Crystallization and partial characterization of Staphylococcus aureus hyaluronate lyase

ARCHIVES

OF

RIOCHEMISTRY

Crystallization

AND

BIOPHYSICS

and

Partial aureus

GOPAL l’he PentrsylvaGa

687-694

168,

(1973)

Characterization Hyaluronate

S. RAUTELAZ

State University,

AND

Lyase’ CARL

Ogontz Campus,

Received

of Staphylococcus

ABRAMSOW A bington,

Pennsylvania

19001

June 6, 1973

Staphylococcus aureus, strain 1801, hyaluronate lyase WRSpurified and crystallized to homogeneity as ascertained by chromatography and disc-gel electrophoresis. Purification procedures included sequential ammonium sulfate fractionation, acetone precipitation, Sephadex gel filtration, and ion exchange chromatography. During its passage through the cation exchange column, the hyaluronate lyase was resolved into two minor and one major fraction. The major peak, which was found to be cationic, was further characterized and designated as Fraction III. Carbohydrate analysis showed the presence of neutral sugars galactose, glucose, and mannose in the ratio of 1:3:6. Amino sugars galactosamine and glucosamine (or mannosamine) were present in a ratio of 1: 1. Quantitative amino acid analysis of the Fraction III showed a relative abundance of the basic amino acids lysine and histidine.

Hyaluronate lyase (EC 4.2.99.1) of Staphylococcus aureus as well as other bacteria has been purified only to a limited extent (l-4), and very few of the physicochemical properties of the enzyme are known (5). The heterogeneity of hyaluronate Iyase has been reported but not substantiated. Thus, the presence of two (l), three (2,5), and four (G-8), or more (9) isoenzymes has been reported. However, these isoenzymes have been demonstrated in crude preparations and have not been characterized as true multiple molecular forms. Complete purification of S. aureus hyaluronat’c lyase to crystallization is described. Results suggest three fractions, but evidence does not substantiate them as isoenzymes. The major fraction is partially charact)erizcd as a protein. EXPERIMENTAL

PROCEDURE

Enzyme assay. For rapid screening purposes the hyaluronate lyase activity was measured by the 1 This investigation was supported in part by Nat,ional Science Foundation Grants GB-6441 and GB-12896. 2 Present address: DuPont Laboratories, Clinical Division, Wilmington, Delaware. 3 Present address: Pennsylvania College of Podiatric Medicine, Philadelphia, Pa. 19107.

d Abbreviations: prevention method; unit; NIX, national phosphate buffer. 687

Copyright .\!I rights

0 1973 Iv hcarienric Press,
Inc. reserved.

mucin-clot prevention (MCP)” method of Harris and Harris (10). The substrate, potassium hyahronate was prepared from human umbilical cords (3). All other enzymatic determinations including those employed for specific activity measurements were performed using the turbidity reducing unit, method (TRU) (3, 11) with a single modification. Four milliliters of albumin reagent instead of nine were used (3). The purified hyaluronate used as a substrate for this procedure was obtained from Worthington Biochemical Corporation. A straight line relationship between subs1 rate concentration and turbidity (expressed as Klett units) was not observed, and an experimental standard curve like the one shown in Fig. 1 was used. A standard enzyme curve (Fig. 2) was also prepared 1,) plotting turbidity reducing units as a function of national formulary units (NFU). The hyaluronat e lyase standard was obtained from National Formulary Standards, Washington, DC. The enzyme: activity was expressed in NFU. Saline phosphate buffer (pH 5.27, I = 0.15), which was found earlier to be optimal for enzyme activity (12), was used throughout the course of this investigation fog enzyme assay. Protein determination. The protein ronrent ra tion of effluent from various columns was montorcd by measuring the absorbance at 280 nm MCP method, mucin-clot TRU, turbidity reducing formulary unit; SPB, salinr

