Partial characterization of reaction products formed by the degradation of hyaluronic acid with ascorbic acid

BIOCHIMICA

BBA 26

ET BIOPHYSICA

385

ACTA

258

PARTIAL

CHARACTERIZATION

THE DEGRADATION R. L. CLELAND*,

OF HYALURONIC

A. C. STOOLMILLER,

From the Department Departments

OF REACTION

of Medical

ACID WITH

L. RODeN

Chemistry,

PRODUCTS

AND

University

FORMED

ASCORBIC

BY

ACID

T. C. LAURENT

of Uppsala,

of Pediatrics and Biochemistry, the LaRabida-University

Uppsala (Sweden) and the of Chicago Institute and the

Joseph P. Kennedy, Jr. Mental Retardation Research Center, University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Received

July ITth, 1969)

SUMJIART

Hyaluronic acid was degraded with ascorbic acid at pH 3.3 for various periods of time. The extent of the degradation was measured by viscometry and by reduction of newly-generated reducing end groups with NaB3H,. Characterization of the products with respect to molecular weight distribution as well as the nature of the reducing terminal end groups showed that the degradation proceeded essentially by random cleavage of glycosidic bonds. The relative proportion of uranic acid and hexosamine end groups was determined, and it is concluded that hexosaminidic linkages were cleaved more easily than uronidic bonds.

INTRODUCTION

The degradation of hyaluronic acid by ascorbic acid has been studied for many years and the extensive literature has recently been reviewedi. Although several parameters of this reaction have been carefully detailed, the exact mechanism of polysaccharide degradation has not been elucidated, nor have the reaction products been completely characterized. It is generally assumed that the reaction involves cleavage of glycosidic linkages by a mechanism involving some free-radical speciesl, but it was recently suggested that the protein moiety of the hyaluronic acid-protein complex may also be a site of the reaction with ascorbic acid2. A major difficulty in the analysis of the cleavage products is the detection and quantitative determination of newly formed end groups which are generated only in minute amounts. Reduction with NaB3H, is a sensitive technique for the determination of reducing end groups in polysaccharides3, and this method has been applied in the present study. It will be demonstrated that the action of ascorbic acid results mainly in cleavage of hexosaminidic linkages but also, to a smaller extent, of glucuronidic bonds. * Permanent U.S.A.

address:

Department

of Chemistry,

Dartmouth

College,

Hanover,

N.

H.,

Biochim. Biophys. Acta, 192 (1969) 385-394

R. L. CLELAND t?t&?l.

386 %lATERIALS

L-Ascorbic

acid was purchased

from Merck and Co., NaB3H,

(zoo mC/mmole)

from New England Nuclear, and L-r,4gulonolactone from Nutritional Biochemicals Corp. L-Gulonic acid was obtained by hydrolysis of the 8-lactone in 0.2 M HCl for I0 min at 100’. amine

n-Glucosaminitol was prepared in the following manner. 1C’-Acetyl-u-glucos(IOO mg) from Sigma was dissolved in 20 ml of cold 0.01 M Tris-HCl buffer

(pH 8.0) and IO mg of NaBH, were added. After 14 h at 4’, excess NaBH, destroyed by addition of acetic acid and the solution evaporated repeatedly

was with

additions of methanol. N-Acetyl-n-glucosaminitol was deacetylated by hydrolysis in 2 M HCl for 2 h at 100’. After removing HCl in vucuo the n-glucosaminitol n-as purified by adsorption

on a column

(1.0 cm

x 5 cm) of Dowex

200-400 mesh). The product was eluted with 0.5 M HCl. 2-Acetamido-2-deoxy-3-O-(B-D-glucopyranosyluronic pared from hyaluronic

acid tetrasaccharide

50-X8

(H+ form,

acid)-n-glucitol

was pre-

(a gift from Dr. A. Telser) by an analogous

procedure. The tetrasaccharide was reduced with NaBH,, hydrolyzed in I M HCl for 3 h at IOO“, and after removal of HCl by evaporation the hydrolysate was used as chromatographic

standard

without

further

purification.

