Studies ,of the Physicochemical and Enzymatic Properties of … · 2003-02-04 · THE JOURNAL OF BIOLOGICAL Cmmsnt~ Vol. 242, No. 24, Issue of December 25, pp. 5715-5723, 1967 Printed

THE JOURNAL OF BIOLOGICAL Cmmsnt~ Vol. 242, No. 24, Issue of December 25, pp. 5715-5723, 1967

Printed in U.S.A.

Studies ,of the Physicochemical and Enzymatic Properties

of Papaya Lysozyme*

(Received for publication, July 31, 1967)

JAMES B. HOWARD AND A. N. GLAZER

From the Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, Cali- f ornia 90024

SUMMARY

Papaya lysozyme, purified by ion exchange chromatography on IRC-50, has been investigated with respect to physicochemical and enzymatic properties. In agreement with earlier findings, it was found that papaya lysozyme has a molecular weight of about 25,000 and that glycine is its sole amino-terminal residue. Molecular weight determinations in dissociating solvents indicate that the molecule consists of a single polypeptide chain. Papaya lysozyme lyses Micrococcus Zysodeikficus cell walls at a rate one-third of that exhibited by egg white lysozyme; however, it displays a chitinase activity toward tetra-hr-acetyl-D-glucosamine 400 times that of the egg white enzyme. No involvement of tryptophan residues in the active site of papaya lysozyme could be demonstrated.

The presence of bacteriolytic activity in plant tissues was first reported by Fleming in 1922 (1). Later, Meyer, Hahnel, and Steinberg, (2) reported lysosyme activity in crude proteolytic enzyme preparations from papaya and fig latex. In 1955, Smith et al. (3) reported the isolation and characterization of a crystalline lysozyme from papaya latex. This protein showed powerful lytic activity toward suspensions of Sarcina lutea (3). However, the molecular weight of papaya lysozyme, 25,000, was strikingly different from that of lysozymes from animal sources (3). The latter enzymes all have molecular weights of about 14,000 (4). In addition, Smith et al. (3) showed that the amino acid composition of papaya lysozyme bore little resemblance to that of the egg white enzyme.

Lysozymes from animal sources and egg white lysozyme, in particular, have been extensively studied in recent years (4). These studies have culminated in the determination of the primary sequence of egg white lysozyme (4, 5) and elucidation of the three dimensional structure of this protein at 2 A resolu-

* This investigation has been aided by Grant GM 11061 and Training Grant GM 00364 from the National Institute of General Medical Sciences, United States Public Health Service.

tion (6, 7). X-ray crystallographic studies of enzyme-inhibitor complexes (8), coupled with chemical and enzymological investi- gations (4, 9, lo), have permitted considerable insight into the mechanism of action of egg white lysozyme (7, 8, 10). Since cases are known in which enzymes of widely diverse origin, e.g. bacterial subtilisins and the pancreatic proteolytic enzymes, trypsin and chymotrypsin, share very similar mechanisms of action (11) , it was of considerable interest to examine a lysozyme the gross physical properties of which differed from those of egg white lysozyme. It should also be emphasized that whereas lysozymes from animal sources have been studied in great detail, information on lysozymes of plant origin is limited.

In the initial stages of this investigation, it was found that preparations of papaya lysozyme, obtained by the method of Smith et al. (3), and crystallized three times, were heterogeneous as judged by acrylamide gel electrophoresis, end group analysis, and ion exchange chromatography. The preparations originally investigated by Smith et al. (3) appeared homogeneous by several criteria; hence, the difference between the earlier preparations and those studied here probably reflects differences in the starting material, i.e. the dried papaya latex. It was possible to obtain papaya lysozyme, homogeneous by a variety of criteria, by ion exchange chromatography of the crystalline material on IRC-50. This paper presents a comparison of the physicochemical and enzymatic properties of such preparations with those of egg white lysozyme. Most notably, papaya lysozyme has been found to exhibit a powerful chitinase activity, sug- gesting that this may be its significant biological function in the latex. Furthermore in contrast to egg white lysozyme, no involvement of tryptophan in the active site of papaya lysozyme could be demonstrated.

EXPERIMENTAL PROCEDURE

Materials

Dried papaya latex was supplied by Wallerstein Laboratories, New York. Crystalline egg white lysozyme (Lot 45291) was obtained from C. F. Boehringer und Soehne GmbH, Mannheim. Crude chitin and chromatographically pure crystalline N-acetyl- n-glucosamine were obtained from Calbiochem. Micrococcus lysodeikticus cells were obtained from Mann, and p-nitrophenyl-

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5716 Studies on Papaya Lysoxyme Vol. 242, No. 24

P-AT-acetyl-n-glucosamine (Lot 15B-5010), from Sigma. Casein was obtained from Matheson Coleman and Bell, as was N-bromosuccinimide. Before use, the latter was recrystallized twice from ethyl acetate-petroleum ether (35-SO0 fraction). IRC-50 (minus-400 mesh), Dowex 50-X2 (50 to 100 mesh), and BioGel P-2 (200 to 400 mesh) were obtained from Calbiochem. The ion exchange resins were recycled (12) before equilibration with the appropriate buffers. All other reagents used were of analytical grade.

