The Hunter Corrective Factor PURIFICATION AND PRELIMINARY CHARACTERIZATION

Abstract Fibroblasts from patients with the Hunter syndrome are deficient in a specific protein, designated "Hunter corrective factor," which is required for the degradation of sulfated mucopolysaccharide. This factor has now been purified 120-fold from normal human urine by (NH4)2SO4 fractionation, gel chromatography on Sephadex G-200, passage through anti-albumin Sepharose to remove albumin, a major contaminant, and finally, preparative polyacrylamide gel electrophoresis. The last procedure separates two "isofactors," which are probably charge isomers; both differ in charge from the Hunter factor derived from fibroblast secretions. The molecular weight of urinary Hunter factor is estimated at 65,000 by polyacrylamide gel electrophoresis and 114,000 by gel filtration. The most highly purified preparation of urinary Hunter factor shows a single protein component in polyacrylamide gel electrophoresis at pH 8, but can be resolved into several bands by isoelectric focusing in polyacrylamide gel. It is free of the common lysosomal glycosidases and sulfatases, as well as of factors effective in other mucopolysaccharidoses (Hurler, Scheie, Sanfilippo A and B, and Maroteaux-Lamy). The Hunter corrective factor accelerates the degradation, by Hunter fibroblasts, of dermatan sulfate labeled in the sulfate or galactosamine moieties, as well as of exogenously added proteodermatan [35S]sulfate. The effect of the factor persists in the recipient cells with a half-life of 2 days.


Fibroblasts
from patients with the Hunter syndrome are deficient in a specific protein, designated "Hunter corrective factor," which is required for the degradation of sulfated mucopolysaccharide.
This factor has now been purified 120-fold from normal human urine by (NH4)$04 fractionation, gel chromatography on Sephadex G-ZOO, passage through anti-albumin Sepharose to remove albumin, a major contaminant, and finally, preparative polyacrylamide gel electrophoresis.
The last procedure separates two "isofactors," which are probably charge isomers; both differ in charge from the Hunter factor derived from fibroblast secretions.
The molecular weight of urinary Hunter factor is estimated at 65,000 by polyacrylamide gel electrophoresis and 114,000 by gel filtration.
The most highly purified preparation of urinary Hunter factor shows a single protein component in polyacrylamide gel electrophoresis at pH 8, but can be resolved into several bands by isoelectric focusing in polyacrylamide gel. It is free of the common lysosomal glycosidases and sulfatases, as well as of factors effective in other mucopolysaccharidoses (Hurler, Scheie, Sanfilippo A and B, and Maroteaux-Lamy). The Hunter corrective factor accelerates the degradation, by Hunter fibroblasts, of dermatan sulfate labeled in the sulfate or galactosamine moieties, as well as of exogenously added proteodermatan [35S]sulfate. The effect of the factor persists in the recipient cells with a half-life of 2 days.
The Hunter syndrome (mucopolysaccharidosis II) is a genetic disorder of mucopolysaccharide metabolism, the only one of the mucopolysaccharidoses to be transmitted as an X-linked recessive (1). Its 3). Fragments of these mucopolysaccharides are also excreted in urine in unusually large amounts.
Fibroblasts derived from the skin of Hunter patients perpetuate the metabolic error in tissue culture and accumulate excessive dermatan sulfate (4,5). The accumulation and increased turnover time of dermatan sulfate can be ascribed to inadequate degradation (6).
The abnormal metabolism of sulfated mucopolysaccharide by Hunter fibroblasts can be corrected, i.e. changed toward normal, by adding to the culture medium a particular protein found in fibroblast secretions or in urine derived from individuals who do not have the Hunter syndrome (7,8). Thus the defect in the Hunter syndrome can be equated with a deficiency of this protein, designated the Hunter corrective factor, which is required for mucopolysaccharide degradation. This paper describes a procedure for the purification of the Hunter corrective factor from normal human urine, some investigations of its properties and function, and comparison with the Hunter corrective factor from fibroblast secretions (9). Similar studies have been reported for the analogous but distinct Hurler corrective factor (10) and Sanfilippo A corrective factor (11).
Goat antiserum against human serum albumin was a gift of Dr. J. Robbins, National Institute of Child Health and Human Development.
Cell Culture-Skin fibroblasts from normal individuals and from mucopolysaccharidosis patients were maintained in culture as previously described (6,7).