METHODS

earlier

Hyaluronic acid was isolated from extracts of rooster comb as described for hyaluronic acid from the vitreous bodp. The protein content was less

than 2% as determined by amino acid analysis after acid hydrolysis’j. are given in Table I (Sample I). Degradation SWANN~. A solution

Analytical

data

with ascorbic acid was carried out essentially as described by of 2.5 g of hyaluronic acid in I 1 of distilled water was dialyzed

overnight against a suspension of Dowex 50-X8 (H+ form) in distilled water. The dialyzed solution (pH 3.3) was diluted to 4 1 and divided into four equal portions. One of these was retained

as a control while the other three were mixed with ascorbic

acid (I mM). The three solutions were kept for 8,19, and 67 h at room temperature and subsequently dialyzed against several changes of distilled water. The polysaccharide from each of the four solutions was precipitated by addition of 3 vol. of ethanol and IO ml of 2 M sodium acetate. The amounts recovered were the same in all samples (approx. 500 mg). The analyses of the preparations, named Samples TABLE ANALYSES

I OF

HYALURONIC

Sample

ACID

BEFORE

AND

Uranic acid

Hexosamine

(%)

(%)

I

39.1

31.’

2 3 4

38.9 45.6 41.’

31.9 36.5 31.4

Biochim.

Biophys.

Acta,

192 (1969) 385-394

AFTER

DEGRADATION

WITH

ASCORBIC

ACID

DEGRADATION

OF HYALURONIC

387

ACID

1-4, are given in Table I. A portion of Sample 4 was fractionated

on DEAE-cellulose5 (see Table III). NaBH, reductions were performed in the following manner. Samples (1-2 mg) of untreated or degraded hyaluronic acid were dissolved in 6 ml of cold (0”) 0.01 M Tris-HCl buffer (pH 8.0). To each solution, I mg of NaB3H, was added in o.2-mg portions over a 24 h period. Excess NaBH, was destroyed by addition of acetic acid to pH 5 and the reduced polysaccharide was dialyzed in the cold for 24 h against six changes of distilled water. After this period of time the specific activity of the labeled polysaccharide was not changed by further dialysis. Control experiments established that the recovery of polysaccharide after dialysis was quantitative, and that prolonged treatment with NaBH, did not yield products with significantly higher specific activity. Samples of radioactive polysaccharide were prepared for ion-exchange chromatography by hydrolysis in I M HCl or 0.5 M H,SO, for 5 h at 100’. The acidic solutions were adjusted to pH 2.0 by use of Dowex 3-X4 (C032- form, 20-50 mesh) for the HCl hydrolysates and a saturated solution of Ba(OH), for the H,SO, hydrolysates. These solutions were then applied to columns (1.0 cm x 12 cm) of Dowex 50-X8 (H+ form, 200-400 mesh) which were eluted with water and subsequently with 0.5 M HCI. The aqueous fractions, which contained the neutral and anionic components, were concentrated, adjusted to pH 8 with NaOH and applied to columns (1.0 cm x 12 cm) of Dowex 1-X8 (Cl- form, 200-400 mesh). The neutral fraction was eluted with water and the anionic fraction with I M HCl. All eluates were lyophilized and the residues dissolved in a small quantity of water. Viscometry was performed as described previously5 in 0.2 M NaCl and the weight-average molecular weight, M,, was determined from a logarithmic plot of the limiting viscosity number [q] against Mw. Radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer (Model 3375) or in a Packard radiochromatogram scanner (Model 7201). The conditions for scintillation counting have been described previously6. Uranic acid was determined by the carbazole method of DISCHE’. Hexosamine was estimated by the modification of BOASTof the Elson-Morgan reaction with omission of the resin treatment. Descending paper chromatography was performed on Whatman No. I paper in the following solvents: A, I-butanol-pyridine-acetic acid-water (15: IO: 3: 12, by vol.) ; B, ethyl acetate-acetic acid-water (3 :I :I, by vol.). Compounds were visualized with ninhydrin or a AgNO, dip reagentg. RESCLTS