Methods

Preparation of Papaya Lysoxyme-Papaya lysozyme was prepared from the dried papaya latex by Procedure I of Smith et al. (3). The protein was recrystallized three times from 0.01 M mercuric chloride in 40 ‘$& saturated ammonium sulfate and was stored as crystals in the mother liquor of the third recrystalliza- tion. Protein prepared in this manner is designated as papaya lysozyme-3X. The enzyme was dissolved by dialysis at 4” at pH 4.0 against 0.1 M acetate buffer, containing 0.02 M cysteine and 0.005 M EDTA, followed by exhaustive dialysis against the appropriate buffer.

Further purification of papaya lysozyme-3X was carried out by chromatography on IRC-50 at pH 7.15 in 0.14 M sodium phosphate buffer. Equilibration of the columns to the proper pH and ionic strength was followed by measurement of the conductivity and pH of the effluent. Analytical columns (0.9 x 30 cm) and preparative columns (2.0 x 135 cm) were run at 8 to 10 ml per cm2 and at room temperature (22-23”). Samples were applied in 0.05 to 0.1 of the total volume of the column at a protein concentration of 1 y0 after exhaustive dialysis against the column buffer. The elution was monitored at 254 rnp with a LKB Uvicord, and fractions equal to the volume in which the sample was applied were collected. These fractions were pooled, exhaustively dialyzed against water, lyophilized, and stored at 4”.

Determination of Protein Concentration-An E:& (280 rnp) of 23.8 + 0.3 (five determinations) was obtained by assuming 100% recovery from amino acid analysis. This value was in agreement with an E:& (280 rnp) of 22.6 + 1.3 (two determinations), as determined by refractive index, assuming a refractive index increment of 0.00187 for a 1% protein solution. The E:?m (280 mp) based on the amino acid analysis was used throughout this paper except for the ultracentrifuge studies where the protein concentration was determined by refractive index. An Et’& (280 rnp) of 26.5 was used for egg white lysozyme (5).

Amino-terminal End Group Analysis-The method of Stark and Smyth (13) was used to examine the amino-terminal residues of papaya lysozyme. Since glycine had been reported to be the sole NHz-terminal residue in papaya lysozyme (3), the analysis was confined to the acidic and neutral amino acids. Protein blanks were run in parallel for each analysis. A significant background of glutamic acid was obtained which was not ap- preciably lowered even when the suggestions of Stark and Smyth (13) for elimination of pyrrolidone carboxylic acid were followed. Quantitation of the end group was done by amino acid analysis, and complete recovery of all amino acids was assumed.

Circular Dichroism and Ultraviolet spectra-The ultraviolet absorption and difference spectra were recorded on the Cary model 14 recording spectrophotometer with matched quartz cells of l-cm light path. Circular dichroism spectra were deter-

mined by means of l-cm quartz cells in the Jasco-Durrum ORD/UV model 5 recording spectropolarimeter. All spectra were recorded at room temperature.

Amino Acid Analysis-Protein hydrolysates, prepared by the method of Moore and Stein (14), were maintained in duplicate at 110 + 2” in sealed evacuated tubes for the periods indicated in the text. Analyses were performed in a Spinco model 120B amino acid analyzer.

Cysteine plus half-cystine, methionine, and proline were determined after oxidation with performic acid by the method of Moore (15). Aminoethylation was carried out by the method of Plapp, Raftery, and Cole (16), and their color value of 0.91 x lysine was used to calculate the recovery of S-aminoethylcysteine.

Chromatography and Detection of Sugars-Descending paper chromatography was run at room temperature on Whatman No. 3MM paper in l-butanol-acetic acid-water (200:30:75, by volume). The usual running time was 44 to 48 hours. The sugars were detected by dipping in 0.5 M NaOH in 95% ethanol and heating to 100-110” for 5 to 10 min. This treatment produced a derivative which fluoresced strongly under ultraviolet light (9). The standard silver nitrate-sodium hydroxide spray for reducing groups was also used (17). However, the increased sensitivity of detection of the fluorescent derivative aided the demonstration of minor components and of oligosaccharides which stain much less intensely with AgNOz-NaOH than simple sugars. Thin layer chromatography on Silica Gel H was performed in I-butanol-pyridine-water (70: 15 : 15, by volume) as described by Osawa and Nakayawa (18). For this technique, only the AgN03-NaOH reagents were used. Quantitative determination of reducing groups was by the ferroferricyanide method (19) which was modified by increasing by 50% the concentration of sodium dodecyl sulfate, sulfuric acid, and ferric ammonium sulfate. Also, the incubation at 100“ was increased from 20 to 30 min.

