Proteoglycans of Bovine Articular Cartilage

’ h E ~ O U H N A I OF . fiIOLO(:ICAI,

CHEMISTHY

Vol. 266. No. 11. Issue of April 15. pp. 7016-7024. 1991 Printed In U S A.

1991 hy T h e American Society for Biochernlstry and Molecular Biolopy. Inc.

m i ’

Proteoglycans of Bovine Articular Cartilage THE EFFECTSOF DIVALENT CATIONS ON T H E BIOCHEMICAL PROPERTIES OF LINK PROTEIN* (Received for publication, November 19, 1990)

Lawrence Rosenberg, HaingU. Choi, Lih-Heng Tang, Subhash Pal, andThomas Johnson From the Montefiore Medical Center, Bronx, New York 10467

Daryl A. Lyons and Thomas M. Laue From the Department of Biochemistry, University of New Hampshire, Durham, New Hampshire03824

In cartilage proteoglycan aggregates, link protein Cartilage proteoglycan aggregates are assembled from prostabilizes the binding of proteoglycan monomers to teoglycan monomers, hyaluronate, and link protein (1-4). In hyaluronate by binding simultaneously to hyaluronate an individual aggregate, many proteoglycan monomers bind and to the G1 globular domain of proteoglycan mon- at regular intervals to a single central hyaluronate chain (5omercoreprotein.Studiesreportedhereinvolving 7). Link protein stabilizes the binding of proteoglycan monmetalchelateaffinitychromatographydemonstrate omertohyaluronate (8-10) by bindingsimultaneouslyto that link protein is a metalloprotein that binds Zn2+, hyaluronate and to the G1’ globular domain of proteoglycan Ni”+, andCo2+.Zn2+and Ni” decrease the solubilityof monomer core protein. link protein and result in its precipitation. However, Studies of the biochemical and functional properties of link link protein is readily soluble and functional in low protein have been hampered by the apparent insolubility of ionic strength solvents from which divalent cations have been removed with Chelex 100. These observa- link proteins in low ionic strength associative solvents. Intions make it possible to study the biochemical prop- deed, the proteoglycan literature is replete with references to erties of link protein in low ionic strength, physiologic experiments undertaken todefine the basic biochemical propsolvents. erties of link protein, which were unsuccessful because of the Studies were carried out to define the oligomeric apparent limited solubility of link protein in associative solstate of link protein alone inphysiologic solvents, and vents (4, 11-13). thetransformationin oligomeric statethatoccurs Recently, we foundthat link protein is soluble inthe when link protein binds hyaluronate. Sedimentation presence of EDTA, even in low ionic strength physiologic equilibrium studies demonstrate that in 0.15 M NaCl, solvents. Thisobservation led us tocarryoutsystematic 5 mM EDTA, 50 mM Tris, pH 7,link protein exists as studies of the effects of divalent cations on the solubility of a monomer-hexamer equilibrium controlled by a forlink protein. The studiesshow that link protein isa metallomation constant of 2 X lo’? ””, yielding a AG‘ of -36 Zn“’, Ni”, andCot+. Zn‘+ drastically kcallmol for the formation of the hexamer from six proteinthatbinds decreases the solubility of link protein. However, link protein monomers. On binding hyaluronate oligosaccharides (HAloor HAIS),link protein dissociates to dimer.Link is readily soluble and functional in associative solvents from protein hexamer is rendered insoluble byZn2+.Greater which divalent cations have beenremoved. These observations suddenly made itpossible to study the than 90% of the protein is precipitated by 2 mol of Zn2+/mo1of link protein monomer. The binding of hy- basicbiochemical properties of link proteinin low ionic aluronate oligosaccharideby link protein strongly in- strength physiologic solvents. Specifically, these observations hibits the precipitation of link protein by Zn2+. The made itpossible to work with readily soluble link proteinover link protein/hyaluronate oligosaccharide complexis a broad concentration range, to carry out sedimentation equicompletely solublein the presenceof 2 mol of Zn2+/mo1 librium studies of the oligomeric state of link protein alone of link protein. At higher molar ratios of Zn2+/link and of link protein bound to its natural ligands, hyaluronate protein, the inhibitory effectof hyaluronate oligosac- and GI, and to carry out studies of the bindingof link protein charide on the precipitationof link proteinis gradually to G1by gel chromatography, ion-exchange chromatography overcome. Hyaluronate oligosaccharide is not dissociated from link protein byZn2+.Hyaluronate remains and sedimentation equilibrium, with definitive results. bound to the link protein which is precipitated Zn2+, by The present study shows that in low ionic strength physiologic solvents, in the absence of diualerzt cations, link protein or to the link protein which binds to Zn”-charged iminodiacetate-Sepharose columns. Hyaluronate oli- alone exists as hexamer and that link protein bound to hyagosaccharides and Zn2+bind to different sites on link luronate exists as dimer. Link protein binds Znn+, Nil+, and CO”. Znn+ hasa profoundly deleteriouseffect on thesolubility protein. of link protein. However, when link protein binds its natural is transligand hyaluronate (HAloor HAI2), and link protein formed from hexamer to dimer, the effect of Zn2+ on the * This work was supported by National Institutesof Health Grants AR 21498 and AR 34614 and National Science Foundation grants BBS 86-15815 and DIR 9002027. The costs of publication of this article were defrayed in part by the payment of page charges. This article must t.herefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: G1, the G1 globular domain of proteoglycan monomer core protein; HA,,, and HA,,, hyaluronate decasaccharide andduodecasaccharide; Mes, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyI)dimethylammonio]-l-propanesulfonic acid EGTA, [ethylenebis(oxyethylenenitril~)]tetraacetic acid.