Molecular weight distribution after degradation

Treatment of hyaluronic acid with ascorbic acid at pH 3 caused a rapid decrease in 31, to about 3% of the initial value after 67 h (Table II). The molecular weight distribution in the degradation product may be estimated from the data in Table III which shows the results of fractionation on DEAE-cellulose according to a method described in detail elsewhere5. Briefly stated, the distribution function of molecular weight in each fraction may be assumed to follow a SCHULZ~Ofunction having M,/M,, = 1.25, where M, is the number-average molecular weight. This value is similar Biochim.

Biophys.

Acta,

192 (19%)

385-394

388

R.

TABLE

L. CLELAND

et al.

II

MOLECULAR

WEIGHT

OF HYALUROKIC

ACID

SAXPLES

calculated number-average molecular weight based on specific activity of the products and the assumption that one reducing end group per chain had reacted with XaB”H,. M, = wcightaverage molecular weight calculated from measurement of [v] (see text). ;I[,

=

Time* (h)

Speczfic activity* * (countslmin per icg wonic acid)

~lfolecular

AI a/.-lI n :If,

M t”

I 800 ooo

I

0

193

2

s

jS0

223 000 74 roe

3 4

19 67

835 1601

51500 26900

* Time of treatment with ascorbic * l Average of two experiments.

TABLE

WEIGHT

ASCORBIC

DISTRIBUTION

OF

THE

PRODUCTS

AFTER

TREATMENT

Recovered material (wt. %)

I

6.8 7.1 9.1 12.0 9.6

; 6

12

2.1

13

0.7

;;

1645 1595 1380 1300 1188 I300 1220

8.3

II

22

35 57

1800 1890

9.2

7.6 5.9

i&f,‘*

3630

IO.1

IO

ACID

HYALURONIC

67 h (Table I, Sample 4) was Schleicher and Schuell, 0.74 Samples (2 mg) of Fractions described in METHODS

Molecular

Specijk activity (countslmin per pg uranic acid)

11.6

L 9

OF

ACID

2

3

1.70 1.36 I.90

acid.

X 338.mg portion of hyaluronic acid degraded with ascorbic acid for fractionated into thirteen fractions on DEAE-cellulose (Cl- form, mequiv/g)5. The total recovery (weight basis) was 369 mg or rag %. I and 3-11 were reduced with NaBaH, and subsequently assayed as

Fraction

8

126000 70000 51000

III

MOLECULAR WITH

weight

100 119

I33 163 170 215

(nf,),,.

=

l

weight.

**

M,“,

II

850

7 400 II 200

23 22 26 26 3’ 32 36 33 35 (38 (40

900 750 ooo 950 150

17600 21600

24

100

250 100 250 000) 000) 800

l

**

25900 30000 35 39 47 49

8oo 2oo 7oo 3oo

61 400 (68

ooo)

(80

ooo)

23 000

* M, = calculated number-average molecular weight based on the specific activity of the products and the assumption that one reducing end group per chain had reacted with NaB”H,. ** M, = number-average molecular weight calculated from M, (obtained from [?I] with the assumption that Mw/M,, = 1.25 for fractions). *** The values in parenthesis were estimated for purposes of calculation of (Mn)av.. the average value of M, for the fractions. to

those

bovine

obtained vitreous

experimentally hyaluronic

for the unfractionated Biochim.

Biophys.

Acta,

for

acid

degradation 192

(1969)

385-394

fractions

from

a large-scale

fractionation

of

The distribution function product was assumed to be given by the weight-

by

the

same

method5.