Preparation of Chitin Oligosaccharides-Crude chitin was pulverized in a glass tissue grinder, centrifuged, and dried at 50” for 24 hours. The chitin was hydrolyzed in HCl as described by Rupley (20). After 13 hours of hydrolysis at 40”, the mixture was condensed to a syrup on a rotatory evaporator at 40 mm of mercury and 40”. Saturated NaOH was placed in the trap to neutralize the HCl. The syrup was diluted 4-fold with water and concentrated again on the rotatory evaporator in order to eliminate HCl. This operation was performed five times. The syrup was taken to dryness in a vacuum over NaOH pellets.

Two grams of the dried hydrolysate were dissolved in 20 ml of water and applied to a column (4 x 150 cm) of BioGel P-2 equilibrated with chloroform-saturated water. Fractions (10 ml) were collected at 30 ml per hour at room temperature. The elution was monitored by measurement of the absorbance at 230 rnp and by quantitative analysis of the reducing power. Frac- tions were pooled and condensed to a syrup by rotatory evapora- tion. The sugars were collected by centrifugation after precipi- tation with excess absolute ethanol. Each oligosaccharide was further purified by paper electrophoresis at pH 1.9 in order to remove partially deacetylated material. The purified sugars were recrystallized three times from water-ethanol and then air-dried at room temperature. A characterization of the products is summarized in Table I.

Ultracentrifuge Studies-Sedimentation velocity was determined with a phase plate at 59,780 rpm in the Spinco model E


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Issue of December 25, 1967 J. B. Howard and A. N. Glazer 5717

ultracentrifuge equipped with a schlieren optical system. Low speed sedimentation equilibrium by the method of Richards and Schachman (21) and high speed sedimentation equilibrium by the

method of Yphantis (22) were determined with the interference optical system of the model E ultracentrifuge. Three double- channel cells (2.7 mm) with sapphire windows were used in all equilibrium determinations. In all cases the results were cor- rected to the viscosity and density of water at 20”.

Determination of Enzymatic Activity-Lysozyme activity on bacterial cell wall substrate was determined by means of a standard assay similar to that used by Smith et al. (3). M. Zysodeikti- cus cell wall preparation (20 mg) was suspended in 100 ml of buffer at the appropriate pH. Ten milliliters of substrate were incubated at 37” with the enzyme (lop4 to 10m3 mg per ml of substrate). After 5 min, duplicate 2-ml aliquots were taken and fixed with 0.2 ml of 4 M NaOH. The absorbance at 440 rnp was determined after 30 min at room temperature. The absorbance corresponding to 100% hydrolysis was determined by incubation of 1 mg of enzyme with 10 ml of substrate for 24 hours at 37”.

Proteolytic activity was determined by the method of Kunitz (23) with heat-denatured casein as substrate, in the presence of 1.6 x 1OP M 2,3-dimereaptopropanol. Blanks for both the enzyme and the substrate were incubated. All proteolytic activity was calculated from the activity curve for papain and was reported as a fraction (w/w) of papain present.

Chitinase activity was determined by incubating N-acetylglucosamine oligosaccharides at the concentration of 1 to 10 mg per ml of final reaction mixture with 2 x lop4 to 1OP M enzyme. The reaction was performed in 0.2 M acetate buffer at either pH 4.7 or 5.5, at 37”. Aliquots (25 to 50 ~1) were taken out at appropriate time intervals, and the reaction was stopped either by diluting to 1 ml with water and freezing at -20” for quantitative reducing group analysis or by adding an equal volume of glacial acetic acid and storing at -20” before paper chromatography.

fi-N-acetyl-n-glucosamidase activity was determined using p- nitrophenyl-P-A-acetyl-o-glucosamine in the assay system of Findlay, Levvy, and Marsh (24). cdl at 405 rnp for p-nitrophenol used for determining the percentage of hydrolysis of the substrate was 1 .S x lo4 (25). The incubation mixture was buffered with 0.2 M acet.ate at pH 4.7 or 6.0 and maintained at 37”.

RESULTS

Ion Exchange Chromatography of Papaya Lysoxyme-The elution pattern obtained on IRC-50 chromatography of papaya lysozyme-3X is shown in Fig. 1. An identical pattern and similar relative proportions of each component were obtained for each of four separate preparations examined. These were obtained from two different batches of dried papaya latex. Furthermore, the pattern was unaltered after storage of the mercuri-lysozyme in the crystalline form in 40y0 saturated (NH4)&04 for 6 months at 4” prior to chromatography. Re- chromatography of the most retarded component (designated as papaya lysozyme-E) indicated a single symmetrical peak with an elution volume identical with that obtained on the original chromatographic separation (see Fig. 1). Component A, which was eluted at the breakthrough volume, also rechromatographed in its original position (Fig. 1). However, rechromatography of this material did not give a symmetrical peak (Fig. 1). At-

TABLE I

Characterization of chitin oliyomers

component

RGlcNAcr solvent II*

Melting Ratio of

RSG,lleiVh&, point reducing

groups after

G Litera- hydrolysis

Litera- to reducing ture Found ture Found groups before (18) (18) hydrolysis

____

Di-N-acetylglucosamine.... 0.56 0.64 0.69 269-262255-2591.8 & 0.1

Tri-N-acetylglu- eosamine... 0.27 0.30 0.34 304306298-3053.1 f 0.2

Tet,ra-N-acetylglucosamine 0.15 0.c 4.2 III 0.1

a Solvent I, I-butanol-acetic acid-water on What,man No. 3MM paper.