7016

Metalloprotein Properties

Protein of Link

7017

Solubiljty-Studies were also carried out in which different amounts of a particular divalent metal ionwere added directly to link protein solutions, and the amountof link protein which was precipitated was determined. Link protein in 4M GdnHCl was dialyzed against Chelex EXPERIMENTALPROCEDURES 100-treated 0.15 M NaCI, 50 mM Tris,pH 7. The solution was Ma(erinls-GdnHC1was from Research Plus Laboratories. CsCl centrifuged at 30,000 rpm for 30 min a t 5 "C to remove small amounts of insoluble link protein. The concentration of soluble link protein in optical density grade was from Gallard-Schlesinger. Phenylmethylthe clear supernatantwas adjusted to 3 or 4 mg/ml with Chelex 100sulfonyl fluoride, Tris, iodoacetamide, and testicular hyaluronidase V 1-5 were from Sigma. Benzamidine hydrochloride was from Aldrich. treated 0.15 M NaCI, 50 mM Tris, pH 7. Link protein (50 nmol) was added to a series of tubes. Varying ('helex 100 was from Bio-Rad. Chelating Sepharose 6B (-iminodiacetate-Sepharose6B) was from Pharmacia LKB Biotechnology Inc. amounts of buffer were added, so that thefinal volumeof the solutions would he constant after the addition of metal ion solution. Varying I'uratronic grade 1 CaCI, .6H,O, MgCI,.6H2O, ZnCL. 2H20, NiCI,. amounts of Ca'+, Mg", Co", Ni'+, or Zn'+ were then added so that (iH,O and CoC12.6H,0 were from Johnson Matthey. Hyaluronate the molar ratio of metal ion/link prot,ein ranged from 0 to 10. Upon oligosaccharides (HA,,, and HA,?) were prepared as described (14). Isolation of Link Protein-Fresh wet bovine articular cartilage from the addition of Zn2+, link protein solutionsbecame turbid instantly. These solut,ions were allowed to stand for 3 h at 5 "C before centrif4-month-old calves was shaved from the occipital condyles and added ugation. On the addition ofNi", turbidity developed more slowly. immediately to ice-cold 4 M GdnHCI, 0.15 M sodium acetate, 50 mM EDTA, pH 6.3, containing 0.1 mM phenylmethylsulfonyl fluoride, 1 The Nil+, Ca'+, Mg'+, and Co'+ solutions were allowed to stand at mM iodoacetamide, and 1 mM benzamidine hydrochloride as protease 5 "C overnight before centrifugation. The solutions were then centriinhibitors. The cartilage was extracted by slow stirring a t 5 "C for 48 fuged a t 30,000 rpm for 30 min a t 5 "C, and the concentrationo f link protein in the clear supernatantwas determined from the A,,,. h. The extract was filtered and dialyzed at 5 "C for 16 h against 20 Studies of the Binding of Divalent Metal Ions to Link Protein hy volumes of 0.15 M sodium acetate, 5 mM EDTA, pH 6.3, containing protease inhibitors. Equilibrium density gradient centrifugation unMetal Chelate Affinity Chromatography-Chelating Sepharose 6B (12 der associative conditions was then carried out in 2.5 M CsCl at 5 "C ml; capacity, 30 rmol of Zn'+/ml gel) was suspended in a column and fully charged with divalent metal ion by washing the column with lhr 60h at 40,000 rpm. The gradient was divided intosixequal excess divalent metal ion. For example, a 12-ml column was charged lractions, called A1 through A6' (15). Fraction A1 was recycled in a second associative gradient. Two volumes of 5.5 M GdnHCI, 5 mM with Zn" by washing the column with 150 ml of ZnCL (1 mg/ml) in EDTA, 0.15 M sodium acetate, pH6.3, containing protease inhibitors water over a 1-h period. The column was then washed with 36 ml of were added to one volume of fraction Al. The solution was stirred 0.5 M NaCI, 50 mM Tris acetate, pH 8. Ten ml of the charged chelating i'or 4 h, then dialyzed overnight against 20 volumes of 0.15 M sodium Sepharose 6B was then repacked into a 1.5 X 14-cm column on the acetate, 5mM EDTA, pH6.3, containing protease inhibitors. Equilib- top of 10 ml of unchargedchelatingSepharose 6B. The layer of rium density gradient centrifugation under associative conditions was uncharged chelating Sepharose 6B at the bottomof the column is to carried out in 3.5 M CsCl a t 5 "C for 60 h a t 40,000 rpm. Fraction bindanydivalentmetal ion which mighthereleased duringthe AlAl from the second associative gradient was used for the prepaelution of link protein with the pH 8-4 gradient, and preventleaching ration of link protein. of the divalent metal ion into the eluant along with the link protein. Fraction AlAl was subject.ed to equilibrium density gradient cenAfter the charged chelatingSepharose6B was layeredon the trifugation under dissociative conditions. The gradient was cut into uncharged Sepharose, the colcmn was washed with 80 ml of 0.5 M six equal fractions, called D l through D6.' Fractions D5 and D6 were NaCI, 50 mM Tris acetate, pH 8. Link protein (60 nmol in 1.0 ml of pooled. The link protein in fraction D5D6 was concentrated on an the same solvent) was applied to the column, and the column was Amicon filter, dialyzed against 4 M GdnHCI, 0.15 M sodium acetate, washed with 60 ml of the same solvent. Thecolumn was then eluted 5 mM EDTA, pH 6.3, and purified by gel chromatography on Sepha- with a pH8-4 gradient formed with 50 ml of 0.5 M NaCI, 50 mM Tris cry1 9-300 in 4 M GdnHCI, 0.15 M sodium acetate, 5 mM EDTA, pH acetate, pH 8, in the mixing chamber and 50 ml of 0.5 M NaC1, 50 6.3. The homogeneity of the link protein was assessed by sodium mM Tris acetate, pH4, in the reservoir. The A,&,, and pHof t'ractions dodecyl sulfate-polyacrylamide gel electrophoresis (16). (2.3ml) collected at arate of' 33ml/h were determined.Control Solubility Studies-Divalent cations were removed from solvent,s experiments were carried out under the same conditions using chehy passing the solvents over 2.7 X 50-cm (300ml) Chelex 100 columns. lating Sepharose 6B which had not been charged with divalent metal The Chelex 100was prepared according to the manufacturer's instruc- ion. Additional experiments were carried out to determine whether a tions (Bio-Rad Technical Bulletin '2020). Briefly, 250 g of Chelex 100, link protein/HA,, complex would bindto Zn"-charged chelating 100-200 mesh, sodium salt, was washed batchwise with 1.5 liter of 1 Sepharose 6 8 columns; and if so, whether HA,, was dissociated from N HCI, 1.5 liters of H1O, 1.5 liters of 0.1 N NaOH, and 5 litersof H,O. link protein or remained hound to link protein when link protein The Chelex 100 was packed into 2.7 X 50-cm columns. Each column hound Znr+. was washed with 3 liters of water. Solvents (10 times concentrated) Studies of the Stoichiometry of Binding of Zn" to Link Protpinto be used in the solubility studies were then passed over the columns. Studies of the stoichiometry of binding of Zn" to link protein were The first four column volumes of each solvent were discarded, and carried outusing "'Zn, according to thefollowing procedure. Solutions the required volume of 10 timesconcentratedsolvent was then were prepared which contained either link protein ( 3 mg/ml), 1 mM collected. ZnCI2 or 2 mM ZnC1, in 0.15 M NaCI, 50 mM Tris, pH 7. A series of Solubility studies were carried out as follows. Link protein in 4 M solutions were prepared in 1.5-ml microcentrifuge tubes in which the GdnHCI, 6 mM EDTA, 0.15 M sodium acetate, pH 6.3, was concen- molar ratio of ZnY*to link protein ranged from 0 to 20. Specifically, trated on an Amicon filter to approximately 4 mg/ml and dialyzed varying amounts of 1or 2 mM ZnCI.?were added toeach tube. Varying against 20 volumes of this solvent for 16 h. The solution was centri- amounts of buffer were added so that the final volume of each would fuged at :30,000 rpm for 15 min, and the concentrat.ion of link protein be 1.3 ml. ""Zn (3 p l , 80,000 dpm) was added t.0 eachtube.Link in 4 M GdnHCI was determined from the ALXll using E"'" = 1.39 (9). protein (50 nM;2.4mg; 0.8 ml) was added to each tube. The tubes The link protein concentration was adjusted to3 mg/ml by dilution were mixed, allowed to stand at 4 "C for 24 h, then centrifuged at with 4 M GdnHCI. Aliquots (1 ml) of the link protein solution in 4 M 12,000 X g at 4 "C for 15 min in a Beckman microfuge. From the (idnHC1 were dialyzed (250 volumes; X 3, 16 h; 8 h; 16 h) against supernatant, 200-pI aliquots were taken in duplicate from each tube Chelex 100-treated 0.15 M NaCI, 50 mM Tris, pH 7, or the same and the radioactivity was determined in aPackardAuto-Gamma buffer containing EDTA, ZnCI,, NiCly, CaCI,, etc. (Table I). After Counter. Aliquots (200 pl) of t,he supernatant were also taken for dialysis, we notedwhetherthesolutions remained clear, became protein determinations using link protein as a standard. The superturbid, or whether the link protein had formed a heavy precipitate. natants were decanted, and the pellets of sedimented link protein The solutions were centrifuged at 30,000 rpm for 30 min a t 5 "C to were drained. The radioactivitv of the sedimented link protein reremove insoluble link protein. The concentration of the soluble link maining in the tubes was determined. The link protein was dissolved protein in the clear supernatant of each solutionwas then determined in 0.1 N HCI, and the amount of link protein in the precipitate was f'rom the A,,,,. determined. Blank tubes which contained ZnCI, and ""Zn" but no Slr,ichiornfZtryof the &/f?c.t of Diuaknt hfftal Ions on Link Protein link protein were included to determine the amount ofZn'+ which was adsorbed to the tubes in the absence of link protein. While the 'The nomenclature A I , AlD1, etc., defines the proteoglycan frac- amount of Zn" adsorbed to the tubes was negligible, the am0unt.s of tionsprepared hy equilibrium density gradient centrifugation, as Zn'+in theprecipitatesandsupernatants were corrected for the adsorbed Zn". Equilibrium binding data in t.he form [Zn'+],,.,,,,,.,as a desrrihed hy Heinegard (15).