Assay of Corrective Factor Actitity---Addition of Hunter factor to the culture medium reduces intracellular accumulation of radioactive mucopolysaccharide in Hunter fibroblasts exposed to 35so4. Correction in Hunter fibroblasts follows the same kinetics as in Hurler (10) or Sanfilippo A (11) fibroblasts; the definition of a unit is analogous, one unit being the factor activity that gives 5456 by guest on March 23, 2020 http://www.jbc.org/ Downloaded from 5457 half-maximal correction in Hunter fibroblasts and is calculated maintained by means of circulating liquid coolant at 0"; gel as previously described (10). electrophoresis was carried out at a constant current of 25 ma Conditions for assay were less stringent than those specified (initial and final voltage were 150 and 250 volts, respectively). for the Hurler factor (10). Several fibroblast lines from well Elution buffer was pumped at a constant rate of 1 ml per min; diagnosed Hunter pat,ients were used, from 1 to 5 days after 2-ml fractions were collected with a Buchler refrigerated fraction subculturing, at cell densities ranging from 0.5 to 1.0 mg of cell collector at 0". protein per loo-mm Falcon Petri plate (denser or older cultures Isoelectric Focusing in Polyacrylamide Gel-The technique used were less sensitive to correction).
However, for any one experi-was that described by Doerr and Chrambach (18), with the folmerit one single subculture was used; where the experiment re-lowing modifications.
Gel solutions were deoxygenated with quired activity determinations at different times, the samples argon gassing. Total gel concentration was 3.5% T, crosswere stored frozen and assayed in one batch. linking = 2% C; gels contained lo/, Ampholine (p1 range 3 to 6) Preparatzon of Anti-albumin Xepharose-The immunoglobulin Gels were stained for protein (15) after removal of Ampholine by G fraction of 124 ml of goat antiserum against human serum diffusion in 12.5c/, trichloroacetic acid (19), while identical gels albumin was prepared by precipitation with 50% saturated am-from the same experiment were sliced transversely and eluted monium sulfate and DEAE-cellulose chromatography (12)) yield-with 0.02 M KC1 to determine the pH gradient. ing 5.1 g of purified IgG. The amount of specific antibody was Preparation of Partially Purijied Proteodermatan [%']&Ifourld to be 795 mg by quantitative immune precipitation (13). fate-Hunter fibroblasts, grown to confluence in 1410 cm2 Bellco The IgG fraction, 140 ml, containing 35 mg of protein and 5.4 roller bottles, were incubated with 150 ml of serum-free medium mg of specific antibody per ml, was dialyzed against 0.2 M sodium (7) containing 1 mCi of 35S04'. After 4 days the medium was citrate buffer, pH 6.0, and coupled to 140 ml of packed Sepharose concentrated in collodion bags (Carl Schleicher and Schuell) to 4-B by the method of Cuatrecasas (14). The Sepharose had been about 2 ml. After dialysis against 6 liters of 0.15 M NaCl, the activated with 300 mg of cyanogen bromide per ml of packed gel; concentrate was applied to a Sephadex G-200 column, 67 x 1.5 coupling was performed in 0.2 M sodium citrate buffer, pH 6.0. cm, equilibrated with 0.15 M NaCl, and eluted with the same salt The product was washed with 20 liters of 0.1 M NaHC03 over a concentration.
Most of the radioactive material emerged with period of 10 hours; only 10% of the protein was found in the the void volume; the peak fractions were pooled and concentrated washes, indicating that about 90% had been coupled.
in a collodion bag to 3.5 ml, containing 7 x lo6 cpm per ml of ra-To test its antigen-binding capacity, the anti-human serum dioactive mucopolysaccharide. The carbohydrate chains, over albumin Sepharose was equilibrated with 0.1 M sodium phosphate 95% dermatan sulfate by criteria of electrophoresis in 0.1 M buffer, pH 7.0, and poured into a Pasteur pipette to give a bed ZnSOd and in barbital buffer, pH 8.6,' are bound to protein (6). volume of 2 ml. A solution of human serum albumin in the same Assays of Lysosomal Enzymes-Aryl sulfatase A and B were buffer (2.28 mg, 0.5 ml) was passed through the small column at assayed by the method of Porter et al. (20); glycosidases were room temperature, at 12 ml per hour, and the absorbance of the assayed as previously described (10). eluate at 280 nm was monitored (A280 for an albumin solution of 1 mg per ml = 0.58). The capacity was found to be 0.69 RESULTS AND DISCUSSION mg of human serum albumin adsorbed per ml of packed Sepharose. Assuming a 2: 1 stoichiometry in the binding of antigen to Purification of Hunter Factor antibody, this corresponds to approximately 16% of the theoretical capacity.