6 Solvent II, l-but.anol-pyridine-water on thin layer Silica Gel H.

.8 - n,

2

.2 -

/ _-___- ‘L- I I , , , , / , , , ,

0 10 20 30 40 50 60 70 80 90

TUBE NUMBER

FIG. 1. Chromatography of papaya lysoayme on a column (2 X 135 cm) of IRC-50 in 0.14 M phosphate buffer, pH7.15. Fractions A to E were pooled as indicated by the solid bars. The dotted line represents rechromatography of Fraction A; the dashed line represents rechromatography of Fraction E.

tempts to chromatograph mercuri-lysozyme, without prior re- moval of the heavy metal by dialysis against cysteine and EDTA, resulted in an irreversible adsorption of the lysozyme to the ion exchange resin.

The lysozyme activity of the material in the peak tube of each of the components, A to E, was determined by the standard assay with M. lysodeikticus as substrate at pH 4.8. The activity of each component was linear with concentration of protein, and all components exhibited the same specific activity within experimental error ( *5%) .I

Since papaya lysozyme is extracted from the latex together with a number of proteolytic enzymes, notably the sulfhydryl proteases papain and chymopapain (3)) the crystalline enzyme as well as the various lysozyme components obtained by ion exchange chromatography were examined for the presence of

1 When lyophilized preparations of Components A to E were stored for several months at 4”, a decrease in the lytic activity toward M. lysodeikticus was observed with A showing the greatest decrease in activity, approximately 32$&, and E the least, approximately 18%.


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5718 Studies on Papaya Lysozyme Vol. 242, )Yo. 24

contaminating proteolytic activity. The assays were performed using casein as substrate, and the activities observed were expressed in terms of those obtained with papain crystallized two times. In the absence of sulfhydryl compounds in the assay mixtures, no proteolytic activity could be demonstrated in the mercuri-lysozyme crystallized three times. Upon the addition of 2,3-dimercaptopropanol, however, various preparations of papaya lysozyme-3X exhibited 1 to 20/, (w/w) proteolytic activity. The requirement for activation suggests that the contaminating protease is indeed one of the sulfhydryl proteolytic enzymes of papaya latex. On ion exchange chromatography, the proteolytic activity was concentrated principally in Peak B in

FIG. 2. Acrylamide gel electrophoresis of papaya lysoayme-E (A) and papaya lysozyme-3X (B) in a 10% acrylamide gel equilibrated with 0.037 M glycine-Tris buffer at pH 9.0. Electrophore- sis was performed at 5 ma per tube for 210 min at 4”.

which it was present to the extent of approximately 15% (w/w). Peak E exhibited less than 0.1% (w/w) of papain-like activity.

Acrylamide gel electrophoresis of papaya lysozyme-3X showed five components, while papaya lysozyme-E appeared to be essentially homogeneous (Fig. 2).

Amino Acid Composition-The amino acid analyses for papaya lysozyme-3X and E are given in Table II. There appear to be no significant differences between the amino acid compositions of these two materials, and, indeed, these compositions were in agreement with those originally reported for this protein (3). Amino acid analyses of protein obtained from Peaks A, B, and C also did not show significant differences from that of papaya lysozyme-3X. Half-cystine and methionine values were obtained from analyses of both performic acid-oxidized and aminoethylated papaya lysozyme (Table II). The value for half-cystine, as cysteic acid, obtained from hydrolysates of oxidized papaya lysoeyme-E was approximately 1 residue higher than the value for S-aminoethylcysteine obtained from the hydrolysate of the aminoethylated protein. The latter value is thought to be the correct one on the basis of the following con- siderations. Cysteine buffers were used to remove mercury from crystalline papaya lysozyme prior to chromatography of the protein on IRC-50 (see “Experimental Procedure”). The exhaustive dialysis and ion exchange chromatography involved in the preparation of papaya lysozyme-E apparently failed to remove all traces of extraneous half-cystine. Thus, free cysteic acid in an amount of approximately 0.5 mole per mole of protein could be demonstrated on paper electrophoresis of perfonnic acid-oxidized papaya lysozyme-E in 6 M urea at pH 4.6. Cysteic acid could no longer be detected if the protein was dialyzed against 6 M urea followed by deionized water, before performic acid oxidation. It would appear, therefore, that the free cysteic acid originated from strongly bound rather than covalently linked half-cystine.

