polyacrylamide gels, in The Protein Protocols Handbook (Walker, J. M., ed),

Humana Press, Totowa, NJ, ), pp. 415–425.

5. Stone, K. L. and Williams, K. R. (1996) Reverse-phase HPLC separation of enzymatic

digests of proteins, in The Protein Protocols Handbook (Walker, J. M., ed.), Humana

Press, Totowa, NJ, pp. 427–434.

6. Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995)

N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature

and immature bovine cartilage. J. Biol. Chem. 270, 20516–20524.

7. Fisher, L. W., Hawkins, G. R., Tuross, N., and Termine, J. D. (1987) Purification and partial

characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin

from the mineral compartment of developing human bone. J. Biol. Chem. 262, 9702–9708.

8. Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1991) Catabolism of

aggrecan in cartilage explants. Identification of a major cleavage within the interglobular

domain. J. Biol. Chem. 266, 8683–8685.

9. Farndale, R. W., Buttle, D. J., and Barrett, A. J. (1986) Improved quantitation and discrimi-

nation of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim.

Biophys. Acta. 883, 173–177.

10. Johnson, H. J., Rosenberg, L., Choi, H., Garza, S., Höök, M., and Neame, P. J. (1997)

Characterization of epiphycan, a small proteoglycan with a leucine-rich core protein. J.

Biol. Chem. 272, 18709–18717.

11. Fernandez, J., DeMott, M., Atherton, D., and Mische, S. M. (1992) Internal protein

sequence analysis: enzymatic digestion for less than 10 µg of protein bound to

polyvinyldifluoride or nitrocellulose membranes. Anal. Biochem. 201, 255–264.

Preparation of High–Specific-Activity Heparan Sulfate 79

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

High–Specific-Activity

S-Labeled Heparan Sulfate

Prepared from Cultured Cells

Nicholas W. Shworak

1. Introduction

Cultured cells are a facile reagent for elucidating the molecular mechanisms that

regulate the biosynthesis of heparan sulfate (HS) (1–3). However, a typical confluent

flask (~20 million cells) produces only a small amount of HS (1–2 µg), which is at or

below the detection limit of many nonradioisotopic techniques. Fortunately, this

limitation can be circumvented by the metabolic labeling of cells with Na

Sulfate from the culture medium is transported into the cytoplasm, where it is incorpo-

rated into the biosynthetic sulfate donor adenosine 3'-phosphate 5'-phosphosulfate

(PAPS), which is transported into the Golgi apparatus (4). Specific biosynthetic enzymes

transfer a sulfonyl group from PAPS onto maturing glycosaminoglycan chains.

The high sulfate content of glycosaminoglycans ensures that metabolic labeling with

is relatively selective and allows for extreme sensitivity. Although PAPS is

the obligate sulfate source of all cytosolic and membrane-bound sulfotransferases, gly-

cosaminoglycan biosynthesis typically accounts for ~70% of PAPS consumption (5–7).

Consequently,

-2

from the culture medium is preferentially incorporated into gly-

cosaminoglycan chains. Cells can even be incubated with virtually carrier-free

(~1.5 × 10

Ci/mol), which allows for the production of

S-labeled HS with

an exquisitely high-specific-activity (theoretically ~1.2 × 10

Ci/mol, given that there

are ~80 sulfates per chain) (3,8). Such extreme measures are beneficial for applica-

tions that require large amounts of radioactive HS. One example is structural analysis

for the identification of rare modifications, such as 3-O-sulfated disaccharides. Such

species can comprise less than 1% of HS sulfate, and their isolation can require exten-

sive manipulations that result in substantial loss of starting material (3,9,10). Conse-

quently, large amounts of input radioactivity are necessary for detection of the final

product. A second application is detection of sulfotransferase enzymatic activity by the

80 Shworak

ligand affinity conversion approach (Chapter 10). Although each assay requires a

small amount of HS as substrate (100,000 cpm), the analysis of a large number of

samples mandates a substantial supply of

S-labeled HS.

This chapter describes protocols for generating ~100 µCi of high-specific-activity

S-labeled HS from cultured L cells. Suggestions are included for adapting this

method to other cell types and for obtaining low-specific-activity material.

2. Materials

2.1. General

1. Water should be deionized, then either distilled or processed through a Milli-Q

(Millipore)-type filtration system. Analytic-grade chemicals are used throughout.

2. Hand-held radioactivity detectors. A Geiger-Müller probe provides the most sensitive direct

detection of unshielded β-emissions. A sodium iodide scintillation probe detects the second-

ary γ-radiation emitted when shielding is bombarded by β-rays (Bremsstrahlung effect).