Step 1. Ammonium Sulfate Fractionation-Freshly collected Analytical Polyacrylamide Gel Electrophoresis-The methodol-morning urine from healthy men in the military (110 liters) was ogy of Rodbard and Chrambach (15), and the buffer system saturated to 70% by addition of 450 g of ammonium sulfate per 1958.8 of Jovin et al. (16), operative at pH 7.95, O", were used as liter, and the precipitate was collected as previously described described previously (9); the sole modification was removal of (10). This and all subsequent steps were performed at O-4".
oxygen from the polymerization mixture (in an ice bath) by bub-The sticky precipitate was suspended in 500 ml of water, stirred bling of argon gas at 20 ml per min for 5 min through the mixture. on a magnetic stirrer for 1 hour, and the suspension centrifuged.
This procedure resulted in somewhat higher Kx values than the One ml of the supernatant fluid was removed for assay, while the previously used deaerat'ion procedure. remainder, 630 ml, was further fractionated by the addition of Preparative Polyacrylamide Gel Electrophoresis-The Buchler 155 g of ammonium sulfate (giving about 50% saturation), the Polyprep 100 apparatus was used as described (17). An elution pH being held at 6.0 by periodic additions of 0.5 M Na2HP04.
chamber of 1 mm width (one-half turn) was employed. After 1 hour, the precipitate was removed by centrifugation and Conditions for polymerization, electrophoresis, and the buffer 147 g of ammonium sulfate were added to 700 ml of the supernasyst,em (1958.8) were the same as used for the analytical scale, tant fluid (ca. 807, saturation), the pH again being maintained with the following modifications. The "Lower Buffer" was made at 6.0. The precipitate was collected by centrifugation after 1 5 times concentrated; 1 liter was recirculated at a rate of 3.9 ml hour and redissolved in a minimal amount (112 ml) of 0.01 M per min. "Upper Buffer," 1 liter, was recirculated at the same sodium phosphate buffer, pH 6.0, in 0.15 M NaCl ("50 to 80% rate. "Elution Buffer" was 0.132 M in 4-picoline, titrated to pH fraction"). Recovery in this and all subsequent steps is listed 6.8 with HCl, and 25(-; (w/v) in sucrose.
in Table I Total gel concentration in the "Separation Gel" was 5% T, Step d. Chromatography on Sephadex G-dOO-The 50 to 80% cross-linking = 2ol, C; the volume of the gel was 40 ml (surface ammonium sulfate fraction was dialyzed overnight against two area = 10 cm2, height = 4 cm). The "Stacking Gel" was 3.12% changes, 6 liters each, of 0.5 M NaCl in 0.01 M sodium phosphate, T, 2Oo/; C; the volume was 12 ml. Deoxygenation was per-pH 6.0, and subjected to filtration on Sephadex G-200. As seen formed by argon gassing, as above; photopolymerization was car-in Fig. 1, top, the Hunter factor is eluted in a peak located at the ried out for 60 min. ascending limb of a large protein peak. The latter contains The temperature in the gel and elution buffer reservoir was 1 J. Derge and E. F. Neufeld, unpublished experiments. Top, 50 to 80% ammonium sulfate fraction, 110 ml, Azso = 32; bottom, Fractions 180 to 210 of above pooled, concentrated to 62 ml, and re-applied to the same column.
predominantly albumin, as determined by electrophoresis and immunodiffusion.
The fractions with the highest specific activity, 180 to 210, were pooled and concentrated to a volume of 62 ml by ultrafiltration on a Diaflo PM-30 membrane in an Amicon 400 ultrafiltration cell (50 p.s.i. pressure from a nitrogen tank over a period of about 4 hours at 4"). The concentrate was rechromatographed on Sephadex G-200 (Fig. 1, bottom); fractions with the highest specific activity, 170 to 210, were pooled and concentrated as above Step S. Removal of Albumin on Anti-albumin Sepharose-Attempts to remove residual albumin by conventional techniques failed because albumin accompanied Hunter factor in all procedures performed at pH 6.0 or higher (chromatography on DEAEcellulose or hydroxylapat,ite, or electrophoresis in polyacrylamide gel), whereas Hunter factor activity could not be preserved in procedures requiring more acidic pH. Removal of albumin by affinity chromatography on anti-albumin Sepharose proved an adequate solution to this problem.