Amino-terminal End Group Analysis-Smith et al. (3) performed end group analyses on papaya lysozyme by the fluoro- dinitrobenzene method of Sanger. They reported a recovery of 0.34 mole of dinitrophenyl-glycine per mole of papaya lysozyme, without correction for loss during acid hydrolysis. No other amino-terminal end groups were found in significant amounts (3). Examination of the amino-terminal residues in papaya lysozyme-3X by the cyanate method of Stark and Smyth (13) has indicated a more complex picture with the present preparations. In addition to glycine, which was recovered in a yield of 0.30 residue per mole of protein, aspartic acid, leucine, tyrosine, and alanine also appeared in significant amounts (see Table III). These five amino acids accounted for 0.88 mole of total end group per mole of protein. In contrast to these findings, papaya lysozyme-E appeared to be essentially homogeneous by end group analysis. As may be seen in Table III, 0.85 mole of glycine was recovered per mole of protein, and only very small traces of other amino acids were found, after correction for background. These results suggest that the heterogeneity of papaya lysozyme-3X may result in part from proteolytic cleavage of bonds in this enzyme, most probably in the latex itself or in the course of the purification procedure. The latter possibility appears to be less likely since the end group heterogeneity was not observed in the study of Smith et al. (3).

Ultracentrifuge Stud&--The s;,,,~ for papaya lysozyme-E, in 0.1 M acetate buffer at pH 4.0, both in the presence and absence of cysteine (0.02 M) and EDTA (0.005 M), was found to be 2.70.


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TABLE II

Amino acid composition of papaya lysozyme

Papaya lysozyme-3x Papaya lysozyme-E Literature (3) .- Amino acid

Lysine .......... Hist.idine. ......... Arginine. Aspartic acid. ...... Threonine .......... Serine ............. Glutamic acid. ..... Proline ............. Glycine ............ Alanine. ........... Valine .............. Methionine ......... Isoleucine Leucine. ............ Tyrosine, ........... Phenylalanine ...... Tryptophan ........ Half-cystine ........ S-Aminoethylcys-

teine .............

Unmodified proteinC S-Aminoethylated proteinb S-Aminoethylated protein” Unmodified proteina

i /.&+%0le

0.053 0.016 0.065 0.113 0.062 0.065 0.058 0.092 0.131 0.097 0.033

0.052 0.060 0.061 0.052

0.038

residuesd 10.2

3.0 12.7 21.7 11.9 12.5 11.0 17.7 25.2 18.6

6.4

(4) 10.0 11.5 11.7 10.0

(7)

7.3

jmole

0.046 0.015

0.104 0.056 0.061 0.053 0.088 0.120 0.086 0.033

0.050 0.059 0.050 0.049

0.037

residuesd

9.5

(l&f 21.8 11.7 12.8 11.0 18.3 25.0 18.0

6.9

(4) 10.5 12.3 10.5 10.2

(7)

7.7

pmole residue& 0.059 9.9 0.016 2.6 0.080 13.0 0.135 22.0 0.082j 13.3j 0.093f 15.2j 0.067 10.9 0.1118 18.10 0.160 26.1 0.115 18.8 0.042i 6.8” 0.024g 3.9g 0.068” ll.li 0.073” 11.9i 0.078j 12.9j 0.067 10.9 0.045i 7.3i 0.052~ 8.50

residuesd 10.2

2.8 12.7 22.9 13.0j 13.8j 11.1 18.3 26.7 18.6

7.oh

(4) 10.5h 11.8’” 11.8 10.5

(7) (8)

pmole

0.041 0.011 0.050 0.090 0.052’ 0.055f 0.045 0.073 0.107 0.076 0.028”

0.042h 0.047h 0.047 0.042

10 3

13 22 13 16 11 18 26 21

8 4

11 12 13 12

7 8

a 20- and 50-hour duplicates. b 24-hour duplicates. c 24., 48-, and 72-hour duplicates. d Assumes 20 residues of ammonia and a molecular weight of 24,745 (3). e The parentheses indicate assumed values for the number of residues. I Extrapolated to zero time of hydrolysis. g 24- and 72-hour duplicates for protein oxidized with performic acid assuming 94’% recovery for cysteic acid and 100% recovery for

methionine sulfone. h Assumed complete release at 50 hours. i Assumed complete release at, 72 hours. i Determined spectrophotometrically.

TABLE III

Amino-terminal residues in papaya lysozyme as determined by cyanate method

Papaya lysozyme-3Xa Papaya lysozyme-Ea Amino acid

T- T Carbamylated Control Carbamylated Control Difference Difference

0.08 0.03 0.05 0.02 0.30 0.18 0.03 0.18 0.14 0.03

- jmole

0.012 0.001 0.009 0.012 0.014 0.005

0.001

mole/mole protein

0.08 0.03 0.02 0.14 0.88 O.Q6 0.03 0.03

mole/?nole protein

0.12 0.03 0.08 0.06 0.35 0.20 0.03 0.18 0.14 0.03

?nole/mole protein

0.04

0.03 0.04 0.05 0.02

0.06 0.03 0.01 0.02 0.85 0.04 0.03 0.03

Aspartic acid. Threonine. Serine Glutamic acid Glycine. Alanine. Valine Leucine Tyrosine Phenylalanine

a Average of two determinations. No corrections for loss during hydrolysis.

