Size distribution of cartilage proteoglycans determined by sedimentation field flow fractionation

Biochimica et Biophysica Acta, 966 (1988) 231-238

231

Elsevier BBA 22945

S i z e distribution of cartilage p r o t e o g l y c a n s d e t e r m i n e d by s e d i m e n t a t i o n field f l o w f r a c t i o n a t i o n L.E. Schallinger a,,, E.C. Arner

a a n d J.J. K i r k l a n d

b

E.1. DuPont de Nemours and Company, a Medica[ Products Department and ~ Central Research and Development Department, Experimental Station, Wilmington, DE (U.S.A.)

Key words: Proteoglycan molecular weight distribution; Sedimentation field flow fractionation; (Cartilage)

Sedimentation field flow fractionation (SFFF), a new, gentle, high-resolution separation method, rapidly separates cartilage proteoglycan subunits and aggregates within 1-2 h with recoveries of greater than 80%. Transmission electron micrographs of SFFF fractions from cartilage proteoglycans showed that subunits and aggregates are effectively separated; the characteristic morphology of these materials is preserved. In addition, the molecular weights of components in these fractions were accurately determined without standards. Separations performed in this study showed that SFFF has the sensitivity to detect small variations in proteoglycan size distribution resulting from differences in animal species, cartilage type, and cartilage treatment. Advantages of SFFF include small sample requirements, high resolution, rapid separations, and high component recovery. Significant potential is indicated for SFFF in proteoglycan research.

Introduction Sedimentation field flow fractionation (SFFF) is a new, high-resolution method for separating and determining the mass or size distribution of a wide variety of colloids and particulates in the 0.005-2/~m diameter range [1-4]. S F F F also can be used to separate and isolate many types of soluble macromolecules, especially those of biological interest in the 106-1012 Da range [5]. In contrast to conventional separation methods based on chemical interactions, SFFF is a very mild, non-interactive technique based on physical properties. The S F F F method imposes low shear forces on components; as a result, the method is

* Permanent address: 3 M / 3 M Center, Bldg. 201-BW-11, St. Paul, MN 55144-1000, U.S.A. Correspondence: E.C. Arner, Medical Products Department, E.I. DuPont de Nemours and Co., Experimental Station E400/2247, Wilmington, DE 19898, U.S.A.

particularly attractive for separating and isolating fragile biomacromolecules. Previous studies have shown that S F F F can fractionate such biological materials as bacteria, whole red blood cells, intact viruses, large D N A and RNA, liposomes, and bacterial cell wall fragments [6-12]. Results with the fragile lambda-viral DNA and smaller supercoiled plasmids have shown that these materials are not altered during S F F F separation; molecular weights, molecular conformation and biological activity remain unchanged during isolations [13]. There are several additional advantages of S F F F in biological fractionations. Typical separations are performed in 1-2 h, and recoveries of isolated materials are high (80-100%). Since SFFF separations of biological molecules normally are done in conventional buffered salt solutions of relatively low ionic strength, isolates often are ready for use without additional clean-up steps. Sample concentration and molecular conformation do not influence the elution process in the

0304-4165/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

232

proper operating range [13,14]. If the density of components is known, accurate molecular weights can be determined without reference to standards. If the density of the components of interest is unknown, this property can be accurately determined with appropriate SFFF experiments [15]. Proteoglycans appeared to be an excellent candidate for fractionation and characterization by SFFF, since the molecular weight ranges for both subunits and aggregates are well within the accessible range of this method ((0.5-2.5)- 10 6 for subunits, 107-108 for aggregates) [16-18]. Methods previously used to characterize proteoglycans have limitations that are not a feature of SFFF. Several studies used Sepharose 2B columns to assess differences in proteoglycan size. Unfortunately, only relative differences were obtained with this approach [19-21]; size-exclusion chromatography (SEC) is not an absolute method without appropriate standards. Other SEC studies used light-scattering measurements to define the molecular weight of subunits in isolated fractions, since no appropriate standards exist for SEC molecular-weight calibration [22]. Furthermore, SEC inherently has lower resolution and imposes much higher shear forces on samples than SFFF [23]. Although sedimentation velocity, sedimentation equilibrium, and low-angle light scattering are absolute methods that can be effective in defining sample heterogeneity, they all lack the resolution of SFFF and the ability or convenience of isolating fractions during the characterization. Materials and Methods