688

RAUTELA

AND ABRAMSON

80 KLETT UNITS

SO

HYALURONATE

(mgl

FIG. 1. Standard substrate curve showing the relationship acid and absorbance (turbidity) measured in Klett units. in a Beckman DU Spectrophotometer. Actual determinations of protein concentration were made by the method of Lowry et al. (13). Enzyme p&cation. All purification procedures were carried at 4°C. The enzyme preparation was concentrated by dialyzing against dry Sephadex G-200 or by pervaporation using an electric fan. Crude enzyme in the form of brown lyophilized powder, prepared by Worthington Biochemical Corporation, was extracted with water (10 g with 1 liter water). By sequential ammonium sulfate fractionation, a precipitate containing most of the enzyme activity was obtained between 0.8 and 1.0% salt saturation. The precipitate was dissolved in minimum quantity of saline phosphate buffer (SPB) (pH 5.27, Z = 0.15). The enzyme was eluted through a Sephadex G-25 Fine column (2.5 X 40.0 cm), in a descending mode, with a flow rate of 45 ml/hr. The column was equilibrated and eluted with SPB. The ammonium sulfate in the effluent was monitored by barium chloride solution. The fractions containing enzyme were pooled and concentrated. The enzyme was then precipitated by adding acetone to a final concentration of 75%. The precipitate was collected by centrifugation and dried to a powder in vacua in a desiccator. Temperature throughout the above procedure was maintained below -8°C. Further purification was achieved by using Sephadex G-100 columns (2.5 X 28.0 cm) equipped with flow adaptors. Saline phosphate buffer was used to dissolve acetone powder, equilibrate, and elute the column. At specified times, 0.2 g acetone powder in 7.5 ml SPB was injected automatically into the bottom of each column. The elution was carried out in an ascending direction at a flow rate of 18 ml/hr. The fractions showing enzyme activity were pooled and concentrated. Prior to ion exchange chromatography, the above preparation was desalted by a Sephadex

between concentration

I 6.0

I

I

I

of hyaluronic

I

I

-

I

( -

./-• 5.0

. /’

TR 40 UNITS

-

3.0

.

-

0

r’

/

20

I 40

I 60 N F

I so

I IO

I 12

UNITS

FIG. 2. Standard enzyme curve showing relationship between national formulary units (N.F.) and turbidity reducing units (T.R.). G-25 Fine column (2.5 X 35.0 cm). The column was equilibrated and eluted with sodium phosphate buffer (pH 5.77, I = 0.07). Effluent was monitored for NaCl by a AgNOa solution. Purified enzyme preparation was then subjected to CMSephadex 50 (a cation exchanger) column (2.5 X 27.0 cm), equilibrated with the sodium phosphate buffer (pH 5.77, I = 0.07). The column was eluted with a linear gradient of sodium phosphate buffer. The first system consisted of the buffer with a pH of 5.77 and I = 0.07, and the second system of the buffer with a pH 6.27 and Z = 0.3. Three hyaluronate lyase fractions I, II, III showing distinr.t activities were pooled separately. Fraction III, most cationic peak containing most of the enzyme activity, was further purified by repeated chromatography on a CM Sephadex 50 column identical to the one previously described. The column was eluted with a linear gradient of sodium phosphate buffer. The first