The concentrate from the second gel filtration column was dialyzed overnight against two changes, 500 ml each, of 0.1 M sodium phosphate, pH 7.0. It contained 54 mg of albumin, as estimated by passage of an aliquot over a small antibody-sepharose column.
A preparative scale affinity column was made by packing 105 ml of settled anti-albumin Sepharose, with a binding capacity for 72 mg of albumin, into a column (2 x 33.5 cm) which was then equilibrated with 0.1 M sodium phosphate buffer, pH 7.0. The G-200 concentrate was passed through this column at room temperature, at 30 ml per hour, and fractions of 5.4 ml were COIlected. Fractions with an absorbance at 280 nm greater than 0.1 were pooled and concentrated by ultrafiltration, as above, to a volume of 6.5 ml. From the absorbance of the eluate, it could be calculated that 55 mg of albumin had been adsorbed.
Complete removal of albumin was demonstrated by immunoelectrophoresis by means of the procedure of Scheidegger (21). The material applied to the anti-albumin Sepharose column exhibited a strong precipitin arc, while the eluate showed none, even at twice the protein concentration.
Step 4. Preparative Polyacrylamide Gel Electrophoresis-The gel filtration step had provided fractionation based on molecular size. Gel electrophoresis was therefore applied at a relatively nonrestrictive pore size, which provided separation based predominantly on net charge. The selected pH of 8.0 was as low as compatible with recovery of activity.
At this pH and at a gel concentration of 5% T the factor mobility was high (RF = 0.8 to 0.9), separation from neighboring bands was adequate, and short fractionation times could be expected to yield increased resolution due to reduced diffusion. Fig. 2 shows that under these conditions Hunter factor activity is separated into two "isofactors," characterized by RF values of 0.91 and 0.76. These isofactors are probably charge isomers, Concentrated eluate from the anti-albumin Sepharose column (6.0 ml, A280 = 15.7,40,000 Hunter factor units per ml) was dialyzed against "Upper Buffer"; 1.5 g of solid sucrose and 0.1 ml of 0.1% bromphe-no1 blue were added and the sample was layered on top of the gel column. Rr was calculated on the basis of elution time. The dye front first appeared in the eluate 240 min after its entrance into the separation gel. The first fraction containing the dye is designated as 1, and corresponds to an RF of 1.0; 120 fractions were collected, the last being eluted after 480 min, corresponding to an RF of 0.5. Aliquots of 10 ~1 were assayed for Hunter factor activity.
since molecular sieving was relatively ineffective under the electrophoretic conditions used; moreover, upon gel filtration on Sephadex, Hunter factor activity appeared homogeneous.
The eluate was reanalyzed in the same electrophoretic system (at 7.5% T) with the results shown in Fig. 3. The activity profile of the faster migrating isofactor is coincident with two broad protein bands (RF, 0.64 to 0.70 and 0.49 to 0.56). Comparison of the relative stain intensities of these bands with the corrective actor activity of the fractions from which they are derived (Fig.   5459 2) shows that the faster of these two bands cannot be the one responsible for the activity.
The peak of the more slowly migrating isofactor activity is coincident with a single band of protein (RF = 0.44; 7.5% T).2 The coincidence suggested that the protein of RF = 0.44 (Pool 4) might be the Hunter factor. This proposition was tested by analytical polyacrylamide gel electrophoresis of Pool 4 at various gel concentrations.
The resulting parameters descriptive of molecular size (Kn) and net charge (Y,) for the Hunter factor activity and the single protein band are listed in Table II, lines A  and B, respectively Although both parameters differ slightly, statistical analysis of the joint 95% confidence limits of KR and Yo revealed that the two curves were not significantly different, i.e. that the Hunter factor activity is indistinguishable from the protein band in the buffer system employed.a However, gel electrophoretic analysis at a single pH does not appear sufficient evidence for a claim to isolation of electrophoretically homogeneous Hunter factor, particularly since analytical gel electrophoresis was carried out in the same buffer system as preparative fractionation.