Theory The theoretical basis for SFFF has been given previously [1-4]. Briefly, separations are carried out in a very thin, open channel with a continuously flowing carrier liquid, under the influence of an external gravitational force field. Samples are slowly pumped into the SFFF channel inlet with an initial force field perpendicular to the channel (centrifuge spinning). During a relaxation period, flow is interrupted while sedimentation equilibrium is reached. Flow is then re-initiated to create a laminar flow profile; carrier liquid velocity is at a maximum in the center of the channel

and approaches zero velocity at the walls because of frictional drag. Particles with smaller effective mass (mass × density) move in faster flow streams that are closer to the center of the channel. Particles with larger effective mass move down the channel in slower flow streams nearer the wall. Thus, in SFFF, species elute in order of increasing effective mass. At a constant force field (rotor speed), the molecular weight of a well-retained component relates to its retention in the following manner:

M= (

R°T6VR

]

VoGW( A3/3~ ) ]

(1)

where M is the molecular weight of the component (g/mol); R 0 is the gas constant (8.31-107 g - c m . s -~ . K - 1 - m o l ~); T is temperature (Kelvin); VR is the retention volume of the eluted component (ml); V0 is the dead volume of the channel (ml); W is the thickness of the channel (cm); and Z~3 is the density difference ( g / c m 3) between the component density 8s ( g / c m 3) and the carrier liquid. The force field G relates to rotor speed according to: ~o~r 2 G=(~6-) r

(2)

where c0 is the rotor speed in rpm and r is the distance (cm) from the center of the rotor to the channel. Eqn. 1 defines the mass of a component eluting in a S F F F experiment in terms of physical parameters that either are known (i.e., rotor speed, channel dimensions, particle and carrier density, etc.) or are measured as a result of the separation (retention volume, VR). Shape features normally do not effect retention, since the shape-dependent frictional coefficients associated with particle movement cancel out in the relationship governing the separation [14]. Therefore, S F F F can directly measure the mass of a component without the need of standards. The separations in this study were carried out with a S F F F technique involving a precisely controlled exponential decrease in the centrifugal field during the separation, rather than a constant centrifugal field. This method, called time-delay, exponential-decay S F F F (or TDE-SFFF), has the

233 distinct advantage of allowing the separation of samples containing components with a very wide mass range to be carried out with good resolution in a convenient time span [3]. For well-retained samples in TDE-SFFF, the mass or molecular weight (g/mol) and retention time t R (min) of the component exhibit a simple log-linear relationship: In M = In A + tR/'r

(3)

where

A={

6R°T"r etoGoW(A~/6s ) }

(4)

and e is the natural log base (2.718); t o is elution time of an unretained component (channel deadtime) (rain); ~- is the time (rain) of the delay and also the time constant of the exponential decay; and GO is the force field initially applied in the separation.

Instrumentation Details of the S F F F equipment used in this study have been given elsewhere [3,4,9,10]. The instrument operates with maximum force fields of about 80 000 gravities, so that most components of greater than about 250000 molecular weight usually can be adequately retained for isolation and characterization. Separations were done with a specially modified Model L5-50B ultracentrifuge (Beckman Instruments, Fullerton, CA) fitted with a high-speed, water-cooled rotating face seal, a Model 850 microprocessor-controlled solvent metering system ( D u P o n t Medical Products, Wilmington, DE), a Varichrom UV spectrophotometric detector (Varian Instruments, Walnut Creek, CA), an air-actuated microsampling valve (Valco Instruments, Houston, TX), and a MINC023 microcomputer (Digital Equipment Corp., Maynard, MA). The centrifuge contained a 'floating' plastic channel assembly of a design previously described [4]. The specially designed face seal in the S F F F instrument provides for smooth, low-shear entry and exit of the carrier liquid to and from the spinning separating channel. Components eluting from the channel are detected by ultraviolet (UV)

absorption, or turbidimetry for particulates. An on-line computer monitors and controls rotor speed, stores data from the detector, computes molecular weights (or particle diameters), and plots information from the separation. Recovery of fractionated material is carried out by collecting the eluent as it exits from the detector. For all types of cartilage studied, we found that an initial constant force field of 80000 gravities for 20 min produced conditions for retaining the subunits and other lower-weight sample components of interest on the channel. A slow exponential decay of this force field then provided adequate resolution of these components from aggregate sub-populations. With these conditions, fractions were conveniently isolated and collected. Total separation time for each sample was 90 min.