CRYSTALLlZATION

S. aureus

system consisted of the buffer with a pH of 5.77 and Z = 0.1, and the second system of the buffer with a pH of 6.27 and Z = 0.3. CrystaZlization. The purified enzyme preparation was enclosed in a dialysis tubing and was allowed to evaporate slowly until the volume was reduced by about 90%. The pervaporation was facilitated by gently blowing a continuous stream of air from an electric fan. Polyacrylamide disc electrophoresis. The enzyme preparations obtained during the successive pt:rificat,ion stages were subjected to polyacrylamide gel electrophoresis according to the procedure of Davis (14). Trisglycine buffer (PH 9.5), gel concentration of 7.5%, and 4 mA current/tube for 1 hr were used. Separated protein bands were stained with aniline blue black in 7% acetic acid, with the latt,er also used for destaining. MoZec&ar weight. Molecular weights of pure enzyme preparation (Fraction III) as well as those of I and II were determined by Sephadex Gel filt,ration technique (15, 16). Sephadex G-200 Superfine column (2.5 X 35.0 cm) with flow adaptors was used. Saline phosphate buffer pH 5.27, Z = 0.15 was used t,o dissolve isoenzymes and authentic protein samples as well as to equilibrate and elute the column. The samples were injected and the column was eluted in automatically, ascending direction with a flow rate of 18 ml/hr. Carbohydrate analysis. Total carbohydrate of three enzyme preparat,ions were determined by the anthrone reaction (17). Qualitative and quantit,ative determinations of neutral and amino sugars was performed only on pure enzyme. For neutral sugars, lyophilized enzyme preparations were hydrolyzed with 2 N HzS04 for 6 hr in a sealed tube at, 100°C. The neutral sugars were separated from the charged molecules by passing the hydrolyzate through a 0.9 X 6.0 cm column of CGC 241, 200-400 mesh (Na form), feeding directly to a second column 0.9 X 5.0 cm of CGA 541, 200-400 mesh (formate form). Both ion exchange resins were obtained from J. T. Baker Co., Phillipsbllrg, NJ. The nelltral sugars were eluted with 30.0 ml of distilled water. The quantitative determination of neutral sugars using paper chromatography followed by Nelson-Somogyi Copper reduction method was done according to t’he procedllre described by Spiro (17). For the det,ermination of amino sugars, lyophiliecd enzyme (Fraction III) was hydrolyzed with 4 N HCl in a sealed tube at 100°C for 7 hr. The amino sllgar was separated by passing the hydrolyzate through a 0.9 X G.0 cm column of CGC 241, 200-100 mesh, Na form. The column was first eluted wit,h 15.0 ml of water; the retarded amino sugars were then eluted with 10.0 ml of 2 N HCl. Qualitative and quantitative determination of

HYALURONATE

689

LYASE

amino sugars was done according to the procedure described by Spiro (17). Amino acid analysis. The purified hyaluronate lyase (Fraction III) was hydrolyzed for 20 hr according to the procedure of Moore and Stein (18). Automatic amino acid analyzer (JLC-5AH) was used for the quantitative amino arid analysis

(18). RESULTS

Enzyme purification. staphylococcal

Scheme 1. The

The purification for hyaluronate lyase is shown in data on enzyme purification

are summarized in Table I. Sequential ammonium sulfate fractionation resulted in a threefold purification. A major protein peak containing all the enzyme activity was successfully separated from ammonium sulfate and other low-molecular-weight compounds by Sephadex G-25 column (Fig. 3). This procedure also resulted in a fourfold purification.

Acetone fractionation apparently did not result in additional purification; however, it was convenient to prepare large amounts of acetone powder at one time. Hyaluronate lyase was purified go-fold by Sephadex G-100 column chromatography. As seen in Fig. 4, the enzyme was eluted as a single peak between two other prottbin peaks which were devoid of any activity. Elution profiles from CM Sephadex-50 column are shown in Fig. 5. A major anionic protein peak emerged wit’hout rrtardat,ion CRUDE

ENZYME

* --pGT;U:MOSIUdjmTE

DISCARD

PRECIPITATE i SEPHADEX

G-25 3 ACETONE

(75%)

SEPHADtX

G-100

SEPHAD)X

G-25

CM-SEPHADEX 4 FRACTIONS

1 I

1

V

=+ CM-SEPHADEX CRYSTALt

IZATION

SCHEME 1. Purification coccal hyaluronate crystallization.

lyase

scheme for Staphylofrom crude enzyme to

690

RAUTELA

AND ABRAMSON TABLE

SUMMARY OF PURIFICATION

. (‘m’y$j

NFU/mg

210 1,200 870 1,360 400 950

840 312 282 276 144 93

6.40 12.30 6.90 11.60 0.15 0.35

33 97 128 124 2,600 2,700

3.6 130 190 180

6.6 22 38 36

Description

Volume W)

Units/ ml

I II III IV V VI VIII

Water extract Ammonium sulfate G-25 column Acetone powder G-100 column G-25 column CM-Sephadex Fraction I Fraction II Fraction III CM-Sephadex (III)

4,000 260 324 200 360 98 170 170 200 200

a Data from a purification

LyasV

Total ~liis~

Fraction No.

IX

I

OF STAPHYLOCOCCAL HYALURONATE

0.018 0.019 0.003 0.002

Yield (%)

PUdication

100 37 34 31 17 11

200 6,800 63,000 90,000

1 3 4 4 80 80

0.7 2.6 4.5 3

6 200 2,000 2,700

operation with 409 of crude enzyme powder as a starting material.