DEGRADATION

OF HYALURONIC

ACID

389

average sum for the fractions, obtained by multiplying the distribution function for each fraction by its weight fraction in the recovered material. The resulting distribution is shown as the points in Fig. I. The solid line represents the theoretical distribution functionll for a product of random cleavage having the same M, as the average M, for the fractions, or M, = 40000, as calculated from [q] values for the fractions. This distribution is the so-called “most probable” distribution,

Fig. I. Molecular-weight distribution data for hyaluronic acid degraded by ascorbic acid. The quantity dw/dM represents the usual normalized differential distribution function. The points (filled circles) on the curve were calculated (see text) from the fractionation data in Table III. The solid line represents the Schultz distribution function for M,/Mn = z (“most-probable” distribution) and the dashed line that for M,/M,, = 312, both for a sample having M, = ~OOOO, the average value for the recovered fractions.

familiar in synthetic polymerization, given by the Schulz distribution for M,/Mn = 2. The dashed line represents the Schulz function for M,/M,, = 1.5. The points are seen to fit better the “most probable” distribution*. In addition the experimental value for M,/Aln was 40 000123 ooo = 1.74 for the recovered fractions, which agrees reasonably well with the expected value of 2. A value of 1.65 is obtained from the average of M,/M, values for the degraded samples in Table II. There appears to be some degradation of Sample 4 on fractionation, since M, of the initial material was 51 ooo and that of recovered fractions only 40 ooo. This degradation presumably broadens the distribution somewhat, so that the initial distribution may be somewhat sharper than that determined from the fractions. Nevertheless, we conclude on the basis of molecular weight distribution that the ascorbic acid degradation of hyaluronic acid proceeds essentially by a random cleavage mechanism. Kimtics

of the degradation

The kinetics of the degradation with ascorbic acid have traditionally been followed viscometrically. Analysis of the degradation as a random cleavage process, involving attack of a cleaving agent of essentially unchanging concentration on chain bonds, also at nearly constant concentration, shows that a plot of r/M, should be linear with timeli. Values of M, may be calculated4J2 from [q], and M, is proportional to M, under the assumption that M,/Mn is constant, as expected for the random cleavage indicated by our distribution analysis. When plotted as l/Mv, * -1 similar calculation with the assumption that fractions were very sharp (M,/M, = 1.05) fitted better the theoretical curve for M,/M, = 1.5. It seems unlikely, however, that this degree of sharpness would be obtained in a single fractionation. Biochim.

Biophys.

Acta,

192 (1969) 385-394

R. L. CLELAND

390

et al.

against time, the viscosity measurements all gave the sigmoidal pattern evident in the curves of Fig. 2 although these curves were not absolutely reproducible from one experiment to another. Since the variation of r/M,+, is decidedly non-linear, the data suggest that unmodified

ascorbic

acid may not be the cleaving

active species may arise during the reaction

agent, but that other

period.

I

ol 0

1

2

3

4

5

1O-3 t (mmutes) Fig. 2. Rate data for the ascorbic-acid degradation of rooster comb hyaluronic acid at the different 0, 0.3 ascorbic acid concentrations (mM) indicated for each curve: 0, 0.025 mM; 0, 0.1 mM; mM; 0, I mM; A, IO mM. Other experimental conditions were 0.01 M NaCl, pH 3.0 (&o.r), and 25’.

At longer times the rate d(r/M,)/dt appears to reach a maximum followed by a gradual decrease, at least for the lower ascorbic acid concentrations studied. Whether the rate actually drops to zero is not clear, even though this appears to be the case at 0.025 mM ascorbic acid, where the final value of M, was about 400000 (M, = 200000). It appears that no absolute limiting molecular weight is reached in the degradation process, but rather that increased concentrations of ascorbic acid yield products with correspondingly lower molecular weights and that the reaction is terminated presumably by depletion of the chain-cleaving agents. If this interpretation is correct, the efficiency of the reaction is low, since 0.025 mM ascorbic acid produces no more than 0.002 mM of new chains. Data at times longer than 2000 min were obtained also at ascorbic acid concentrations of I and IO mM, which indicated molecular weights below 30000, but the viscosity values were so near those of the solvent that the values were not considered quantitatively reliable. The previous indication that ascorbic acid is not the primary cleaving agent is supported by the observation that the degradation rates, d(r/M,)/dt, are not proportional to ascorbic acid concentration. This is especially true at times less than about 500 min. Reduction of degradation jwoducts with NaBH, NaBaH, reduction of undegraded hyaluronic acid and the degradation products obtained after 8, 19, and 67 h resulted in formation of radioactive products of increasing specific activity (Table II, Samples 1-4). These data indicate that the degradation proceeded with cleavage of glycosidic bonds and liberation of new reducing groups or formation of other groups sensitive to NaBH, reduction. If the incorporation Biochim.