3. Materials for radioactive work. Disposable bench coat and latex gloves, plastic shielding

(Owl Scientific) wrapped with sufficient lead foil to capture secondary Bremsstrahlung

photons. Collection of radioactive waste is best performed with a vacuum aspiration setup

comprised of two serially coupled side-arm flasks. The first is the fluid reservoir and the

second collects accidental overflow.

4. Plastic ware. All plastic tubes/flasks for containment of radioactivity should have lids

that provide a complete seal. Required items include 25- and 75- cm

flasks (Nunc), 15-mL

polypropylene conical tubes (Fisher # 05-539-5), and 2-mL screw-cap polypropylene

tubes with a cap that contains a sealing Neoprene O-ring (Sarstedt # 500 65 716).

5. Pipetters. A full complement of micropipetters (Rainin) and aerosol-resistant disposable

tips. An Eppendorf Repeater (VWR # 53512-500).

6. Bottle-top filters. For sterilization use 0.22-µm, and for removal of particles use 0.45 µm.

7. Liquid scintillation. Vials, 7-mL capacity (Fischer Scientific), aqueous compatible

scintillant (e.g., Ecolume, New England Nuclear), and a liquid scintillation counter.

8. Centrifuges. A microfuge and a low-speed tabletop centrifuge.

9. A liquid-nitrogen source.

2.2. Labeling Cell Surface Proteoglycans

1. Tissue culture setup. Apart from standard equipment, a specialty gas mix of 5% CO

balanced with air should be placed near the laminar-flow hood. A plastic tube from the low-

pressure regulator is connected onto a plugged 5- or 10-mL pipet, which acts as sterile filter.

2. Appropriate sterile medium. L cells use Dulbecco’s modified Eagle’s medium contain-

ing 10% fetal bovine serum, 100 units/mL of penicillin G, and 100 µg/mL of streptomy-

cin sulfate (Life Technologies, Inc.).

3. PBS (phosphate-buffered saline) and trypsin/EDTA. PBS is 0.22-µm filtered and stored

at room temperature (Sigma # P4417). Sterile trypsin/EDTA (# 25300-062, Life Tech-

nologies) is distributed in 50-mL aliquots. Reserve stocks are stored at –20°C and work-

ing stock is stored at 4°C.

4. PBS/Na

and Trypsin/EDTA/Na

. Supplement the above solutions to 1 mM Na

5. Labeling medium base. A sulfate-free base mixture is generated by combining the ingre-

dients of Table 1 in the ascribed order (the 100 × sulfate-free salt mixture is 4% KCl, 2%

MgCl

·7H

O and 1.4% NaH

·H

O, autoclaved and stored at room temperature). The

mixture is 0.22-µm filtered into an autoclaved 500-mL bottle. The gassing pipet (item 1),

is fully inserted into the bottle and flow is set to initiate light bubbling. The medium is

Preparation of High–Specific-Activity Heparan Sulfate 81

fully CO

equilibrated when the indicator dye turns a stable orange color. The bottle is

tightly capped and stored at 4°C (see Note 1).

6. Carrier-free Na

(1,495,000 Ci/mol, ~1 mCi/µL) (ICN). Centrifuge the vial before

opening to pool radioactivity away from the lid. (see Note 2).

7. 0.1 M Na

(10 mL). Dilution of stock from Table 2; sterilize by 0.22-µm filtration.

8. Ice-chilled grate. To generate a stable, level, chilled work surface, place a metal grate

(VWR # 54848-316) on a level bed of ice in a large, flat Styrofoam tray.

2.3. Purification of Proteoglycans by Preparative

DEAE Microchromatography

1. The stocks listed in Table 2 are required to generate the DEAE column buffers listed in

Table 3. The stocks and DEAE column buffers are stored at room temperature unless

stated otherwise. All column buffers are 0.45-µm filtered.

2. Preparative wash buffer. A 50-mL solution of 150 mM NaCl, 0.03% Triton X-100, and

30 µg/mL BSA is made from Table 2 stocks, then stored at 4°C.

3. Preparative elution buffer. A 50-mL solution of 1 M NaCl, 0.03% Triton X-100, 30 µg/

mL BSA, and 2.5 µg/mL glycogen is made from Table 2 stocks, then stored at 4°C.

4. GUTE (100 mL). 4 M guanidine

·HCl (Fluka # 50935), 50 mM Tris pH 7.4, 1 mM EDTA.

(see Note 4).

5. Chondroitin sulfate (from shark cartilage, Sigma C-4384). Make 2 mL of a 20-mg/mL

solution in water, store at –20°C.