solubility of link protein is strongly inhibited. The results of these studies are described in this report.


7018

Protein of Link

f'itnction of [Zn"],,,,;,l were fit to various models using LIGAND (18). M~ X 10-3 .5'(Jdimcvzlation Eyuilihrium and Sedimentation Velocity StudiesShort column (20 pl of sample volume) sedimentation equilibrium experiments were conductedusinganAN-Drotorina Reckrnan -200 -200 model E analytical ultracentrifuge equipped with interference optics, >I 35 milliwatt He-Ne Laser light source (19), and an on-line data -116 acquisition system(20). Link protein was examineda t concentrations -97 -116 -97 of' 2.0, 1 5 , 1.0. 0.5, 0.25 and 0.125 mg/ml in 0.15 M NaCI, 5 mM -66 ISDTA, 50 mM Tris,pH 7 ' , a t rotorspeeds of 16, 20, 30, and 36 -66 thousand rpm. The procedures of Yphantis (21) were followed. -45 Longcolumn (3-mmcolumnheight)sedimentationequilibrium experiments were also carried out using long interference cells (30mm centerpieces) with side-wedge windows, in an AN-J rotor,a t link -31 -45 protein concentrations of0.215,0.464,0.916,1.348, and 1.861 mg/ml. -31 Runs were made at 12, 14, and 16 thousand rpm at 20 "C. Following the runs,cells were washed,filled with water, and run under the same conditions, so thatblankcorrections could be made. Interference patterns were recorded on Kodak Technical Pan plates, and read with an automated plate reader. The fringe displacement data were FIG. 1. Coomassie Blue-stained 5-20% gradient slab gelsof analyzed using NONLIN (22), yielding a z average molecular weight (M,) for each sample (2.3). For the determination of stoichiometries calf articular cartilagelink protein, reduced ( l e f t )and unreduced (right). and association constants, up to 15 data sets at different loading concentrations, radial positions and rotor speeds were analyzed simultaneously. Selection of the model and calculation of the molar TABLE I association constants were performed as described previously (24). E/fect of divalent cations on the solubility of link protein The partial specific volume of link protein (0.715)was calculated All solvents were treated with Chelex 100 before the addition o f from its amino acid and carbohydrate composition. T o demonstrate divalent cations, chelating agents or CHAPS. The concentration of the dissociation of link protein hexamer to dimer when link protein binds HA,,,, aliquots of link protein solutions were titrated with HA,,,, link Drotein before dialvsis was 3 mdml. r ;so1ul)le Solvent so that the molar ratio of HAJlink protein in the solutions was 0.5, 1, 2, 3 , 4, and 5. In the solutions in which the molar ratio of HA,,,/ 0.15 M NaCI, 50 mM Tris, pH 7 885, 92.9 link protein was 0.5, 1, and 2, the final link protein concentration 89.5 +5 mM EDTA was 1.66 mg/ml. At molar ratios of HAl,,/link protein of 3, 4, and 5, 89.6 +-5mM EGTA the link protein concentration was 1.83 mg/ml. An equal amount of 89.6 +1 mM EDTA, 1 mM EGTA HA,,,was added to thereference solution to minimize its contribution to the interlerence fringes. Experiments in which link protein was 89.3 +15 mM CaCI, titrated with HA,,, were analyzed a t rotor speeds of 10,000, 12,000, 92.0 +i5 mM CaCl, 16,000 and 20,000 rpm. 95.7 +I50 mM CaCI, Sedimentation velocity studies were carried out in an AN-E rotor using long cells with :Wmm double sector centerpieces, as previously 98.1 +10 pM MgCI, described (10). Values for S2,,,3,,,vt.,,l were extrapolated to zero protein 98.4 +lo0 pM MgC1, concent ration to obtain Sl:,l,.G,lv,.,,,,which was corrected to standard conditions (S~,,.,;,,,.,),using a measured buffer density of 1.008158 g/ 84.5 +1 p~ ZnCI, m i and tabulated viscosity data (25). The frictional coefficient ratios 23.0 +IO pM ZnCl, (///,,,,,,),the I'errin shape factor ( f l f , ) ,and the axial ratio ( a / h ) were 1.2 +I00 p~ ZnCI, determined as descrihed previously (24).