EFFLUENT

VOLUME.

ml

FIQ. 3. Desalting of hyaluronate lyase fraction previously precipitated between 0.8 and 1.0 ammonium sulfate fractionation. Sephadex G-25 equilibrated and eluted with sodium phosphate buffer pH 5.27, I = 0.15, in a descending mode, flow rate 45 ml/hr.

1 li

li

480 400 240 320 160 80

EFFLUENT

VOLUME.

2’3*

mi

FIG. 4. Elution profiles from Sephadex G-100 column of acetone powder dissolved in so-

dium phosphate buffer, pH 5.27. This step resulted in an eighty fold purification luronate lyase.

of hya-

CI~YSTALLIZATION

S. uwew

followed closely by a second protein peak. Three distinct enzyme activities named I, II, and III were also eluted. The fraction I was least cationic, was not retarded in the column, and was eluted with the buffer between 1 = 0.10 and 1 = 0.12. The fraction II, moderately cationic, was eluted between I = 0.13 and I = 0.15. The fraction III, most active and most cationic was eluted with the buffer between I = 0.21 and I = 0.24. Fraction III when applied to a second column of CM Sephadex was eluted as a single peak between1 = 0.21 and I = 0.25 (Fig. 6). TKO very small inactive protein peaks mere also eluted. Further passage of Fraction III through CM Sephadex columns did not result in any measurable increase in the specific activity, however, the contaminating minor protein peaks were eliminated (Fig. 7). Crystallization. The very tine hyaluronate

TUBE

FIG. sodium luronate eluting II (I =

NUMBER

(VOL.

HYALUHONATR

LYAHE:

691

lyase crystals began to appear, during slow evaporation, after 3 days when the original volume was approximately reduced by 80 %. The crystals were more abundant but not larger in size by the time 90% reduction in the original volume occurred. A photomicrograph of the crystals is shown in Fig. 8. Polyacrylamide disc electrophoresis. The results are presented in Fig. 9. The data shorn that, for the enzyme preparation at a given stage of purity, the number of proteins resolved by disc electrophoresis was in agreement with the number of protein peaks eluted by column chromatography. Pure enzyme showed homogeneity both by ion exchange chromatography (Fig. 7) and disc electrophoresis (Fig. 9). Molecular weight. Pure enzyme had a molecular weight of 84,000 corresponding to a K,, (13, 14) value of 0.426 (Fig. 10). The molecular weights of two other enzyme

7.8 ml EACH1

5. Elution profiles from CM-Sephadex 50 column eluted with a linear gradient of phosphate buffer pH 5.77 to pH 6.27, Z = 0.07 to Z = 0.3, respectively. The hyalyase was resolved into three fractions I, II and III. The ionic st,rengths (Z) of buffer corresponding to maximum enzyme activities; fraction I (I = 0.11); fraction 0.14); fraction III (I = 0.22).

Fro. 6. Further purification of hyaluronate lyase (Fraction III) on second CM-Sephadex 50 column rlsing a linear gradient of sodium phosphate buffer pH 5.77 to pH 6.27 with Z = 0.1 to Z = 0.3, respect,ively. Maximum enzyme activity was eluted at 0.22 ionic strengt,h.

692

RAUTELA

AND ABRAMSON

fractions (I and II) were also 84,000 corresponding to K,, values of 0.425 and 0.432, respectively. Carbohydrate-protein analysis. Analysis suggested that pure hyaluronate lyase was a glycoprotein. Total protein to total carbohydrate ratio of the partially purified enzyme preparation (Stage VI, Table I, before application to ion exchange column) was 50 %/