In view of the fact that the factor is poorly soluble and readily inactivated below pH 5, a meaningful variation of fractionation pH values was not possible. Therefore, isoelectric focusing in polyacrylamide gel was carried out on the most purified material as an additional test for homogeneity.
It was found that Pool 4 could be fractionated into 5 species (with apparent p1 values of 3.00, 4.10 (major), 4.50 (ma-2 Fig. 3 shows that RF values, with the exception of those for Fractions 30 to 40, are not characteristic constants (15) for each component, but appear to vary in regular fashion as a function of elution volume.
Such variation has been previously observed in preparative electrophoresis (R. A. Yadley and A. Chrambach, manuscript in preparation) and is ascribed to the fusion of two adjacent band distributions in different proportions. 3 KR and YO values for fibroblast factor in line F in Table II differ from those in line E, because deoxygenation of the gel solution was achieved by evacuation in the first instance and argon gassing in the second (see "Materials and Methods").
Since the KR values of urinary and fibroblast factor are indistinguishable when measured under the same conditions (lines C and D, respectively) the two factors must be of the same molecular size. Aliquots of 100 ~1 of the designated fractions from the preparation of Fig. 2 were applied to each gel. The gels were stained for protein with Coomassie blue, and RF values of bands were determined.

II
Parameters descriptive of molecular size (K R, Kay) and of net charge (Yo) of Hunter factor preparations Ten rg of protein, or 20 to 30 units of Hunter factor activity were subjected to electrophoresis at various polyacrylamide gel concentrations.
Gels were stained for protein, or sliced and assayed for activity (9); RF values were determined and Kz and Yo values calculated as described (15 Hunter corrective factor activity could not be recovered from the gel, and it is not known to which of these bands it corresponds. Nonetheless, these results indicate that Hunter factor has not yet been purified to homogeneity.

Molecular Size and Net Clbarge of Hunter Corrective Factor
The physical parameters determined by polyacrylamide gel electrophoresis and by gel filtration for the urinary Hunter factor activity are listed in Table II and compared with the corresponding values for the preparation derived from fibroblasts. The activity of Pool 4 and that of crude urinary concentrate from Step 1 are indistinguishable with regard to mclecular size (RR) from the factor derived from fibroblast cultures (lines A, C, and E, respectively).
The molecular weight of fibroblast Hunter factor had been estimated in earlier experiments (9) at 65,000, with 95yc confidence limits between 32,000 and 115,000 (line F in Table II) . 3 The free mobility (Y,J appears similar for crude urinary factor (line C) and the purified activity of Pool 4 (line A), whereas Hunter factor in fibroblast secretions appears to be a more negatively charged species (line E).
These conclusions, based on Ka and Yo values and their standard deviations, are supported by a more rigorous t,reatment, the consideration of joint 95yG confidence limits for KR and Y0.4 The observed difference in free mobility, Yo, between Hunter factor from fibroblast secretions and from urine may be due to tissue-specific differences.
In addition, the urinary factor itself is heterogeneous, as shown by electrophoretic resolution of isofactors.
Since the factor preparation from fibroblasts was derived from cells of one individual, whereas that of the urinary factor was derived from a pooled population, genetic polymorphism is a possible cause for the heterogeneity.
However, secondary alterations of the factor after release from the cell, or during purification, are at present equally plausible explanations. 4 Dr. David Rodbard provided unpublished methods and computer programs for testing the identity of proteins on the basis of joint, 95y0 confidence envelopes of Kn and Yo, for calculating confidence limits of molecular weight from gel filtration data, and for optimization of conditions in preparative gel electrophoresis.
Gel filtration of the urinary Hunter factor at either 0.01 or 0.5 ionic strength, pH 6.0, gives a higher molecular size estimate than polyacrylamide gel electrophoresis at pH 8.0 and 0.015 ionic strength (114,000, with 95yc confidence limits of 77,000 and 170,0004; line D in Table II).
The reasons for this discrepancy are unknown.
In contrast to the Hurler correct,ive factor (lo), the Hunter corrective factor shows no aggregation at low ionic strength.

Spec$city
Purified Hunter factor has no effect on fibroblasts of individuals with other mucopolysaccharidoses. Fifty units of Hunter factor applied to cells of the Hurler, Scheie, Sanfilippo A and B, and Maroteaux-Lamy genotypes revealed less than 0.5 unit toward any of these. The Hunter factor has no effect on sulfated mucopolysaccharide accumulation of normal cells. The factor was equally effective on fibroblasts of patients with the mild (adult) or severe (juvenile) form of the Hunter syndrome (22).