--

w"

-

RESULTS

Link protein has a variable and limited solubility in associative solvents prepared from commercial buffers (4,9, 1113) using water from which divalent cations have not been removed. Recently, we found that linkproteinis readily soluble in the presence of EDTA, even in low ionic strength, physiologic solvents. This observation motivated us to carry out a systematic study of the effect of divalent cations on the solubility of link protein, to determine which divalent cations have a deleterious effect on the solubility of link protein. The Effect of DivalentCations on the Solubility of Link Protein-Link protein was isolated from calf articular cartilage as described under "Experimental Procedures." The homogeneity of the link proteinwas assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1).The effect of divalent cations on the solubility of link proteinwas determined as follows. Link protein in4 M GdnHC1,0.15 M sodium acetate, 5 mM EDTA, pH6.3, was concentrated on anAmicon filter, centrifuged, and adjusted to 3 mg/ml in 4 M GdnHC1. Aliquots of the link protein solution were exhaustively dialyzed against Chelex 100-treated 0.15 M NaCI, 50 mM Tris, pH 7 , or the same buffer containing various divalent cations (Table I). The solutions were centrifuged at 30,000 rpm for 30 min at 5 "C to remove insoluble link protein. The concentration of the soluble link protein in the clear supernatantof each solution was then determined from theA,*,,.

-

--

+1 p M NiCI, +10 pM NiCI, +100 pM NiCI,

86.0 82.4 47.i

+ l o p M COCI:! +lo0 pM CoCI? + 1 mM CoCI, +5 mM CoCI,

92.5 90.2 61.1 25.8

+ l % CHAPS + 1 CHAPS, 100 p~ ZnCI,

102.0 51.1

0.15 M NaCI, 10 mM NaH,I~O.l, pH7 0.15 M NaCI, 10 mM KH,PO,,

87.1 89.9

nH 7 '

As shownin Table I, link protein is approximately 90% soluble a t 3 mg/ml in Chelex 100-treated 0.15 M NaC1, 50 mM Tris, pH 7, in the presence or absence of EDTA. Ca')+,Mg'+, and Mn" donotdecreasethe solubility of link protein. However, Zn')+, NF+, and Co" decrease the solubility of link protein and result in its precipitation. Dialysis against 10 p M ZnCI,! decreases the solubility of link protein by over 7.5'36, and, on dialysis against 100 p~ ZnCI,, a precipitate of link protein quickly forms and essentiallyall of the link protein is rendered insoluble. As shown at the bottom of Table I, the solubility of link

Metalloprotein Properties of Link Protein

7019

protein in Chelex 100-treated low ionic strength solvents is approximately 100% in the presence of 1% CHAPS. In these experiments, link protein in4 M GdnHCl at 3 mg/mlwas dialyzed against Chelex 100-treated 0.15 M NaCl, 50 mM Tris, 1% CHAPS, pH 7 . However, CHAPS will not prevent the precipitation of link protein by Zn2+.As shown at the bottom of Table I, if link protein in 0.15 M NaC1, 50 mM Tris, 1% CHAPS, pH7 , is dialyzed against the same solvent containing 1%CHAPS and 100 p~ ZnCl,, 50% of the link protein is precipitated. In studies of the biochemical properties of link protein, if CHAPS is used to enhance the solubility of link protein, solvents should neverthelessbe treated with Chelex 100 to remove divalent cations such as Zn2+and Ni". In the solubility studies described above, link protein at 3 0 1 2 3 4 5 6 7 8 9 1 0 mg/ml in 4 M GdnHCl was dialyzed against each low ionic Moles Metal lonlMoie Link Roidn strength solvent. To determine the maximum solubilityin FIG. 2. Stoichiometry of the effect of divalent metal ions of mg/ml of link protein in low ionic strength associative solthe solubility of link protein. Divalent metal ions were added vents, link protein a t much higher concentrations in 4 M directly tosolutionscontaining 50 nmol of link protein,andthe GdnHCl was dialyzed againstthesamesolvents,andthe amount of link protein which was precipitated was determined. The solubility was determined (Table 11). As shown in Table 11, solubility of link protein was not decreased by the addition of Ca'+, were obtained for these the solubility of link protein in Chelex 100-treated solvents is Mg'+, or Co'+, and essentially identical curves three divalent metal ions. Therefore, the curves for Mg" and Co'+ greater than 6 mg/ml. Stoichiometry of the Effect of Zn2+ onLink Protein Solubil- are not shown. ity-Studies were also carried out inwhich different amounts of divalent cations were added directly t o link protein solu- on the solubility of link protein. Studies of the Bindingof Divalent Metal Ionsto Link Protein tions, and the amountof link protein which was precipitated was determined. Specifically, link protein (50 nmol) was added by Metal Chelate Affinity Chromatography-Control experit o a series of tubes. Varying amounts of Ca2+, Mg", Co2+, ments were first carried out inwhich link protein was applied 6B columns which hadnotbeen Ni"', or Zn'+ were then addedso that themolar ratio of metal tochelatingSepharose ion/link protein ranged from 0 t o 10. The tubes were mixed, charged with divalent metal ion. Link protein does not bind allowed to stand at 4 "C, centrifuged t o sediment insoluble t o uncharged columns, elutes in the wash, and yields chrolink protein, and the concentration of the soluble link protein matograms which are identical to thoseshown in Fig. 3A and B. Experiments were then carried out in which link protein in the clear supernatantwas determined from the AZRO. When Ca", Mg", or Co2+ was added, no precipitate was was applied to chelating Sepharose 6B columns which had formed, and the solubility of link protein was not decreased been charged with Ca2+, Mg2+, Zn'+, Ni", or Co2+.The col(Fig. 2). When Zn'+ was added, precipitates formed instantly umns were washed with 0.5 M NaCl, 50 mM Tris acetate, pH at molar ratios of Znz+/link protein20.6 and the % solubility 8, then eluted witha pH 8-4 gradient. When link protein was of link protein decreased progressively as the molar ratio of subjected to metal chelate affinity chromatography on Ca2+Zn'+/link protein was increased. At a molar ratio of 4, essen- or Mg2"charged columns, none of the link protein bound to the column and all of the link proteinwas eluted in the pH 8 tially 100% of the link protein was rendered insoluble (Fig. wash (Fig. 3, A and B ) . Under the conditionsof these exper2). In the Ni"-containing solutions, precipitates formed more iments, link protein does not bind Ca2+ orMgz+. In contrast, allof the link protein bound to Zn'+, Ni2+-,or slowly, and 48% of the link proteinwas rendered insoluble at Co2+-chargedcolumns, indicating that link protein binds Zn2+, a molar ratio of 10. asingle Thus, of the divalent metal ions studied, only Zn'+ and Ni"+ Nit+, and Coz+. Linkproteinissharplyelutedas at low concentrations decrease the solubility of link protein. unimodal peak from the Zn2+-iminodiacetate-SepharosecolZn'+ results in the complete precipitation of link protein on umn (Fig. 3C). The elution begins at pH 7.6 and is complete dialysis against 10 WM ZnC1, and on the direct addition of 4 at pH 4.4. In the case of Ni", 88% of the link protein elutes mol of Zny+/mol link protein. Of the divalent metal ions so as a broad peak between pH 7.4 and 4.2 (Fig. 3 0 ) . Eleven far studied, Zn'+ has the most pronounced deleteriouseffect percent of the link protein is eluted aassecond peak between pH 4.1 and 3.2. In the case of Co2+,none of the link protein bound to the column could be eluted from the column even TABLE I1 by elution at pH 3. Thus, whereas link protein binds Zn2+, Maximum solubility of link protein in low ionic strength Ni", and Co", thereare largedifferencesin thebinding associatiue soluents affinities of link protein for these three divalent metalions. c o n ~ ~ ~ ~ $ iFinal o n conc. in Stoichiometry of Binding of Zn2+ to Link Protein-Studies Solvent associative in 4 M were also carried out in which the amount of Zn2+ bound to solvent GdnHCl link protein precipitated by Zn'+ was determined. A series of 0.15 M NaCI, 50 mM Tris, pH 7 8.75 mg/ml 6.65 mg/ml solutions was prepared that contained 50 nM link protein, "Zn, and in which the molar ratio of Zn2+ to link protein 0.15 M NaC1, 8.75 mg/ml 7.30 mg/rnl ranged from 0 to 20. The tubes were mixed, allowed to stand 5 mM EDTA, 50 mM Tris, pH 7 for 24 h, and the link protein precipitates were sedimented. The amounts ofZn'+ and link protein in the supernatants 0.15 M NaC1, 1% CHAPS, 8.75 mg/ml 8.68 mg/ml 50 mM Tris, pH 7 and precipitates were determined. A t a molar ratio of Znn+/ link protein = 3, 97.5% of the link protein was recovered in 0.1520M NaC1, 11.23 mg/ml mg/ml the precipitate, and ata molar ratio of Zn'+/link protein 24, 10 mM NaH,PO,, pH 7 all of the link protein was recovered in the precipitate. There-