This is in reasonable agreement with the value of 2.57 S reported taming cysteine (0.02 M) and EDTA (0.005 M), at a protein for the original preparations of crystalline papaya lysozyme (3). concentration of 0.2 to 1.0 mg per ml, both by the high and low An apparent molecular weight of 27,500 f 1,000 was obtained speed sedimentation equilibrium methods (21,22). A molecular for papaya lysozyme-3X in 0.1 M acetate buffer at pH 4.0, con- weight of 24,600 f 1,500 was obtained for papaya lysozyme-E


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TABLE IV Comparison of enzymatic properties of papaya and egg

white lysozymes”

Substrate Activity of papaya lysozyme compared

to egg white lysozyme”

S. lutea...................................... 0.21 (3) M. lysodeikticus............................... 0.35 * 0.05 Tetra-N-acetyl-n-glucosamine. 430 p-Nitrophenyl-p-iV-acetyl-n-glucosamine.. c

5 Details of assay mixtures and incubation conditions are found in the text.

b This ratio is obtained by comparing the maximal activity of each enzyme at its pH optimum toward each of the substrates listed.

c No detectable hydrolysis of this substrate was observed either at pH 4.6 or 6.0.

FIG. 3. Time course of hydrolysis of tetra-N-acetyl-D-glucosamine by papaya lysozyme, at pH 4.6, and by egg white lysozyme at pH 5.5, at 37”, at a substrate concentration of 9 mg per ml. I, papaya lysozyme; 11, egg white lysozyme.; 111, control. A, B, C, and D show the positions of mono-, dl-, tri-, and tetra-N- acetylglucosamine at the end of 44 hours descending chromatography in 1-butanol-acetic acid-water (4:1:5, by volume). The chromatogram was developed with alkaline AgNOa.

under the same conditions. The reason for the 10% difference between the apparent molecular weights of papaya lysozyme-3X and E is not yet clear.

A preparation of papaya lysozyme-3X was reduced for 2 days at 4” in 0.5% /?-mercaptoethanol and 6 M guanidine HCl at pH 8.0, and then exhaustively dialyzed against 0.1 M acetate buffer at pH 4.0, containing 0.5% P-mercaptoethanol and 6 M guanidine HCl. Determination of the molecular weight of this preparation,

in the last mentioned solvent, over a protein concentration range of 0.2 to 1.0 mg per ml, by the high speed sedimentation equilibrium method, gave a molecular weight of 28,000 + 800 (assuming no change in partial specific volume), indicating that papaya lysozyme most probably consists of a single polypeptide chain.

Enzymatic Activity: Lysis of M. Zysodeikticus-Smith et al. (3) reported that papaya lysozyme exhibited a high lytic activity toward Sarcina lutea. Similarly, it was found in this study that this enzyme readily lysed cells of M. lysodeikticus. A value for the maximum lysis of the latter substrate by papaya lysoayme was obtained by exposing a suspension of cells to a high concentration of enzyme at 37” at pH 4.6 for 24 hours. The absorbance at 440 rnp remaining at the end of this exhaustive lysis was taken to represent 100% hydrolysis, and this value was used in the determination of the kinetics of breakdown of M. lysodeikticus

by papaya lysozyme. It may be added, parenthetically, that the maximum decrease in absorbance at 440 rnp obtained with papaya lysozyme was 85% of that obtained with egg white lysozyme under the same conditions. As had been reported earlier for 8. lutea (3), the lysis of M. lysodeikticus followed second order kinetics for the first 25% hydrolys’is. The observed second order rate constant was linear with the concentration of papaya lysozyme over the concentration range 7.5 x lop5 to 10m3 mg per ml of substrate solution. A pH optimum of 4.7 was observed for the lysis of M. lysodeikticus. The same pH optimum had been found with S. lutea (3). The optimum pH for the action of egg white lysozyme on these substrates is 6.0 (4). As shown in Table IV, the maximal lytic activity of papaya lysozyme toward M. lysodeikticus is about 35% of that exhibited by the egg white lysozyme.

Chitinase Actitity-Preliminary experiments with suspensions of powdered chitin showed that papaya lysozyme-E was capable of releasing reducing sugars from this substrate. The chitinase activity was considerably higher at pH 4.6 than at pH 6.0, in agreement with the pH dependence of the lytic activity on M. lysodeikticus. The chitinase activity was investigated further with tetra-N-acetylglucosamine as substrate. Representative results are shown in Fig. 3. Within 10 min at pH 4.7, papaya lysozyme had released significant amounts of mono-, di-, and tri-N-acetylglucosamine. By the end of 53 hours, under the conditions chosen, the hydrolysis of the tetrasaccharide was almost complete. In striking contrast, the same molar concentration of egg white lysozyme at pH 5.5 had released only traces of dimer as the major detectable product by the end of 24 hours (Fig. 3).

In order to estimate the rate of hydrolysis of the tetrasaccharide, the release of reducing groups was measured quantitatively as a function of time. The results are plotted in Fig. 4 as the relative increase of reducing power with time at pH 4.8 for both enzymes at a substrate concentration of 8.5 x 10e4 M.