40%. The total protein to carbohydrate ratios of the three enzyme preparation (Stage VIII, Table I) is shown in Table II. Pure enzyme had a protein to carbohydrate ratio of 5: 1. Analysis for neutral sugars in pure enzyme revealed that the molar ratio of galactose: glucose:mannose was 1: 3: 6. Amino sugars galactosamine and glucosamine (or mannosamine) were present in a ratio of 1: 1. The combined amounts of I I I I I I I I amino sugars were approximately equal to I ‘0 23 the amount of galactose. I+ r\ 1 1&O Amino acid analysis. The relative molar d 2 120$ concentrations of amino acids are presented 2 8. $ in Table III. Extremely high concentration 8/ o G 4” of ammonia and low concentrations of amino P I a -I $05 4~ 2 acids in general suggests that destruction of P i 2 amino acids occurred during hydrolysis de0 I+-+-&.2 32 48 24 40 0 spite adequate precautions. The data are inTUBE NUMBER IVOL 718ml EACH1 eluded here, however, merely to suggest that there was a relative abundance of basic Fro. 7. Complete purification of hyaluronate histidine, and argilyase on a third CM-Sephadex 50 column using a amino acids-cystine, linear gradient of sodium phosphate buffer pH 5.77 to pH 6.27 with 1 = 0.1 to I = 0.3, respectively. A homogenous enzyme peak without any contaminating proteins is apparent. The maximum enzyme activity was eluted with the buffer system of 0.23 ionic strength.

FIG. 8. Photomicrograph

nine. DISCUSSION

In this publication, we characterize purified Fraction III of staphylococcal hyaluro-

(X 400) of hyaluronate

lyase crystals following pervaporation.

CRYSTALLIZATION

S. aureus

HYALURONATR

natc lyase as a protein. (Characterization of cnzymat,ic properties will be published.) Purification of S. aweus hyaluronat’e lyase to homogeneity and crystallization as well as its partial characterization has been accomplished.

08

li93

LYASIC

yl&c-j . i \ I

\

07

06

’

II111111

I

CHYMOTRYPSINOGEN

A

OVALBUMIN M w 45,000

05

1

1

K0V

HYALURONATE LYASE M w 84.000

04

’

;

03

\

ALDOLASE M.W 158,000

--I

.

02

01

I

I

I

I

2

3

4

MOLECULAR

lllll/

I

6’310

20

WEIGHT x lO-4

FIG. 10. Molecular weight of purified hyaluronate lyase determined by gel filtration of a Sephadex G-200 superfine, column eqrtilibrated with pH 0.9 sodium phosphate bnfler. Column calibrated with reference proteins chymotrypsinogen, ovalbumin, and aldolase. TABLE FIG. 9. Polyacrylamide disc electrophoresis (using Tris-glycine buffer, pH 9.5, gel concentjrat)ion 7.5r/, 4 mA current/tube for 1 hr) of hyaluronate lyase preparations at various stages of purificat,ion. (A) acetone powder stage, 12 protein and/or glycoprotein bands are evident. (B) Enzyme preparation following Sephadex G-100 gel filtration, O protein and/or glycoprotein bands. (C) Pnrified preparation following second ion exchange chromatography, resolved a single band sttggest.ing homogeneity. TABLE

11

PKOTEIN .\iVl) ~.\RIKIHYDHATI:, li.\TIOS" PURIFWLI H~.\IAJR~N.~TK LY.\SIC FK.~CTIONS Fraction

Protein (%I

I

70

II

88

III n IIpaluronate separation into bohydrate ratio

OF

Carbohydrate (%I

30

12 17 -__ lyase preparation before its fractions had a protein to carof GO-40yc, respectively. 83

III

~~ELATIVIC MOIAN ~ONCESTILATIONS OF !NI~O ~bxus PREHEST IN THF: H~DROLYS.\TI: OF FaacTIoN 1111, Amino acid

Tryptophan Lysine Histidine Arginine Ammonia Aspartic acid Threonine Serine Glrttamic acid Proline

~ Moles 1 % Present 9.40 0.02 0.11 79.10 0.04

O.il 0.03

’/ /

Amino acid

~Moles SC

(ilycinr Alanine Half-cyst inc Valine Methioninc Isoleucine Lrrlcinr Tyrosine Phenylalaninc

0 .3'1 0.44 1 .20

a Pure hyaluronate lyase. histidine and cystine occurred stittttents of the hydrolysate.