Biofihys.

Acta,

Igz

(1969)

385-394

DEGRADATION

OF HYALURONIC

ACID

391

of radioactivity occurs exclusively into one reducing end group per molecule, Mn values of the reduced preparations may be calculated from their specific activities. Surprisingly, the undegraded hyaluronic acid (Sample I) incorporated about 2.5 times as much radioactivity as expected for the M,, value of 550 OOO, calculated from analysis of its molecular-weight distribution by DEAE-cellulose fractionation12. It appears that as much as 60% of the radioactivity in the undegraded hyaluronic acid may have been incorporated into positions other than reducing terminal groups or into impurities present in small amounts. The M, values calculated for the degraded Samples 2-4 were much closer to the M, values as shown in Table III. In fact, reasonably good agreement between the M, values estimated for the fractions of Sample 4 and those calculated from specific radioactivity is seen in Table III, particularly for the lower molecular weight fractions. The better agreement can be explained simply on the basis that the extra reducing groups present in the undegraded molecule influence the calculation of molecular weight much less, as a large number of new reducing end groups are formed during the degradation. If we assume that only one new group which is reduced by YaBH, is introduced by each cleavage due to ascorbic acid treatment, that there are 1.5 extra tritium-labeled groups per undegraded molecule, and that 20 degraded molecules result from each undegraded molecule, there should by only 21.5/20 = 1.08 tritium-labeled groups per degraded molecule. The extra reducing groups should therefore introduce only minor errors in the M, values for the degraded materials. The lower M, values calculated from radioactivity determinations for the fractions of higher molecular weight in Table III are quite possibly due to this effect, which should be more pronounced in the case of larger molecules. of reducing terminal end groups Reduction of reducing terminal glucuronic acid and N-acetylglucosamine moieties of hyaluronic acid with NaB3H, produces radioactive gulonic acid and N-acetylglucosaminitol end groups, respectively. Subsequent acid hydrolysis yields several labeled compounds including gulonic acid and its lactone, N-acetylglucosaminitol, and the reduced disaccharide, 2-acetamido-a-deoxy-3-0-(p-n-glucopyranosyluranic acid)-n-glucitol. The relative proportions of uranic acid and hexosamine end groups can be determined by chromatography of these compounds on ion-exchange resins. The radioactive polysaccharides which resulted from NaB3H, reduction of hyaluronic acid and the degradation products (Table II, Samples 1-4) were hydrolyzed in HCl or H,SO,. Labeled compounds present in these hydrolysates were separated by chromatography on Dowex 50 and Dowex I (Table IV). The distribution of radioactivity after chromatography revealed that a major portion of the radioactivity was present in the cationic fraction, while significantly less appeared in either the neutral or anionic fractions. Paper chromatography of the cationic fraction revealed that approx. 60% of the radioactivity migrated in Solvents A and B as the reduced disaccharide, 15% as free glucosaminitol, while the remainder of the radioactivity migrated more rapidly than glucosaminitol but as a single compound. The nature of this material has not been investigated further. The radioactive compounds present in the anionic and neutral fractions were identified as gulonic acid and &gulonolactone, respectively, Characterization

Biochim.

Bio$hys.

Acta,

Igz

(1969) 385-394

R. L. CLELAND

392 TABLE

IV

CHARACTERIZATION ACID

d d.