Comparison of the initial rates of hydrolysis of the tetrasaccharide shows the turnover rate for papaya lysozyme to be 400 times that for the egg white lysozyme.

P-N-Acetyl-o-glucosamidase A&&y-Papaya lysozyme-3X, papaya lysozyme-E, and egg white lysozyme were all incubated with p-nitrophenyl-P-N-acetyl-n-glucosamine as substrate at both pH 4.6 and 6.0. Less then 1% hydrolysis of the substrate was observed at the end of 20 hours at 37”, even at enzyme to substrate ratios as high as 6:l (w/w). Berger and Weiser (26) had reported earlier on the failure of egg white lysozyme to hydrolyze phenyl-@-N-acetyl-n-glucosamine.


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Circular Dichroism Spectra-Qualitatively and quantitatively, papaya lysozyme-3X and E had the same circular dichroism spectra. As might have been expected from the significant differences in amino acid composition, these spectra were considerably different from those obtained with egg white lysozyme (27, 28). In the aromatic absorption region, only a single posi- tive band at 293 rnp with a shoulder at about 287 rnE.1 was seen in the papaya lysozyme (see Fig. 5A). In contrast to the results obtained with egg white lysozyme, the circular dichroism spectrum of papaya lysozyme was insensitive to the presence of high concentrations of N-acetylglucosamine (see Fig. 5A) both at pH 4.8 and 6.5, even though this compound was found to inhibit the lysis of M. Zysodeikticus by papaya lysozyme. Thus, the interaction of N-acetylglucosamine with papaya lysozyme does not appear to involve significant perturbation of the aromatic chromophores.

E$ect of N-Acetylglucosamine on Ultraviolet Absorption Spec- trum of Papaya Lysozyme-One of the early indications of the involvement of tryptophan residues in the substrate binding site of egg white lysozyme was the appearance of a typical tryptophan difference spectrum upon the addition of the sugar derivative to the enzyme (29, 30). Such a difference spectrum produced by the addition of 0.1 M N-acetylglucosamine to egg white lysozyme may be seen in Fig. 5B. Addition of N-acetylglucosamine to papaya lysozyme at pH 4.8 failed to produce any significant difference spectrum (Fig. 5B).

Oxidation of Papaya Lysoxyme with N-Bromosuccinimide and Iodine Monochloride-Egg white lysozyme and papaya lysozyme- E were exposed to varying concentrations of N-bromosuccinimide and iodine monochloride. The extent of the oxidation of tryptophan residues was calculated by the method of Patchornik et al. (31). The difference spectra obtained by comparing the native with the oxidized proteins displayed the minimum at 307 rnp and the maxima at 288 rnE.1 and 280 rnp, in agreement with the results of Patchornik et al. (31). Treatment with N-bromo-

succinimide at pH 4.0 inhibited the activity of egg white lysozyme

.6

0 2 4 6 8 IO

TIME (hrs)

FIG. 4. Release of reducing groups from tetra-N-ace@-n- glucosamine by papaya lysozyme (3.5 X lo+ M) (A), egg white lysozyme (8.75 X 1OW M) (B), and (3.5 X 1OW M) (C), at a substrate concentration of 8.5 X 10e4 M in 0.1 M ammonium acetate buffer at pH 4.8 and 37”.

0 0 x

2 ‘-I

w

u a

0.0 - I I I

280 290 300

WAVELENGTH (m,ct)

FIG. 5. A, circular dichroism spectrum of papaya lysozyme (0.76 mg per ml) in 0.05 M phosphate buffer, pH 6.5. Spectra obtained in presence of 0.25 M N-acetyl-n-glucosamine, or, 0.25 M glucose were superimposable on the spectrum shown. Light path, 1 cm. B, ultraviolet difference spectra obtained by comparing (a) egg white lysozyme (0.41 mg per ml) in the presence of 0.2 M N-acetylglucosamine with the enzyme alone, (5) papaya lysozyme (0.95 mg per ml) in the presence of 0.2 M N-acetylglucosamine with the enzyme alone, and (c) N-acetylglucosamine with water. All solu- tions were in 0.1 M acetate buffer at pH 4.8.

\ \ 20 \+

‘ox- ---O-- A

01 ’ ” ” ” ” ’ ” 1 0 4 .8 1.2 ’ 1.6 2.0 2.4

MOLES TRYPTOPHAN MODIFIED PER MOLE PROTEIN

FIG. 6. Effect of oxidation of tryptophan residues with N- bromosuccinimide at pH 4.0 on the lytic activity of egg white lysozyme (A) and papaya lysozyme (B).

as previously reported (32). In agreement with earlier reports, our results (Fig. 6) indicated 80% inactivation concurrent with the destruction of 0.8 to 0.9 residue of tryptophan per mole of egg white lysozyme. However, the inactivation of papaya lysozyme with N-bromosuccinimide only reached a level of 80% after oxidation of 2.7 tryptophan residues.