6. Column blocking mix. To 1.8 mL of fetal bovine serum add 0.2 mL of the above chon-

droitin sulfate solution. Spin in a microfuge at maximum speed for 10 min and transfer the

supernatant to a clean tube.

7. DEAE microcolumn assembly. A gravity-flow microcolumn is assembled in the barrel of

a plastic 1-mL syringe. Onto the tip, fit a Luer adapter with ~4 cm of tubing (trim a

“butterfly” catheter; VWR # VT7251). This outflow tract can be plugged by inserting the

point of a “yellow” tip (seal the wide end by flame-melting), whereas the top is sealed

Table 1

Base Mixture for Cell Labeling

Stock solutions Final

Composition Source, sterilization, storage concentration Volume

O 211 mL

1.5 M

D-glucose 0.22-µm filtered, 4 or –20°C 5.54 mM 0.92 mL

5 M NaCl Autoclaved, room temperature 100 mM 5.0 mL

4.4% NaHCO

0.22-µm filtered, 4°C 0.22% 12.5 mL

1 M HEPES, pH 6.5 0.22-µm filtered, 4°C 12.5 mM 3.13 mL

1 M HEPES, pH 7.9 0.22-µm filtered, 4°C 12.5 mM 3.13 mL

11% Sodium pyruvate 0.22-µm filtered, –20°C 0.011% 0.25 mL

0.75% Phenol red Room temperature 0.00038% 0.13 mL

100 × Sulfate free salts 0.22-µm filtered, room temperature 1× 2.5 mL

50 × MEM amino acids Life Technologies, #11130-051, 4°C1 × 5.0 mL

100 × BME vitamins Life Technologies, #21040-050, –20°C1 × 2.5 mL

200 mM

L-glutamine 0.22-µm filtered, –20°C1 mM 1.25 mL

2% CaCl

Autoclaved, room temperature 0.02% 2.5 mL

Total volume: 250 mL

82 Shworak

with a microstopper (Thomas Scientific, # 8751-K10). A bottom frit is punched out of

porous polyethylene (Labconco # 4330115) with a #2 cork bore, then inserted into the

barrel. Flush the syringe with water, then add 320 µL of 50/50 slurry of DEAE Sepharose

Fast Flow (Pharmacia #17-0709-01). Adjust to generate a 160-µL matrix bed.

8. Blocking the DEAE column (see Note 5). Drain the column by gravity, then apply 200

µL of column blocking mix. After 10 min, wash the column by the sequential application

of 1 mL of equilibration buffer, three applications of 1 mL of column wash, 1 mL of

equilibration buffer, 1 mL of GUTE, and 1 mL of equilibration buffer. Store as plugged

columns containing ~1 mL of equilibration buffer.

9. Final column preparation. Before sample application drain the column. Resuspend the

bed by forcefully squirting, in rapid succession, two 0.5-mL volumes of equilibration

buffer directly at the matrix. Allow the bed to repack by gravity flow, then repeat. Pass

through 1 mL of column wash, followed by 1 mL of urea/acid wash. The column is ready

for loading (see Subsection 3.2, step 1).

2.4. Isolation of

S-labeled HS.

1. 10 M NaOH.

2. 10 M NaBH

suspension. Wear gloves and a mask when weighing out this very toxic

powder. NaBH

is extremely volatile, and open solutions should be manipulated in a

fume hood. Resuspend 3.78 g in 10 mL (final volume) of 0.1 M NaOH, then distribute into

0.5-mL aliquots, snap-freeze on liquid nitrogen and store at –80°C. When aliquoting,

mix the stock frequently to ensure even suspension.

3. NaOH/NaBH

. Thaw an aliquot of 10 M NaBH

on ice, mix briefly and then transfer 9

Table 2

Stock Solutions for Generating DEAE Column Buffers

Stock Comments

1.0 M Tris (pH 7.4), 500 mL Autoclave sterilize.

1.0 M NaC

(pH 5.0), 500 mL

5.0 M NaCl, 500 mL Autoclave sterilize.

0.5 M EDTA, pH 8.0, 500 mL Insoluble at low pH, add NaOH pellets while

stirring and monitor pH.

18% (w/v) CHAPS, 100 mL Sigma (# C3023) To 80 mL of stirring water,

gradually add solid. Cover with Parafilm, stir

overnight, adjust volume, 0.45-µm filter. Lot

variability see Note 3.

10 M Urea, 500 mL A saturated solution. Complete dissolution requires

heating at 37°C. If crystals form upon storage,

dissolve by reheating.