OF

LABELED

COMPOUNDS

OBTAINED

UPON

ACID

HYDROLYSIS

OF

HYALUROSIC

NaB3H,

PREPARATIONSAFTERREDUCTIONWITH

Samples of labeled hyaluronic acid corresponding to Samples 1-4 (see Table II) were subjected to hydrolysis in I IM HCl (series A) or 0.5 M H,SO, (series B) and fractionated by chromatography into a cationic, a neutral, and an anionic fraction as described in METHODS. Fraction

Radioactivity after hydrolysis

Distribution of radioactivity after chromatography on Dowex

Counfslmin

Cationic

Neutral

(%I

(%I

(%)

(%)**

66.0 64.8

18.9 19.8

‘5.1

x

10-b

(%)*

Radioactivity vecoveved after chromtograph

ilnionic

3-h 4-A

j.80 10.64

61

65.0 66.5

‘7.4

73

18.9

17.6 15.6

4’ 7’ 76 85

I-B Z-B 3-B 4-H

2.31 6.50 5.99 IO.77

50 61 62 74

jX.8

53.8 55.5 64.4

21.7 22.6 15.1 14.8

‘9.5 23.6 29.4 20.8

47 72 75 83

I-A

2.54 6.76

2-A

16.4

~_ * Percent of radioactivity in the unhydrolyzed polysaccharide. l * Percent of radioactivity in the deacidified hydrolysates.

by their co-migration

with authentic

compounds.

These data indicate

that cleavage

of hyaluronic acid occurs at both glucuronidic and hexosaminidic linkages and, furthermore, that the latter bonds are more susceptible to the action of ascorbic acid. In Table

IV, the recoveries

are also given. Although

of radioactivity

the hydrolysis

at the various

and subsequent

fractionation

stages

of analysis

of the products

is a multi-step procedure, it would seem that the losses of radioactivity were inordinately large, particularly in the analysis of the undegraded polysaccharide. For example, of the radioactivity present in the labeled polysaccharide fractions, only 50-74~~ was recovered in the deacidified hydrolysates. After chromatography of Fractions z-4, 71-85 y0 of the radioactivity applied to the columns was recovered in the labeled compounds, whereas 53 and 59% of the radioactivity present in the two hydrolysates of undegraded hyaluronic acid was lost. Thus, the overall recovery of radioactivity from the undegraded hyaluronic acid was only 23 or 24%, while approx. 45% was recovered from Fractions 2 and 3, and more than ho;/, from the most degraded material (Fraction 4). By comparison, a control sample of i3H]glucosaminitol which was hydrolyzed in HCl or H&SO,, deacidified, and subjected to the resin fractionation was recovered in 87-89% yield. One trivial explanation for the low recoveries would be incomplete destruction of NaBsH, upon acidification after reduction of the polysaccharide and the possible retention of minute quantities of radioactivity by the products during dialysis. Such a phenomenon could account, in part at least, for the anomalously high radioactivity in the undegraded control sample, but it will be recalled that dialysis for extended periods of time failed to significantly decrease the specific activity of the labeled polysaccharide. Although the cause of the observed discrepancies is not yet clear, the Biochim. Biophys. Acta, Igz (1969) 385-394

DEGRADATION OF HYALURONIC ACID fact that more than 607; was

recovered,

substantiates

acid upon hyaluronic

393

of the radioactive

products

our conclusions

regarding

in the most degraded

fraction

the mode of action of ascorbic

acid.

DISCUSSION Extensive

studies

of the

ascorbic

acid

degradation

of hyaluronic

acid

by

is dephysical-chemical methods 13-15 have clearly shown that the polysaccharide graded to products of lower molecular weight, although dialyzable products are not usually formed2*r6. No serious attempts have been made to characterize chemically the mode of cleavage. In a recent report

SWANN~ suggested

that

specific

linkages,

possibly

in the

protein moiety of the proteinpolysaccharide, are preferentially cleaved by ascorbic acid and that the degradation stops when the polymer fragments have a limiting viscosity number of 90-100 ml/g (corresponding to a molecular weight5 of approx. 35 ooo). The present results do not support the hypothesis of Swann. The fractionation of the degraded polymer shows that it has been randomly cleaved. The molecular weights from end-group determinations with NaBH, reduction of the fragments are in good agreement with the physico-chemically main action of ascorbic acid must therefore Hexosaminidic

linkages

determined molecular weights and the be the cleavage of glycosidic linkages.

seem to be more susceptible

than glucuronidic

linkages.