Duration of Corrective Effect
When Hunter factor is removed from the culture medium, its effect persists with a half-life of 2 days (Fig. 4), a figure conparable to that found for Sanfilippo -1 factor, 2 days (II), but shorter than that for Hurler factor, 9 days (10). Fig. 4 shows no significant difference in the persistence of corrective activity of the two urinary isofactors.

Function of Hunter Corrective Factor
Acceleration of sulfated mucopolyeaccharide degradation by Hunter factor has been documented previously (7). It was shown that addition of the factor to Hunter fibroblasts not only reduces the accumulation of intracellular [Y3]mucopolysaccharide, but also reduces its turnover time.
The increment metabolized in the presence of fact,or can be quantitatively recovered in the medium as dialyzable fragments.
Further evidence has now been obtained with proteodermatan [35S]sulfate supplied exogenously.
As seen in Table III, the substrate is degraded to a much greater extent in normal cells than in Hunter cells. The addition of factor to Hunter cells by guest on March 23, 2020 http://www.jbc.org/ increases the breakdown to a normal level; control experiments showed that it has no effect on normal cells. The ethanol-soluble breakdown product behaves as inorganic sulfate on Bio-Gel P-2.
The degradation requires entry of the proteodermatan sulfate 0.  into the cells, since it is not produced by medium previously incubated with normal or Hunter cells. An effect of Hunter factor on the uptake (pinocytosis) of the substrate is excluded by the data shown in Table III. Incubation of the substrate with Hunter factor (followed by boiling to inactivate the factor) did not render the substrate more degradable by Hunter fibroblasts.
Full restoration of the degradative process by the factor is confirmed by examining the effect of the factor on mucopoly saccharide labeled in the hexosamine moiety.
Cells were incubated with [6-3H]glucosamine for 3 days, after which radioactive medium was replaced by unlabeled.
The presence of factor in the chase medium caused the disappearance of two peaks of radioactive material, as demcnstrated by chromatography on Sephadex G-200 (Fig. 5). These correspond in elution position to two peaks of [35S] mucopolysaccharide, the chase of which is likewise accelerated by Hunter factor. The major peak (Fractions 35 to 48) has been identified as containing primarily dermatan sulfate by the criteria of chromatography on DEAE-Sephadex, electrophoresis in 0.1 M ZnS04, and hydrolysis followed by co-chromatography of the radioactive hexosamine with D-gak&OSamkE on Dowex 50 (23). The Hunter corrective factor, therefore, assists Hunter cells in degrading ciermatan sulfate of exogenous or endogenous origin. This suggests that the factor may be a catabolic enzyme.
If so, it must be distinct from the common lysosomal enzymes, since there was no detectable activity toward the p-nitrophenyl 5462 derivatives of a-and P-u-galactose, OL-and P-n-glucose, cr-n-mannose, a-n-fucose, N-acetyl-fl-n-glucosamine and N-acetyl-p-ngalactosamine.
Nor was there detectable aryl sulfatase A or B activity.
For these experiments, 9 units of the most purified preparation, Pool 4, were used. The absence of detectable p-nitrophenyl-fi-n-galactosidase activity in purified Hunter corrective factor, as well as in purified Hurler and Sanfilippo A corrective factors (10,11) should lay to rest the once popular hypothesis (e.g. 24-27) that a deficiency of that enzyme is the primary defect of the three mucopolysaccharidoses.
Enzymatic activity related to mucopolysaccharide degradation has been attributed to corrective factors in three instances.
The corrective factor for Sanfilippo A fibroblasts removes sulfate from the mucopolysaccharide stored by these cells, probably heparan sulfate with a high N-sulfate content (11). The corrective factor for cells from a patient with an atypical mucopolysaccharidosis (28) appears to be /3-glucuronidase.5 The factor corrective for Hurler and Scheie fibroblasts has been identified as cw-n-iduronidase (29). Prolonged incubation of Hunter factor with dermatan sulfate isolated from Hunter cells (labeled in the sulfate, uranic acid, or galactosamine moieties) has so far failed to reveal enzymatic activity of the factor.
Such negative results must be interpreted with caution, since incubation conditions may have been inappropriate.
One must not, however, ignore the possibility that Hunter factor affects mucopolysaccharide degradation by some indirect mechanism.