0. 13 0.05

O.Oi 0.08 0 07 0.07

Ammonia, Iysinr, as the major con

Although it is tempting to suggest the existence of t,hrcv distinct isocnzymw, the enzyme peaks I and II may bc artifacts. The elution of these peaks immcdiatt~ly following

694

RAUTELA

AND

two different nonenaymatic protein peaks (Fig. 5) suggests that they were strongly bound to these proteins and were not fully separated in the cation exchange column. The major peak III, containing most of the enzymatic activity, however, represented that portion of the hyaluronate lyase which was completely separated from the contaminating proteins. Further experimental work is needed to either prove or disprove the existence of hyaluronate lyase isoenzymes. Although the quantitative amounts of protein and carbohydrate present in pure enzyme were determined, the manner in which the carbohydrate portion is bound to the protein and its contribution to the catalytic activity as well as the sequence of amino acid and sugar residues are not known. The data on amino acid analysis is far from conclusive owing to a significant destruction of amino acids during hydrolysis. However, the data do suggest a relative abundance of basic amino acids. This finding is consistent with the basic nature of hyaluronate lyase as has been shown during this as well as past investigations (5, 7, 19). It should also be pointed out that a rapid inactivation of all purified enzyme preparations, despite normal precautions was encountered. Fraction III in solution lost as much as 40% of its activity in 24 hr at 4°C and about 25% even when frozen and reassayed within a week. It should be noted that reported degree of purification as well as percent yields (Table I) could have been significantly higher if the enzyme preparations were stable at later stages of purification. A specific activity of only 200 for enzyme peak I ww evidently due to inactivation during purification. Inactivation may also have been responsible for the failure to obtain an increase in specific activity of Fraction III during repeat,ed ion exchange chromatography. The inactivation kinetics of the enzyme has been reported elsewhere (20). ACKNOWLEDGMENTS

We are indebted to Dr. A. Tucci of Albert Einstein Medical Center, Philadelphia, and Dr.

ABRAMSON

Michael F. Sheff of Pennsylvania Hospital, Philadelphia, for the use of amino acid analyzer and disc electrophoresis apparatus, respectively. We also thank Mr. S. E. Mujorra for his technical assistance. REFERENCES 1. ROGERS, H. J. (1948) Biochem. J. 43, 633. 2. DAVISON, M. M., DEROW,M. A., AND WALKER, B. S. (1949) J. Bacterial. 68, 717. 3. ABRAMSON, C., AND FRIEDMAN, H. (1968) J. Bacterial. 96,886. 4. TIRUNARAYNAN, M. O., AND LUNDBLAD, G. (1968) Acta Pathol. Microbial. &and. 78,211. 5. ABRAMSON, C. (1972) International Sympo-

sium on Staphylococci and Staphylococcal Infections, II, S. Karger, Basic and Polish Medical Publishers (In Press). 6. GREILUNG, H., STUHLASATZ, H. W., AND EBERHARD, T. (1965) 2. Physiol. Chem. 340, 243. 7. ABRAMSON, C. (1967) Arch. Biochem. Biophys. 121, 103. 8. ABRAMSON, C., AND FRIEDMAN, H. (1964) Fed. Proc. Fed. Amer. Sot. Exp. Biol. 23,191. 9. VESTERBERG, O., WALDSTROM, T., VESTERBERG, K., SVENSON, H., AND MALMGREN, B. (1967) Biochim. Biophys. Acta 133, 435. 10. HARRIS, T. N., AND HARRIS, S. (1949) Amer. J. Med. Sci. 217, 174.

11. Worthington Biochemical Corporation (1968) Hyaluronidase (3.2.1.35), Freehold, NJ. 12. ABRAMSON, C., AND RAUTELA, G. S. (1971) Bacterial. Proc . , 81. 13. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 14. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404. 15. ANDREWS, P. (1964) Biochem.

J. 91, 222.

16. Sephadex Gel Filtration Instruction Manual For Protein Molecular Weight Determinations, Pharmacia Fine Chemicals, Upsalla, Sweden. 17. SPIRO, R. G. (1966) inMethods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol VIlI, p. 3, Academic Press, New York. 18. MOORE, S., AND STEIN, W. H. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol VI, p. 819, Academic Press, New York. 19. ABRAMSON, C., AND FRIEDMAN, H. J. (1969) J. Bacterial. 97,715. 20. ABRAMSON, C., AND NAGARAJAN, Bacterial. Proc.. 136.

K.

(1969)