10% (w/v) Sodium azide, 100 mL Extremely toxic. Wear mask, gloves, and coat when

weighing out powder.

1.0 M Na

, 100 mL

20% (v/v) Triton X-100, 100 mL Extensive stirring required. Store at 4°C.

10 mg/mL BSA, 10 mL RIA grade bovine serum albumin (BSA) (Sigma #

A7888). Reserve aliquots (2 mL) stored at –80°C,

working aliquots stored at –20°C.

20 mg/mL Glycogen, 1 mL Boehringer Mannheim (# 901 393)

Preparation of High–Specific-Activity Heparan Sulfate 83

Table 3

Recipes for Most DEAE Column Buffers (100 mL each)

Preparative loading buffer

Equilibration buffer Urea/acid wash Column wash (PLB)

Table 2 Stock

Final Stock Final Stock Final Stock Final Stock

concentration volume concentration volume concentration volume concentration volume

Tris 50 mM 5 mL

Sodium acetate 50 mM 5 mL 50 mM 5 mL 69.4 mM 6.9 mL

NaCl 150 mM 3 mL 150 mM 3 mL 1558 mM 31 mL 222 mM 4.4 mL

EDTA 1 mM 200 µL 1 mM 200 µL 1 mM 200 µ

L 1.39 mM 278 µL

CHAPS 0.6% 3.3 mL 0.6% 3.3 mL 0.6% 3.3 mL 0.833% 4.63 mL

Urea 6 M 60 mL 6 M 60 mL 8.33 M 83 mL

Sodium azide 0.02% 200 µL 0.02% 200 µL 0.02% 200 µL 0.028% 280 µL

1 mM 100 µL 1 mM 100 µL 1.39 mM 139 µL

O 88.3 mL 28.2 mL

84 Shworak

µL of suspension to 89.5 µL of 10 M NaOH, mix, and use immediately. The 10 M NaBH

stock is snap-frozen and returned to –80°C.

4. 8.54 M ammonium formate/1.7 M HCl. To 8.75 mL of 10 M ammonium formate, add 1.5

mL of concentrated HCl (11.6 M). Store at room temperature.

5. PCI-phenol /choroform/isoamyl alcohol, 25/24/1 (v/v/v) (100 mL). Phenol is extremely

corrosive and toxic. PCI preparation and precautions are described extensively by Sambrook

et al. (11).

6. Absolute ethanol (500 mL). Store at –20°C.

7. 1 M ammonium acetate, pH 7.0 (50 mL). Store at room temperature.

8. 5 M NaCl. see Table 2.

9. 10 mg/mL BSA. see Table 2.

10. 0.0005% Triton X-100 (15 mL). Diluted from Table 2 stock.

11. 2-mg/mL glycogen (2 mL). Diluted from Table 2 stock.

12. 5-U/mL chondroitinase ABC (Sigma # C-2905) (1 mL). Dissolve in PBS; aliquots of 100

µL are generated, snap-frozen on liquid nitrogen, and stored at –20°C (see Note 6).

13. Set incubator or oven at 46°C.

3. Methods

3.1. Labeling Cell Surface Proteoglycans

1. Cell maintenance. L cell cultures are maintained in exponential growth at 37°C, under

5% CO

in DMEM with 10% FBS.

2. Prepare an exponentially growing culture for

S-labeling. A 75-cm

flasks is inoculated

at 38,000 cells/cm

. Two days later the flask should be ~50% confluent.

3. Prepare the laminar-flow hood. Turn off the laminar-flow hood so as to prevent aero-

solized radioactivity from entering the hood’s inner machinery. Cover the work surface

with disposable bench coat. Assemble and organize all required hardware, including

appropriate shielding, within the hood. A stable rack is required to hold the test tube

containing the labeling medium. Double-glove, see Note 7.

4. Prepare labeling medium. In a 15-mL conical tube containing 4 mL of labeling base

mixture (see Table 1), add 0.4 mL of fetal bovine serum, 4.6 µL of sterile 0.1 M Na

and then 200 mCi of carrier-free Na

(~200 µL), but do not mix yet. Gently blow

5% CO

gas over the surface and then cap the tube shut. (see Notes 8–10).

5. Remove culture medium. Hold the flask at an angle and aspirate off as much medium as

possible. Gently pipet 5 mL of PBS down the side of the flask so as not to disrupt the

monolayer, swirl to wash the monolayer surface, and aspirate as above. Repeat with a

second PBS rinse and aspirate.