7020

FIG. 3. Demonstration of the binding of Zn'+ and Ni" to link protein by metal chelate affinity chromatography. Linkprotein(60nmol) was applied to columns which had been charged with Ca'+ ( A ) , Mi'+ ( B ) ,Zn'" (C), Ni" (D). The columnswere washed with 60 ml of 0.5 M NaCI, 50 mM Tris acetate, pH 8, then eluted with a pH 84 gradient. Link protein did not bind t,o Ca'+- ( A )or Mgy+-( E )charged columns. Link protein bound to the Zn"-charged column (C) and was eluted as a single unimodal peak by the pH 8-4 gradient.


FRACTION

FRACTION

FRACTION

FRACTION

fore, at molar ratios of Zn'+/link protein 2 4, there is no link protein in the supernatant, theZn2+in the supernatant is free 4 , I ligand and theZn2+ inthe precipitate is bound ligand. Fig. 4A 0 shows the binding of Zn'+ to link protein in the link protein 0 precipitates, ina plotof mol bound Zn2+/mo1total link protein uersus mol of free Zn2+. The binding data presented in Fig. 4A demonstrate that link is a metalloprotein with a moderately high affinity for Zn2+.The equilibrium binding datawere fit to various models using LIGAND (18). The datacould not be fit to a model that consisted of a single class of binding sites. However, a model consisting of a single class of binding sites and including "nonspecific" or unsaturable binding (18, 26) fit the datawell (Fig. 4B).The results obtained using this 1 model indicate that link protein has two high affinity sites for Zn2+ ( n = 2.3 k 0.1; K, = 3.0 (f0.2) X lo7 M-') and that nonspecific binding accounts for less than 8% of the total Zn2+ in the pellet. A modelconsisting of two classesof binding 0 20 40 60 80 100 sites fit these data equally well, as it should (26), the indicated FREEZn2+(Mx 108) tight binding ( n = 2.2 -t- 0.2; K, = 3.5 (k0.5) X lo7 K ' ) sites and much weaker binding ( n = 5 k 3; KO= 4 k 3 x lo5M") sites. Clearly the same description applies to the tight binding sites for either model. Determination of the existence and further characterization of the weaker class of sites will require more detailed experimentation. The Oligomeric State of Link Protein inLow Ionic Strength, Physiologic Solvents-Sedimentationequilibrium and sedimentation velocity studies were carried out as described under "Experimental Procedures." Fig. 5 shows the M , of link pro0.8 tein as a function of concentration in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris,pH 7. M,, decreasesslightly asthe concentration of link protein isdecreased from 2 to 0.125 mg/ 0 24 48 72 96 120 ml, suggesting the presence of a mass-action association. At [Bound] (nM) high protein concentrations, link protein exists asa hexamer with anaverage M , of approximately 260,000. These data sets FIG. 4. Binding of ZnZ+to link protein. A , plot of moles bound Zn"+/molof totallinkprotein versus mol of free Zn" X loH.E , (experiment 11), as well as those frommeniscus depletion Binding of Zn" to a single class of link protein binding sites. E was experiments made using 30-mm pathlength centerpieces (ex- obtained by fitting the data to a model for a single class of binding periment I), were modeled using NONLIN (22) and the results sites, after determining and correcting for nonspecific, unsaturable are presented in Table 111. The model providing the best fit binding (18, 2 6 ) . The results obtained using this model indicate that link protein has two high affinity sites for Zn'+ ( n = 2.3 k 0.1;K., = consisted of a monomer ( M , = 45,000) in equilibrium with a 2.0 (kO.2) X 10' M") and that nonspecific binding accounts for less hexamer. A model which neglected association provided poor than 8% of the total Zn'+ in the pellet. A value of 0.07 k 0.004 was fits to data, asdid any model which did not include monomer obtained for B, the parameter describing unsaturable binding, such (not shown). Models which included intermediate oligomers that ateach free Zn2+ concentration, the concentration of bound Zny+ due to nonspecific binding is B X [Zn2+],,,,. (dimers, trimers, etc.)provided no improvement in the fit, and resulted in apparent association constants near zero for

I*

7021

Metalloprotein Properties of Link Protein 900

400

r

I

900

0 x

200

B

A-

100

0 0.00

1.00

0.80

1.60

LinkProtmlnConcontratlon

2.00

0 ' 0

2.60

mglml

1

2

9

4

1

s

Yolo ratlo HA,, : Link proteln

FIG. 5. The quaternary structure of link protein in low ionicFIG. 6. HAlo-induced dissociation of link protein hexamer strength buffer. Short column sedimentation equilibrium experi- to dimer. Z average molecular weights of link protein to which HAI,, ments were conducted at 23.3 "C as described in"Experimental was added to a total concentration sufficient to provide the molar I'rocedures" and at thecell loading concentration indicated on the x ratios(HAI,,/linkproteinmonomer) shown onthe x axis. Each a Z average molecularweights were determinedfromthecomolecular weight was determined from coanalysis of data from four analysis of dat.a from experiments conducted a t three or more rotor rotor speeds. The error bars indicate the 95% confidence interval. speeds. The error on the molecular weight was symmetrical, and the errorbarsindicatethe 95% confidence interval.Indistinguishable molecular weights were obtained from individual data sets acquired at different rotor speeds (not shown), indicating that the sample is homogeneous.