S F F F separation A 0.5-ml sample volume of freshly prepared proteoglycan solution equivalent to 300-400/~g of uronic acid was pumped into the spinning channel (32000 rpm, 80000 × g ) at a flow rate of 0.1 m l / m i n . During this step, flow was stopped to establish sedimentation equilibrium within the channel. After an additional 5-min period, mobile phase (0.05 M sodium acetate, pH 6, 0.15 M sodium chloride) flow was re-established at 0.5 m l / m i n . After 20 min under these conditions, an exponential force field decay was initiated with a time constant y = 12 min. For fraction collection, 2-ml portions were taken in sterile, graduated sample tubes. BenzamidineHC1 and E D T A were added to the collected fractions at final concentrations of 0.05 M and 0.01 M, respectively. Significant dilution of the injected sample occurs during the isolation as a result of the S F F F separation process. Extraction and preparation of proteoglycans Rabbit articular cartilage was obtained from the knee joints of male New Zealand white rabbits, and calf articular cartilage from calf ankle joint capsules. Both were taken fresh at the time of slaughter. The cartilage was sliced from the joints and placed in ice-cold saline. Calf noses also were obtained fresh at slaughter; cartilaginous septa were excised, sliced, and placed in ice-cold saline.

234 Proteoglycans were extracted by stirring the cartilage for 48 h at 4 ° C in 10 vol. of 4 M guanidine-HC1 ( S c h w a r t z / M a n n , Cambridge, MA) in 0.05 M sodium acetate, pH 5.8, containing the proteinase inhibitors, 0.01 M EDTA, 0.1 M 6-aminohexanoic acid, and 0.05 M benzamidineHC1 [24]. The extract was centrifuged at 10 000 × g for 30 min at 4 ° C to remove residual cartilage. To re-associate the extracted proteoglycans, the supernatant from this treatment was dialyzed using Spectra/por 6 dialysis tubing with a molecular weight cutoff of 2000 (Spectrum Medical Industries, Los Angeles, CA) for 48 h at 4 ° C against 200 vol. of 0.05 M sodium acetate, pH 6.8, containing the proteinase inhibitors listed above [24]. Proteoglycan subunits were isolated from the 4 M guanidine-HC1 extract by equilibrium density gradient centrifugation in cesium chloride (d~ = 1.50 g / m l ) by the method of Hascall and Sajdera [25]; the solution was centrifuged at 100000 × g for 62 h (4 ° C). The bottom of this gradient (d = 1.54 g / m l ) containing the subunits was dialyzed at 4 ° C against 200 vol. of 0.05 M sodium acetate (pH 6.8) with the proteinase inhibitors listed above. In one experiment, we divided calf articular cartilage into two pools. The first pool of cartilage was extracted with 4 M guanidine-HC1 in 0.05 M sodium acetate, p H 5.8, containing the proteinase inhibitors listed above. The second pool of cartilage was extracted with 4 M guanidine-HC1 in 0.05 M sodium acetate, pH 5.8, containing the proteinase inhibitors, 0.01 M EDTA and 1.54. 10-7 M bovine pancreatic trypsin inhibitor. Re-association of proteoglycans from the first pool was accomplished by dialysis as described above. The extract from the second pool was dialyzed using Spectra/por 6 dialysis tubing with a molecular weight cutoff of 10000 for 48 h at 4 ° C against 0.05 M sodium acetate, pH 6.8, containing EDTA and bovine trypsin inhibitor at the same concentrations used in the extraction.

