ANALYTICAL BIOCHEMISTRY 96, 474-480 (1979) A Specific, Sensitive Method for the Determination of Hyaluronatel GEORGE W. JOURDIAN, MELINDA WOLFMAN, ROBERT SARBER,* AND JACK DISTLER Rackham Arthritis Research Unit, and the Departments of Biological Chemistry and Internal Medicine. The University of Michigan Medical School, Ann Arbor, Michigan 48109 Received November 29, 1978 A sensitive and specific assay for hyaluronate was devised. Hyaluronate contained in biological mixtures was digested with a commercially available microbial hyaluronate lyase. The P-A4,5-eneglucopyranuronic acid residues contained at the nonreducing termini of the resulting oligosaccharides were oxidized with periodic acid to yield, among other products, formyl pyruvic acid. The latter compound reacted with thiobarbituric acid to yield a chromo- phore with an absorption maximum at 549 nm. Optimal conditions for quantitative assay of hyaluronate are described. At present there is no simple method for glycans by density gradient centrifugation, the rapid and specific detection and quanti- column chromatography on agarose, and tation of microamounts of hyaluronate. determination of the proteoglycan-hyaluro- Classically, the concentrations of hyaluro- nate complex elution profile by measure- nate in biological solutions have been deter- ment of uranic acid concentration (7). mined by turbidimetric methods (I), or by An alternative approach, utilized by a quantitative determination of its constituents number of investigators for the determina- after acid hydrolysis (2). However, these tion of hyaluronate, has been the application procedures are not specific for the deter- of microbial hyaluronate lyases. In contrast mination of hyaluronate. Other methods to animal hyaluronidases (hyaluronate depend upon isolation and chemical char- glycanohydrolase EC 3.2.1.35), the micro- acterization and involve ion-exchange bial hyaluronate lyases (hyaluronate lyase chromatography (3), cetylpyridinium chlo- EC 4.2.2.1) are eliminases and hydrolyze ride-cellulose microcolumns (49, and hyaluronate to oligosaccharides containing cellulose acetate electrophoresis (6). While terminal A4,Sunsaturated glucopyranuronic generally satisfactory, these methods are acid3 units (8); the unsaturated compounds time-consuming and are compromised if have a characteristic absorption maximum a large excess of other glycosaminoglycans at 232 nm. This property served as the basis are present in the sample. A highly sensitive for the development of direct spectrophoto- and specific proteoglycan-binding assay for metric means for the measurement of hy- hyaluronate contained in mixtures of gly- aluronate. However, biological fluids and cosaminoglycans has been reported but tissue extracts usually contain high levels requires prior isolation of cartilage proteo- of ultraviolet-absorbing compounds such as protein and nucleic acids which interfere ’ This work was supported by Grant HL 19685 with the quantitative measurement of the from the National Institute of Heart and Lung Diseases, National Institutes of Health, and in part by the Arthri- A4,5-unsaturated uranic acid residues by tis Foundation, Michigan Chapter. * Postdoctoral trainee, Training Grant AM 07080 a A4,SUnsaturated glucopyranuronic acid oligo- from the National Institute of Arthritis, Metabolic saccharides = (3-O-P-A4,5-ene-D-glucopyranuronic and Digestive Diseases, National Institutes of Health. acid-2-acetamido-2-deoxy-D-glucose),,. OO03-2697/79/10O474-07$0200/O 474 Copyright 8 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. SPECIFIC SENSITIVE METHOD FOR HYALURONATE DETERMINATION 475 this methodology (9). The latter problem standards and were generous gifts of Dr. has in part been circumvented by colori- M. B. Mathews, University of Chicago: metric measurement of liberated N-acetyl- hyaluronate (human umbilical cord), chon- glucosamine end groups by a modification droitin 4-sulfate (sturgeon cranial cartilage), of the Morgan-Elson reaction (10). How- chondroitin 6-sulfate (sturgeon notochord), ever, crude tissue extracts produce spurious dermatan sulfate (hog mucosa), keratan color and the calorimetric determination sulfate (bovine cornea), and heparin and is affected by the presence of even moderate heparan sulfate (beef lung). Hyaluronate protein concentrations (2). The enzymatic was also prepared from human synovial determination of hyaluronate in biological fluid by electrodeposition (15). Chondroitin fluids is further limited by the observation was prepared from chondroitin 4-sulfate that heparin, chondroitin 4-sulfate, and by solvolytic desulfation with dimethyl dermatan sulfate competitively inhibit some sulfoxide (16). hyaluronate lyases (2). Ohya and Kaneko Hyaluronate lyase. Hyaluronate lyase (11) have isolated a novel hyaluronate lyase purified from Streptomyces hyalurolyticus from Streptomyces hyalurolyticus which (specific activity 3000 turbidity reducing is reported not to be inhibited by glycos- units (TRU)/mg protein)4 was obtained aminoglycans. The enzyme is specific for from Calbiochem. hyaluronate, catalyzes the formation of Reagents. The following compounds oligosaccharides terminating in A4,5-unsat- were obtained from the indicated sources: urated glucuronic acid units, and is stable at sodium periodate and n-butyl alcohol, J. T. elevated temperatures. Baker Chemical Company; sodium arsenite Hascall et al. (12) observed that a perio- and p-dimethylaminobenzaldehyde, Baker date-thiobarbituric acid reagent reacted and Adamson; ammonium dihydrogen ortho- with A4,Sunsaturated uranic acid-terminat- phosphate, BDH Chemicals Ltd.; and thio- ing oligosaccharide chains (found on treat- barbituric acid, Eastman Chemical Com- ment of bovine nasal cartilage proteoglycan pany. All other reagents were of analytical with microbial chondroitinases) to yield a grade or of the highest purity commercially chromophore with an absorption at 549 nm. available. Dialysis tubing with a molecular This observation served as the basis for weight cutoff of 3500 was purchased from development of a convenient assay for the Arthur H. Thomas Company. degradation of bovine nasal proteoglycan by chondroitinase ABC and AC activities. METHODS Koseki et al. (13) applied a similar periodate- thiobarbituric acid assay procedure, de- Standard Assay Procedure scribed by Warren (14), to the microdeter- Incubation with hyaluronate lyase. Reac- mination of chondroitin 4- and 6-sulfate tions were performed in 15 x 125-mm Pyrex and dermatan sulfate after prior digestion tubes. Enzymatic digestions contained the with microbial chondroitinases. following components in a total volume of The above observations served as the 0.2 ml: sodium acetate buffer, pH 5.0, 4.0 basis for development of a specific and pmol; 1.66 pg hyaluronate Iyase (5 TRU); sensitive enzymatic-calorimetric assay and 0 to 100 pg of sodium hyaluronate. for hyaluronate described in this manuscript. Control tubes contained the same compo- nents but lacked either substrate or enzyme. MATERIALS The reaction mixtures were incubated at Glycosaminoglycans. The following a Enzyme activity is expressed in turbidity units glycosaminoglycans served as reference (TRW as defined by Tolksdorf er al. (17). 416 JOURDIAN ET AL. 60°C for 5 h and enzyme action was termi- 1.6t 4 nated by the addition of 0.25 ml of 0.04 M NaIO, contained in 0.08 N H.&SO,. Measurement of eliminase products. The conditions used were similar to those described by Hascall et al. (12). After addi- tion of the periodate-H,SO, reagent the reactants were mixed and the tubes placed in a 37°C water bath for 1 h to allow oxida- tion to proceed. Excess periodate was destroyed by addition of 0.5 ml of 3% (w/v) NaAsO, contained in 0.5 N HCl. The react- ants were mixed, allowed to stand at room temperature for 30 min, and 4 ml of 0.3% HOURS thiobarbituric acid contained in 0.012 N FIG. 1. The formation of periodate-thiobarbituric HCl were added to each tube. The contents acid reactive compounds from hyaluronate as a func- were again mixed thoroughly and the tubes tion of time of incubation with Strepromyces hyaluro- capped with marbles, heated at 100°C for nate lyase: effect of substrate concentration. The 15 min, and cooled to room temperature. incubation conditions were those described for the standard assay procedure except the hyaluronate con- In contrast to the procedure of Hascall centrations (expressed in &incubation) were as et al. (12) we routinely extracted the chro- follows: (A) 10, (0) 20, (A) 35, (0) 50, (X) 75, and mophore with 4 ml of 5% HCl in n-butyl (cl) loo. alcohol to enhance the sensitivity of the assay procedure. Absorbance of the butanol hyaluronate lyase in each incubation. Lower layer was determined at 549 nm. amounts of hyaluronate lyase, i.e., less than 3 TRU per incubation, resulted in sig- RESULTS AND DISCUSSION nificantly lower initial rates and decreased Enzyme Incubation Conditions product formation (Fig. 2). Variation of absorbance values on a day-to-day basis Ohya and Kaneko (11) previously estab- using the same standard hyaluronate solu- lished that Streptomyces hyaluronate lyase tion was within 25%. is stable over a wide pH and temperature range; the presently described assay pro- Enzyme Specificity cedure was conducted at the established pH and temperature optima, pH 5.0 and Ohya and Kaneko (11) reported that 6O”C, respectively. Routinely, the enzyme Streptomyces hyaluronate lyase specifically was dissolved in 0.04 M sodium acetate catalyzes the hydrolysis of hyaluronate and,