I-

TABLE I11

I

Link protein hexamer formation NO."

(TI

Monomer M,"

K,;

AG' 1 4 "

cm - 2

g/md

x10-2h M"

kcai/rnol

f;;s

I

0.837/ 47.100 9.20.034 -36.2 (0.805 0.869)' (45,300 48,900) (2.8 34.8) (-35.5 -36.9) I1 1.32" 42,300 21.1 -37.0 0.017 (1.301.34) (41,600 42,900) (6.3 81.0) (-36.3 -39.2) '' Experiment I: 20.0 "C, 3-mm columns, co-fit of five concentrations (0.215-1.86 mg/ml)andthreerotor speeds (12, 14, and 16 krpm). Experiment 11: 23.3 " C ,0.7-mm columns, co-fit of six concentrations (0.125-2.00 mg/ml) and four rotor speeds (16, 20, 30 and 36 krpm). ' Calculated from (T using p = 1.00815xg/ml and V = 0.715 ml/g. "Determined from values ofIn Ks returned by NONLIN, with a model of 6 L ++ L,; and coverting units from fringesP to M-' using Kb,,(;= K\-,(;(M,/6)"(Y,r/G)', where M , is the monomermolecular weight and Y,r/Gris the specific increment (fringes"1). Y T / Cequals ~ 9.45 for experiment I and 3.52 for experiment 11. Free energy of formation of the hexamer fromsix monomers. " Root mean square of the variance of the fit. 'Determined at 12,000 rpm. 65% confidence interval returned by NONLIN. " Determined a t 16,000 rpm.

2 3 CONCENTRATION (rng/ml)

1

0

FIG. 7. Concentration dependence of sedimentation coefficients of the link protein hexamer(closed circles)and dimer (open circles) in 0.15 M NaC1, 5 mM EDTA, 50 mM Tris, pH 7.In the link protein dimer solutions, the molar ratio of HA,,, to link protein was 3:l. TABLEIV

Link motein sedimentation coefficient and frictional coefficient ratios ~

I'

the intermediate oligomers (not shown). This suggests that under these conditions link protein exists primarily as hexamers and monomers,with hexamer favored by 36 kcal/molhexamer over monomer. As shownin Fig. 6, when link protein hinds HAlo, link protein dissociates from hexamer to dimerwith M, =: 98,000. This dissociation is almost complete at a molar ratio of HAlo/ link protein = 21. The link protein dimer formation constant is extremely large, and certainly greater than 1 X 10' "I. This suggests that HAlo binding strongly stabilizes link protein dimers, aswell as destabilizing hexamers. Hydrodynamic Analysis of the Link Protein OligomersSedimentation coefficients of the link protein hexamer and dimer were determined (Fig. 7, Table IV), and the hydrodynamic propertiesof these oligomers were analyzed (Table IV). Both forms of the molecule sediment as globular proteins of similar, moderate asymmetry. The extent of asymmetry calculated depends on the assumeddegree of hydration ( 6 ) . For most proteins, values of 6 are near 0.3 (27), and glycosylation will alter this value only slightly (27, 28). The calculations presented for 6 = 0.5 g H,O/p protein, then, represent an

4

Oligomer"

Hexamer' Dimerd

S L

(s)

10.32 5.3~

f/fe.lr>h 1.48 1.42

f/hl a t mo:3

f/h, at r,,l

1.35 1.31

1.29 1.24

~

Oligomeric state suggestedbymolecularweightsmeasured by sedimentation equilibrium. *Frictional coefficient ratios calculated assuming M , = 260,000 (hexamer) or M , = 90,000 (dimer + 2 HA,,,), U = 0.715 ml/g and, for f/f,,calculations, either 0.3 or 0.5 g H20/g protein. ' In 0.15 M NaC1, 5 mM EDTA, 50 mM Tris, pH 7. 0.15 M NaC1, 5 mM EDTA, 50 mM Tris, pH 7, with HA,,,/link protein = 3 1 .

extreme. Even at the higher assumed hydration, both forms of link proteinmodel as prolateellipsoids of revolution having axial ratios of about 5:1, whereas at 6 = 0.3 the axial ratios are closer to 6:l. The Binding of Hyaluronate Oligosaccharides by Link Protein Competitively Inhibits the Precipitation of Link Protein by Zn2+-Taken together, the observations described so far show that in low ionic strength solvents, link protein exists as hexamer and the hexamer binds Zn2+, forms aggregates in the presence of Zn2+, andis precipitated by Zn". When link protein binds HAlo, link protein dissociates from hexamer to dimer. Additional experiments were carried out to examine the effect of Znz+ on thesolubility of the link protein/hyaluronate oligosaccharide complex, in which link protein exists


7022

as a dimer. The results were surprising. When link protein binds hyaluronate oligosaccharides (HAlo or HA,,), the precipitation of link protein by Zn2+is strongly inhibited. In the studies presented in Fig. 2, the effect of Zn2+on link protein solubility was examined at a single link protein concentration of 2.4 mg/ml. Fig. 8 provides more detailed information on the effect of Zn2+on link protein concentration when the initial link protein concentration isvaried from 0.2 to 1.6 mg/ml. In these experiments, Zn2+was added directly to the link protein solutions at a molar ratio of Zn2+/link protein of either 2 (closed circles, Fig. 8 ) or 4 (triangles, Fig. 8 ) , and both % solubility (Fig. 8 A ) , and the concentration in mg/ml of the soluble link protein remaining in the supernatant (Fig. 8 B ) were determined. The % solubility decreases progressively as the initial link protein concentration is increased, as shown in Fig. 8A. At a molar ratio of Zn2+/link protein = 2, the concentration of link protein remaining in the supernatant plateausat approximately 350 pg/mlat initial link protein concentrations of 0.6-1.6 mg/ml. This soluble link protein is precipitated if additional Zn2+is added. As the molar ratio of Zn2+/link protein is increased to 4 (triangles, Fig. 8 B ) , the concentration of link protein remaining in the supernatant decreases from 141 pg/ml at 0.2 mg/ml, to 42 pg/ ml at 1.6 mg/ml, to 17 pg/ml at 2.4 mg/ml (Fig. 2). When link protein binds HAlo, the effect of Zn2+on link protein solubility described above is abolished. Solutions were prepared in which the concentration of link protein was varied from 0.2 to 1.6 mg/ml (Fig. 8). HAlo was added at a molar ratio of HAlo/link protein = 2. Zn2+was added at a molar ratio of Znz+/link protein = 2. The % solubility of the link protein/HAlo complex is shown by the curve (open circles) at the top of Fig. 8A. The link protein/HAlo complex is almost completely soluble in the presence of Zn2+,over the entire concentration range. Another experiment was carried out to determine the amount of Zn2+required to overcome the inhibitory effect of the binding of hyaluronate oligosaccharides by link protein on the precipitation of link proteinby Zn2+.Link proteinwas mixed with HAl2 at a molar ratio of HAlz/link protein of 2. Zn'+ was then added directly to aliquots of the link protein/ HAlz solution at molar ratios of Zn2+/link protein which ranged from 0 to 100. The solutions were mixed, allowed to 1w