Quantitation of proteoglycans Proteoglycan content of the SFFF fractions was determined by measuring uronic acid with the carbazole method [26].

Determination of proteoglycan density To calculate the mass (molecular weight) of the

proteoglycan (see Eqn. 1), we required an accurate measurement of the proteoglycan density. Such determinations on re-associated proteoglycans were carried out with a Mettler/Parr Model DMA 60 densimeter (Mettler Instrument Co., Hightstown, N J). After determining the percent solids in the sample, a value of 1.62 g / c m 3 was calculated by taking the difference between the measured density of the equilibrium dialyzate and a dialyzed proteoglycan solution.

Transmission electron microscopy Fractions collected during SFFF separations were prepared for microscopy by a previously described procedure [17]. The solution (hyperphase) used for spreading the proteoglycan sample on the grid was formulated with 333 ffl of 3 M ammonium acetate (pH 5), 12.5 ffl of 1 m g / m l of cytochrome c, and 650 ffl of isolated proteoglycan fraction. The prepared hyperphase solution contained 1-4 ffg/ml of proteoglycans. To prepare the sample for microscopy, 1 ml of a 0.3 M ammonium acetate solution (pH 5) (hypophase) was placed in a plastic petri dish. Surface tension effects maintained this solution as a large, single drop on the plastic surface. Onto this drop was delivered 25 ffl of the hyperphase solution using an Eppendorf pipette. A 200-mesh copper grid previously coated with a 1% formvar solution was immediately touched to the surface of the coated drop. Excess liquid was removed by carefully touching the edge of the grid to bibulous paper. The treated grid was placed for 30 s in a uranyl acetate solution (diluted 1/1000 with 90% ethanol from a 0.05 M stock solution containing 50 mM HC1), then rinsed for 15 s with 90% ethanol. After drying, the grid was shadowed with platinum at an angle of 8 ° in a BAF 400 coating system (Balzers AG, Ftirstentum, Liechtenstein). The grids were then mounted in a Model H600 electron microscope (Hitachi Scientific Instruments, Tokyo, Japan) to obtain micrographs. Measurement of the lengths of proteoglycan subunits and aggregates (subunits plus filamentous backbones) were made by tracing their patterns on a transparent plastic sheet placed over the micrograph. Contour lengths of the particles were then calculated using an image analyzer (IBAS, New York, NY).

235 Results

Fig. 1 shows typical S F F F separations of the proteoglycans from calf articular cartilage, using both UV detection at 280 nm and uronic acid content as monitors. Eluting directly after the channel dead volume, V0, were low-molecularweight (Mr) components that were weakly retained under the conditions of the experiment. The very large UV absorbance peak for these materials overlaps the peak for the proteoglycan subunits. The subunit peak is more obvious when ehition is monitored by the determination of the uronic acid content of S F F F fractions. Next, a low-M r subpopulation of aggregates eluted, followed by a higher-Mr subpopulation of aggregates. In all samples, the M r range for these components corresponded closely with those previously reported [17,26,27]. When isolated proteoglycan subunits were subjected to SFFF, a single, polydispersed peak eluted at a molecular weight centralized at about 1 • 1 0 6. N o additional peaks were seen in the fractogram. This shows that materials eluting at retention times representative of higher weights were not due to self-associating monomers induced by the S F F F separation method. Transmission electron micrographs (TEM) of S F F F fractions from calf articular cartilage showed that the proteoglycan subunits and aggregates were effectively separated from one another; the characteristic morphology of these aggregates was pre-