6. Add labeling medium. Stand the flask upright. Using a 5-mL pipet, carefully mix the

labeling medium by pumping up and down once, then transfer to the bottom surface of the

culture flask. To prevent accidental contamination, avoid generating bubbles — do not

blow out the final drop from the pipet tip. Carefully place the pipet back into the empty

labeling medium tube, then remove the pipet from the pipet gun. Monitor your gloves for

radioactivity; if they are contaminated, replace the outer glove.

7. Gas and incubate the flask. Place the 5% CO

pipet just into the neck of the flask and

blow a gentle gas stream of over the medium for about 10 s. Do not generate bubbles.

Tightly cap the flask and tilt to spread the medium gently over the cell monolayer. Moni-

tor gloves and flask with a β-probe, and if clean place flask in incubator for ~16 h, (see

Note 11). Hang a “Radioactivity in Use” sign on the incubator. Monitor and clean the

culture hood.

Preparation of High–Specific-Activity Heparan Sulfate 85

8. Wash monolayer. Chill the flask on an ice-chilled grate (see Subheading 2.2., item 9)

for 10 min. The resulting negative pressure prevents expulsion of radioactive vapors.

Remove the lid. Aspirate off the radioactive medium and, using a repeat pipetter (see

Subheading 2.1., item 5), rinse the flask with 5 mL of ice-cold PBS containing 1 mM

(as described in step 5). Repeat for a total of 10 rinses, (see Note 12).

9. Release extracellular proteoglycans by trypsinization. Add 2 mL of typsin/EDTA/Na

cap flask, and distribute solution over the monolayer. Incubate at room temperature for 30

min, redistribute the solution, and incubate at 37°C for 10 min. Transfer the cell suspension

to a polypropylene 15-mL conical tube. Rinse the flask with 0.5 mL of PBS and combine

with the cell suspension. Cells are pelleted by centrifugation at 1500g and the supernatant

(~2.5 mL), which contains cell surface proteoglycans, is collected.

3.2. Purification of Proteoglycans by Preparative

DEAE Microchromatography

1. Column loading. Proteoglycans are partially purified and concentrated with a DEAE

microcolumn, prepared by Subheading 2.3., steps 7–9. All effluents are to be consid-

ered as radioactive and disposed of accordingly. The proteoglycan-containing superna-

tant is combined with 6.5 mL of PLB (see Table 3). A pipetman is used to load 1-mL

portions. Apply slowly so that the sample runs down the wall and only minimally dis-

turbs the gel bed. see Note 13.

2. Column washing. Wash with 4 mL of urea/acid wash, 2 mL of equilibration buffer, and 2

mL of preparative wash buffer.

3. Elution of labeled proteoglycans with high salt. A 2-mL polypropylene screw-cap tube is

positioned under the column. Let 200-µL of elution buffer run through the column, then

repeat 4 more times to generate 1.0 mL of eluate, (see Note 14).

4. Determine radioactivity. Close the tube with an O-ring-containing lid (see Note 15).

Briefly vortex and centrifuge. Remove 0.5 µL for liquid scintillation counting.

3.3. Isolation of

S-labeled HS

1. Cleave GAGs from proteoglycans. Add 10 µL of NaOH/NaBH4 mix to the eluate, vor-

tex, and centrifuge briefly. Incubate in a 46°C oven for ~16 h (see Note 16) .

2. Reaction neutralization. Breakdown of borohydride liberates hydrogen gas, which can

lead to excessive foaming and radioactive contamination. To prevent this outcome, chill

the reaction on a bed of ice for 10 min. Uncap slowly to gently release pent up gases.

Carefully add 60 µL of 8.54 M ammonium formate/1.7 M HCl to the sample meniscus.

Bubbling will occur as the residual borohydride degrades. Cap, vortex, and spin briefly,

then return to ice and slowly uncap. Transfer 0.5 mL to a second screw-cap tube.

3. Phenol extraction. Add 0.75 mL of cold (taken directly from 4°C) PCI to each tube, then

cap. Vortex the tubes at maximum for 30 s each. Separate the phases by centrifugation at

10,000g, 4°C, for 10 min. Remove as much top (aqueous) phase as possible without

taking any interphase material (denatured proteins) and transfer into a single 1.5-mL

microfuge tube, (see Note 17).

4. Ethanol precipitation of GAG. Distribute the aqueous phase among four 2-mL screw-cap

tubes and add water so that each tube contains ~260 µL total volume. Add 1.25 mL of

absolute ethanol per tube, and vortex briefly, (see Note 18). Mark a line down the side of

each tube. Place samples in a microcentrifuge with the line toward the outside, so as to

indicate the anticipated site of pellet formation. Spin at 10,000g, room temperature, for

30 min (see Note 19). Gently remove the tubes from the centrifuge and place in a rack so