/ : "

i.: & 3

stand at 4 "C,centrifuged, and theamounts of link protein in the supernatants and precipitateswere determined. In Fig. 9, the curve on the left is for link protein alone, in the absence of hyaluronate oligosaccharide. The curve on the right is for link protein in the presence of HAl2. In the case of link protein alone, 99.3% of the link proteinis precipitated by the direct additionof 4 mol of Znz+/molof link protein. In the case of the link protein/HA12 complex, only 10.3% of the link protein is precipitated at 4 mol of Zn2+/mol of link protein. As the molar ratio of Znz+/link protein is progressively increased, the inhibitory effect of HAlz on the precipitation of link protein is gradually overcome. However,50 mol of Zn2+/mo1of link protein are required to precipitate 98.2% of the link protein. Thus,the molar ratio of Zn2+/linkprotein must be 10 times greater to.completely precipitate link protein to which a hyaluronate oligosaccharide is bound. The observations presented above suggest that hyaluronate oligosaccharides and Znz+might bind to thesame link protein binding site, that HAlz binds with a much higher affinity than Zn", and that at much higher molar ratios of Zn2+,HAlz may be dissociated from link protein, replaced byZn", and the link protein is precipitated. If HAl2 and Zn2+compete for the same binding site, the binding of HAlzto link protein should be abolished when link protein is completely precipitated by Zn2+,at high molar ratios of Zn*+/linkprotein. However, this is not the case. The amount of HAI, present in the link protein precipitated by Zn2+was determined by analyses of uronate contents. HA,, bound to precipitated link protein does not decrease as the molar ratio of Zn2+/link protein is increased. Specifically, 0.9 mol of HA12/molof link protein remains bound to theprecipitated link protein as the molar ratio of Zn2+/link proteinis increased from 10 to 100. The amount of HA,, which is bound to link protein does not decrease, even when the link protein is completely precipitated at molar ratios of Znz+/link protein ranging from 40 to 100. Thus, hyaluronate oligosaccharides remain bound to link protein when link proteinbinds Zn2+. This conclusion is further supported by studies involving metal chelate chro-

20

0

-

0-

I

0.5

1.o

1.5

INITIAL CONCOF LINK PROTEIN, m d m l

-

0

0.6 1.o 1.5 INlTlAL CONCOF LINK PROTEIN.W m l

FIG. 8. Effect of hyaluronate oligosaccharide (HAlo) on the precipitation of link protein by Znz*. A, % solubility of link protein as a function of initial concentration of link protein. B , link protein solubility in mg/ml as a function of initial concentration of link protein. In A, the open circles show the solubility of the HAlo/ link protein complex (link protein dimer) in the presence of Zn2+;the molar ratio of Zn"+/link protein = 2. The closed circles and triangles show the solubility of link protein hexamer at molar ratios of Zn2*/ link protein of 2 and 4, respectively. The link protein/HAlo complex (open circles) is almost completely soluble in the presence of Zn2+ over the entire concentration range.

1 8 10

20

30

40

MOLAR RATIO, Zn" : LINK PROTEIN

FIG. 9. Demonstration of the amount of Znz+ required to overcome the inhibitory effect of HAlz on the precipitation of link protein by Znz+.Solutions were prepared which contained 50 nmol of either link protein alone (closed circles) or link protein/HAtz (open circles) at a molar ratio of HAlz/link protein = 2. Zn2+was then added, at molar ratio of Znz+/link protein ranging from 0 to 100. The final concentration of link protein in the solutions was 2.4 mg/ml. The solutions were mixed, allowed to stand at4 'C, centrifuged, and the percentage of link protein which wasprecipitated was determined. Compared with link protein hexamer (closed circles), over 10 times as much Zn2+is required to completely precipitate the link protein/ HAlz complex, in which link protein exists as a dimer.