served during separation (Fig. 2). T E M grids made with eluent containing unretained components of the separation (V0 < 7 - 1 0 5 Mr) showed only a trace of subunits and no aggregates. The T E M in Fig. 2A of a fraction taken at an elution time interval corresponding to ( 1 - 2 ) - 1 0 6 M r shows a much larger population of subunits, but no aggregates. Image analyzer measurement on 113 of these subunits produced an average length of 268 _+ 53 nm, with a range of 100-400 nm. These results correlate well with those previously reported for bovine articular cartilage, which showed an average subunit length of 226 nm (n = 465), a range of 100-400 nm, and an average M r of 1.5 • 106 [17]. Micrographs taken of S F F F fractions collected in a retention interval representing components in the ( 3 - 7 ) - 1 0 6 M r range showed a marked decrease in the subunit population and an increase in a population of 'star-like' aggregates, as illustrated by the micrograph in Fig. 2B. Using 268 n m as the average length of a subunit, and assuming an average M r of 1.5-106/subunit, we estimate that the two most easily measured 'star' aggregates in Fig. 2B have M r values of about (5.5-7.5) 1 0 6, which is well within the range measured by S F F F (see Fig. 1A). TEMs of fractions collected at retentions representing 1-107 M r or greater showed m a n y examples of aggregates with the characteristic 'fern-like' morphology of these proteoglycans. As expected, most of these materials when spread on grids for •

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Fig. 1. Separation of calf articular cartilage. (A) SFFF separation of proteoglycans prepared with benzamidine-HC1, EDTA and 6-aminohexanoic acid. (B) SFFF separation of proteoglycans prepared with bovine pancreatic trypsin inhibitor and EDTA. The solid line in the fractogram is on-line detection by absorbance at 280 nm; the dashed line represents the uronic acid content.

236

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Fig. 2. TEM of calf articular proteoglycans from SFFF fractions collected at various retention (Mr) intervals. (A) (1-2). 106 Mr; (B) (3-7)" 106 Mr; (C) >_ l" 107 M r.

TEM examination exhibit some degree of entanglement or overlapping. However, Fig. 2C is a TEM of an isolated aggregate. Using the relationships described above, and assuming a value of 416 as the M r per nm of the hyaluronic acid backbone [17], we calculated a 34, value of about 2 . 1 0 7 for this aggregate. This value again agrees with those measured by S F F F (see Fig. ]A). SFFF data can be conveniently used to compare the relative population of subunit and aggregate populations. The uronic acid content of each collected fraction was used for this purpose, rather than the less-selective absorbance at 280 nm. UV absorbance, while representative in the higher-molecular-weight region, is anonymously large near the void volume, V0, because of the large UV response of benzamidine-HC1 and other

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proteinase inhibitors found in that part of the separation. The output of both detection systems are shown in Figs. 1, 3 and 4. Previously reported values, together with estimates from micrograph data, suggest that subunits and aggregates have molecular weights of (0.5-2.5). 1 0 6 and > 3 - ] 0 6, respectively [17,26,27]. Using these as guidelines, we calculate that the aggregates from calf articular cartilage prepared with benzamidine-HC1, EDTA, and aminohexanoic acid as preservatives (Fig. 1A) represented 30% of the total preparation; 70% was composed of subunits. In contrast, aggregates from the same cartilage preparation, but with bovine pancreatic trypsin inhibitor and EDTA as preservatives (Fig. 1B), comprised only 11% of that preparation. Subunits constituted 89%, and appeared as a bimodal distribution. Preparations from calf nasal cartilage contained 24% aggregates and 76% subunits (Fig. 3). The uronic acid assay data showed that the total amount of proteoglycan recovered from the S F F F separation of the calf articular cartilage samples in Fig. 1A and B was 89 and 85%, respectively. Recovery for the bovine nasal cartilage sample in Fig. 3 was 81%. These experiments showed that a bimodal distribution existed for the aggregate populations in two of the three cartilage specimens separated. Both calf and rabbit articular cartilage displayed a bimodal distribution of aggregates, while calf nasal cartilage did not. For the calf articular cartilage preparations (Fig. ]), the benzamidine-preserved