The

nonenzymic cleavage of 0-glucuronides” and nitrophenylglycosidesls by ascorbic acid has recently been shown and presumptive evidence for cleavage of hexosaminidic bonds exists in the case of ~-nitrophenyl-ili-acetyl-D-glucosaminidels. The kinetics of the degradation show a lag-phase before the maximal rate is obtained, and the rate is proportional to a power of ascorbic acid concentration less than one. This would suggest that ascorbic acid is transformed into or causes formation of a glycoside-cleaving that diketo-L-gulonic hyaluronic

reagent, which recalls the observation by NIEDERMEIER et a1.20, acid, which is formed from ascorbic acid, also degrades

acid.

ACKNOWLEDGEMENTS This project was supported by grants from the Swedish Medical Research Council (B-69-13X-4-o5A); the Swedish Cancer Society (53-B68-03XC); AB Pharmacia, the National Institutes of Health (AM-05996); and the American Heart Association. We gratefully ackowledge the technical assistance of Mrs. Lucila Penas and Miss Mary Lou Spach. REFERENCES I L. SUXDBLAD 2 3 4 5 6

AND E. A. BALAZS,in E. A. BALAZS AND R. W. JEANLOZ, The Amino Szlgars, Vol. IIB, Academic Press,New York, 1966, p. 229. D. A. SWANN, Biochem. J., IW (1967) 42C. XI. ABDEL-AKHER, J. K.HAMILTON AND F. SMITH, J.Am.Chem.Soc., 73 (1951) 4691. T. C. LAURENT, M. RYAN AND A. PIETRUSZKIEWICZ, Biochzm. Biophys. Acta, 42 (1960) 476. R.L. CLELAND,M.C.CLELAND,J.J.LIPSKYANDV.E.LYN, J.Am. Chem.Soc.,go(1968)3’41. X. C. STOOLMILLER AND A. DORFMAX, J. Bid. Chem., 244 (1969) 236. Biochim.

Biophys.

Ada,

192 (1969)

385-394

R. L. CLELAND et al.

394

7 Z. DISCHE, J. Biol. Chem., 167 (1947) 189. 8 N. F. BOAS, J. Biol. Chem., 204 (1953) 553. g I. SMITH, Chromatographic and Elactrofihoretic Techniques, Vol. I, Interscience Publishers, New York, 1960, p. 252. IO G. V. SCHULZ, Z. Physik. Chem., B43 (1939) 25. II C. TANFORD, Physical Chemistry of Macromolecules, John Wiley, New York, 1961, Chapter 9. 12 R. L. CLELAND, contribution to NATO Advanced Study Institute on The Chemistry and Molecular Biology of the Intercellular Matrix, 1969. 13 R. SKANSE AND L. SUNDBLAD, Acta Physiol. &and., 6 (1943) 37. 14 C. W. HALE, Biochem. J., 38 (1944) 362. r5 W. PIGMAN, S. RIZVI AND H. L. HOLLEY, Arthritis Rheumat., 4 (1961) 240. 16 W:. PIGMAN AND S. RIZVI, Biochem. Biofihys. Res. Comma&n., I (1959) 39. 17 Y. YAMANE, M. MIYAZAKI AND K. SAKAI, Yakugaku Zasshi, 87 (1968) 191. 18 J. C. CAYGILL, Biochim. Biophys. Acta, 170 (1968) I. xg J. KOJIMA AND J. W. HESS, Anal. Biochem., 23 (1968) 474. 20 W. NIEDERILIEIER, C. DOBSON AND R. P. LANEY, Biochim. Biophys. Acta, 141 (1967) 366.

Biochim.

Biophys. Acta, 192 (1969) 385-394