At acid pH, the primary effect of ICl on egg white lysozyme is the oxidation of tryptophan.2 As shown in Fig. 7, once again 80% inactivation was observed when 0.8 mole of tryptophan was modified by ICl at pH 4.0. The difference spectra obtained by comparing the native with ICl-treated egg white lysozyme were identical with those obtained with the N-bromosuccinimide- treated enzyme. When the parallel experiment was carried out with papaya lysozyme-E, no inactivation was observed at 10

2 A. N. Glazer, unpublished results.


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MOLES ICI PER MOLE PROTEIN

FIG. 7. Effect of treatment of papaya lysozyme and egg white lysozyme with iodine monochloride at pH 4.0 on the lytic activity and tryptophan residues. 0, lytic activity of papaya lysozyme; A, residues of tryptophan modified per mole of papaya lysozyme; 0, lytic activity of egg white lysozyme; A, residues of tryptophan modified per mole of egg white lysozyme.

moles of ICl per mole of protein. At 20 moles of ICI per mole of enzyme, papaya lysozyme was inactivated to the extent of 80%; however, even under these conditions no oxidation of tryptophan was observed. In view of the fact that N-bromosuccinimide did oxidize tryptophan residues in papaya lysozyme, the absence of oxidation with ICl suggests that these residues are more accessible to the smaller oxidizing agent, Br+, than they are to the larger 1+.

DISCUSSION

By ion exchange chromatography on IRC-50 and by acrylamide gel electrophoresis, it could be readily demonstrated that papaya lysozyme-3X consisted of at least five components. These components appeared to be indistinguishable from each other insofar as molecular weight, amino acid composition, enzymatic activity, and circular dichroism spectra were con- cerned. However, papaya lysozyme-3X was heterogeneous with respect to the amino-terminal residue, whereas papaya lysozyme- E appeared to have glycine as the sole amino-terminal residue. This observation suggests that the heterogeneity of crystalline papaya lysozyme is a consequence of limited proteolysis. Since Smith et al. (3) found glycine as the sole amino-terminal residue in papaya lysozyme, the most reasonable conclusion would appear to be that the disparity between the present results and those obtained 15 years ago resides in differences between preparations of dried papaya latex. Certainly, the properties of papaya lysozyme-E agree in every respect with those originally reported for crystalline papaya lysozyme (3). It is noteworthy that the heterogeneous preparation of papaya lysozyme-3X has a specific activity indistinguishable from that of the homogeneous Com- ponent E.

Papaya lysozyme appeared to be a single polypeptide chain of approximately 25,000 molecular weight as judged from end group analysis and from the molecular weight determination performed in 6 M guanidine containing mercaptoethanol. Al- though papaya lysozyme displays a lower activity toward bacterial substrates than does the egg white enzyme, it is certainly as active toward these substrates as many other lysozymes of

animal origin (4,33). A striking finding is the very high chitinase activity of the papaya enzyme. This suggests that papaya lysozyme might better be classified as a chitinase rather than as a lysozyme. Chitinase is reported to have some bacteriolytic activity (34). Furthermore, papaya lysozyme is unlike most lysozymes in molecular weight, but is similar in this respect to chitinase (35).

The involvement of tryptophan in the active site of egg white lysozyme has been demonstrated by a variety of approaches (7, 8, 27, 29, 30, 32, 36). Tryptophan residues are involved in the binding of the substrate (7,8), and, indeed, the binding con- stants for a number of N-acetylglucosamine oligosaccharides have been determined (30). Examination of the circular dichroism spectra and ultraviolet spectra of papaya lysozyme in the presence of high concentrations of N-acetylglucosamine indicated no perturbation of the tryptophan chromophores by this monosaccharide. Moreover, essentially no oxidation of tryptophan by iodine monochloride was observed, even at rela- tively high concentrations of the oxidizing agent. Since under identical conditions, iodine monochloride readily oxidized the tryptophan in the substrate binding site of egg white lysozyme,2 it is clear that the tryptophan side chains are not as accessible to this reagent in the papaya enzyme as they are in egg white lysozyme. Further, oxidation of 3 of the 7 tryptophan residues of papaya lysozyme by N-bromosuccinimide led to only 80% inactivation. This would tend to suggest that the side chains of these tryptophan residues contribute to the over-all conforma- tional stability of papaya lysozyme, but are unlikely to be di- rectly responsible for the binding of substrate. These results lead to the tentative conclusion that tryptophan residues play only a limited role, if any, in the binding of substrate to papaya lysozyme.

Acknowledgments--We are greatly indebted to Mr. Douglas M. Brown for carrying out the ultracentrifugal determinations. We also thank Miss Dorothy McNall for performing the amino acid analyses. We are grateful to Dr. Emil L. Smith for his interest in this work and for his helpful discussions.

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James B. Howard and A. N. GlazerStudies of the Physicochemical and Enzymatic Properties of Papaya Lysozyme

1967, 242:5715-5723.J. Biol. Chem.

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