7023

Metalloprotein Propertiesof Link Protein

by gel chromatography, matography, shown in Fig. 10. As shown in Fig. 10A, HA]:, ometry of binding of link protein to GI alone does not bind toa Zn'+-charged chelating Sepharose 6B ion-exchange chromatography, or sedimentation equilibrium. In 1 M NaC1, 0.01 M Mes, pH 8, link proteinboundto column. Link protein was mixed with HAl2 a t a molar ratio of HA12/link protein= 2. The solution was applied to a Zn2+- hyaluronate oligosaccharides (HAlo-le) exists asa dimer (13). charged column, washed with pH 8 buffer, then eluted witha In associative solvents, link protein alone appears to exist as a higher oligomer, but the molecular weight and exact oligopH 8-4 gradient. All of the link protein bound to the column, and was eluted in a characteristic fashion by the pH 8-4 meric state of native, intact link protein in high or low ionic gradient. When link protein was bound to Zn'+ on the Zn2+- strength solvents has not previously been defined (9,131. This aggregate and charged column, HAl2 remained boundto thelink protein at is because thetendency of linkproteinto a molar ratio of HA12/link protein of 0.7:l. Thus, Zn'+ and precipitate at moderate link protein concentrations hasmade hyaluronate oligosaccharides appear to bind to differentlink it difficult to determine valid molecular weights of the link protein-binding sites. protein oligomer by sedimentation equilibrium or light scattering. DISCUSSION The results of this study indicate that link protein's repuNumerous references in the proteoglycan literature have tation as an insoluble protein is decidedly undeserved. Link alluded to the limited and variable solubility of link protein protein has a variable and limited solubilityinassociative (4, 11-13, 29). The apparent insolubility of link protein in solvents prepared from commercial buffers using water from low ionic strength-associative solvents has hampered studies which divalent cations have not been removed. Commercial of the fundamental biochemical properties of link protein buffers are known to contain appreciable amounts of divalent such as the stoichiometry of binding of link protein to hya- cations (31). However, if divalent cations are removed, link luronate and G1, the oligomeric state of link protein alone in protein is readily soluble and functionalin low ionic strength, low ionic strength, physiologic solvents and the transforma- physiologic solvents. These observations make easy it to work tion in the oligomeric state of link protein that occurs when with link protein at concentrations of 3mg/ml, study the link protein binds its naturalligands, hyaluronate and GI. binding of link protein to GI, the oligomeric state of link Biosynthetic studies have suggested that in proteoglycan protein alone under different environmental conditions, and aggregates the molar ratio of link protein to proteoglycan the transformation in the oligomeric state of linkprotein monomer is 1:l and that link protein exists in a monomeric or G1. which occurs when link protein binds to hyaluronate form (30). However, this suggestion has never been substanThesedimentation equilibrium analysis of link protein tiated by biochemical studies of the stoichiometryof binding described above indicates that a stable hexamer is formed in of link protein to G1 in theabsence of hyaluronate. Because the low ionic strength buffer. Examination of data over a of the variable and limited solubility of link protein, it has concentration range from0.125 mg/ml to 2 mg/ml shows that heretofore been impossible to carry out studiesof the stoichi- there is little or no dissociation upon dilution. At the lowest concentration examined, the total molar concentration of link protein (in terms of monomers) is less than 3 FM, indicating that the energy stabilizing the hexamer is fairly high. However, when the hexamer is titrated with HAlo it iseffectively dissolved to dimers, demonstrating that the hexamer consists of a trimer of dimers. This indicates that at leasttwo types of subunit contacts are involved in stabilizing the hexamer. It is notpossible on the basisof the present data to determine whether the HAi,,-dependent dissolution of the hexamer re0 0 sults from competition for binding sites or by a conforma50 FRACTION tional change resultingfrom HAlo binding toa distinct site. B Thus, when linkproteinbindshyaluronate oligosaccharides, there is a transformation in theoligomeric state of link A530 protein from hexamer to dimer. Data from a previous study indicates that when a chain of high molecular weight hyaluronate is saturated with link protein, the molar ratio of link protein to HAlo segments2:1, is suggesting link protein bound to high molecular weight hyaluronate also exists as a dimer (14). This changein the oligomeric state of link protein must be taken into account in studies of the conformational changes in link protein which occur when link protein binds to hyaluronate. FRACTION In such studies, one must distinguish between conformaFIG. 10. Demonstration that HAI, remains bound to link tional changes directly associated with the bindingof hyaluprotein when link proteinis bound to Zn". Chelating Sepharose ronate by link protein, and those changes which are associated GB columns were charged with Zny+. In A , 122 nmol of HA,, were applied to a column, which was washed with 0.5 M NaC1,50 mM Tris with the dissociation of link protein from higher oligomer to acetate, pH 8. HA,, (open circles) does not bind to the Zn'+-charged dimer. The information contained in this reportis also needed column, and elutes in the wash. In B , 61 nmol of link protein were to demonstrate the change in the oligomeric state of link mixed with 122 nmol of HA,,, allowed to stand overnight at 4 "C, protein which occurs when link protein bindstotheG1 then applied to the column. The column was washed0.5with M NaC1, 50 mM Tris acetate, pH 8, then eluted with a pH 8-4 gradient. Link globular domain of proteoglycan monomer core protein.

0,5r-

protein (closed circles) bound to the column, and was eluted by the p H 8-4 gradient. Thirty-five 76 of the HA,, remained bound t o the linkproteinwhichwasboundtothe Zn"'-charged column,and coeluted with the link protein.

REFERENCES 1. Hascall, V. C., and Kimura, J. H. (1982) Methods Enzyrnol. 82, 769-800

7024


2. Hardingham, T. E., and Muir, H. (1972) Biochim. Biophys. Acta 279,401-405 3. Hardingham, T. E., and Muir, H. (1974) Biochem. J. 139, 565581 4. Heinegird, D., andHascall, V. C. (1974) J. Biol. Chem. 249, 4250-4256 Storrs, 5. Rosenberg, L., Hellmann, W., and Kleinschmidt, A. K. (1975) J . Hiol. Chem. 250, 1877-1883 6. Buckwalter, J. A,, and Rosenberg, L. C. (1982) J.Bid. Chem. 257, 9830-9839 7. Buckwalter, J. A,, and RoseEberg, L. C. (1983) Coll. Rel.Res. 3, 489-504 8. Hardingham, T. E. (1979) Biochem. J . 177, 237-247 9,Tang,L.-H,, Rosenberg, L,,Reiner, A,, and Poole, A, R. (1979) J . Riol. Chem. 254,10523-10531 10. Tang, L.-H., Rosenberg, L. C., Reihanian, H., Jamieson, A. M., and Blackwell, J. (1989) Connect. Tissue 19, 177-193 11. Tengblad, A. (1981) Biochem. J. 199,297-305 12. Pkrin, J.-P., Bonnet, F., Thurieau, C., and Jolles, P. (1987) J . Hiol. Chem. 262, 13269-13272 13. Bonnet, F., Dunham, D. G., andHardingham, T. E. (1985) Biochem. J. 228, 77-85 14. Rosenberg, L., Tang, L.-H., Pal, S., Johnson, T. L., and Choi, H. U. (1988) J . Biol. Chem. 263, 18071-18077 15. Heinegard, D. (1972) Biochim. Biophys. Acta 285, 181-192 16. Laemmli, U. K., and Favre, M.(1973) J . Mol. Biol. 80,575-599 17. Bitter, T., and Muir, H. (1962) Anal. Biochem. 4, 330-334

Protein of Link 18. Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107,220239 19. Williams, R. C., Jr. (1972) Anal. Biochem. 48, 164-171 20. Laue, T. M. (1981) Rapid Precision Interferometry for the Analytical Ultracentrifuge. Ph.D.dissertation,University of Connecticut, CT 21. Yphantis, D. A. (1960) Ann. N. Y. Acad. Sci. 88,586-601 22. Johnson, M. L., Correia, J. C., Yphantis, D. A,, and Haivorson, H. R. (1981) Biophys. J . 36, 575-588 23. Arakawa, T., Yphantis, and D. A. (1987) J. Bid. Chem. 262, 7484-7485 24. Laue, T . M., Johnson, A. E., Esmon, Yphantis, and C. T., D. A. (1984) Biochemistry 23, 1339-1348 25. Weast, R. c. (ed) (1986) ofChemistry and 67th ed., Chemical Rubber Publishing Co., Cleveland, OH 26. Johnson, M. L., and Frasier, S. G. (1985) Methods Enzymol. 117, 302-342 27. Kuntz, I. D., Jr., and Kauzmann, W. (1974) Adu. Protein Chem. 28,239-345 28. H@iland,H, (1986) in ~ h ~Data f o~r Biochemistry ~ and ~ Technology (Hinz, H.J., ed) pp. 129-147, Springer-Verlag, New York 29. Lyon, M., and Nieduszynski, I. A. (1983) Biochem. J . 213, 445450 30. Kimura, J . H., Hardingham, T. E., and Hascall, V. C. (1980) J . Biol. Chem. 255, 7134-7143 31. Crouch, T. H., and Klee,C. B. (1980) Biochemistry 19, 36923697

d

~