237

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sample had a major sub-population of larger aggregates centered at 2 . 1 0 7 M r , and a second population of aggregates centered at 8.0.10 6 M r . The trypsin inhibitor-preserved sample also exhibited a minor population at 2 . 1 0 7, but the major aggregate population was focussed at 5.0. 1 0 6 M r . In contrast, aggregates from calf nasal cartilage preserved with benzamidine-HC1, EDTA, and aminohexanoic acid (Fig. 3) showed a continuous monomodal distribution, with a mean at about 4.107 Mr. The bimodal distribution of aggregates for the calf articular cartilage was reproducibly observed for articular cartilage preparations from additional animals. Here, the lower- and higher-M r aggregate fractions were focussed at about (6.1 + 0.7). 10 6 M r (mean + S.E., n = 4) and (2.3 + 0.3). 1 0 7 M r (mean + S.E., n = 4), respectively. A second identically prepared calf nasal sample showed a monomodal aggregate pattern by SFFF, with the center of mass again at about 4- 1 0 7 M r. As with the calf articular cartilage, rabbit articular cartilage preparations exhibited a marked bimodal aggregate distribution, with masses focussed at about 5.1.10 6 and 1.3-10 7 M r . AS shown in Fig. 4, UV absorbance suggests about equal amounts of each population (uronic acid assays were not done on these samples). Discussion

TEM data showed that SFFF is capable of separating proteoglycan extracts into highly en-

riched fractions. Each of these fractions contains primarily of one of the three morphological forms present in renatured samples: subunits, 'stars', and the larger 'fern-like' aggregates. Fractionations are highly repeatable because of precise instrumental control over the physical parameters used to develop the separation (i.e., gravitational force field, flow rate). Further, the M r values for components in these fractions can be accurately determined without standards. Molecular weights estimated from TEM data show that the masses calculated for subunits, 'star' aggregates, and 'fern' aggregates from calf articular cartilage, are proper for the M r ranges covered by the collected S F F F fractions. Furthermore, M r values observed here for both monomers and aggregates compare well with estimates made by electron microscopy, sedimentation velocity, and light scattering [16-18]. The separations in this study prove that the SFFF method is sufficiently sensitive to detect variations in aggregate size distribution resulting from differences in species, cartilage type, and cartilage treatment. Concerning differences observed between benzamidine- and trypsin inhibitor-preserved samples, it appears likely that the lower M r values obtained for aggregates prepared with trypsin inhibitor were because of sample degradation. This conclusion is suggested not only by the predominance of lower-M r aggregates, but also because a portion of the uronic acid-containing material in the monomer region was present as an additional lower-M r peak at _< 5 • 10 5, suggesting breakdown of the subunit population. It is highly unlikely that differences in aggregate profiles between samples prepared with benzamidine and trypsin inhibitor were artifacts of the S F F F separation. Separations with benzamidinepreserved samples were reproducible for the same sample, and for different samples of fresh cartilage. The measured amounts of subunits and aggregates in samples are subject to modest error, because of the continuous nature of M r distributions for these materials, particularly in the low-M r region containing the subunits. These separations were carried out well below the maximum resolution of the S F F F method. Separation conditions were chosen to show that subunit and aggregate

238

separations could be rapidly accomplished. Higher-resolution separations of aggregate subclasses, such as 'stars' or 'ferns', could be carried out by decreasing the carrier flow rate, or by decreasing the rate of decay of the centrifugal force field (increasing ~-) during the separation. Either of these procedures would result in increased separation time. Although not primarily designated as a preparative method, SFFF has been shown to be of merit in this regard for some applications, for example, purifying plasmid DNA [11]. Proteoglycan aggregates in 0.1-0.5 mg quantities are easily collected in a single separation. Therefore, enough purified material can be obtained in a single or a few runs to do a variety of characterizations. Significantly larger amounts could be obtained in a single run, if desired, but with some sacrifice in product purity. A special advantage of aggregate isolation by SFFF is that unwanted proteinases with very small M r values are eluted first in the channel void volume and are, therefore, effectively removed from the proteoglycan samples. These studies have shown the significant potential of SFFF in proteoglycan research. Advantages include minimum sample requirements, high resolution, rapid separations, and high component recovery yields. Such a tool should be especially useful in pharmacological studies of proteoglycan metabolism involving small animals where the amount of cartilage is limited and the number of samples requiring characterization may be large.

Acknowledgements We thank M.J. Van Kavelaar of DuPont for the transmission electron microscopy, and R. Rappe of 3 M for the image analyzer study. References 1 Giddings, J.C., Yang, F.J.F. and Myers, M.N. (1974) Anal. Chem. 46, 1917-1923.

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