ofactivity with Sarcina lutea as the test organism. The new enzyme proved to have an activity on a...

ISOLATION AND PROPERTIES OF A CRYSTALLINE MERCURY DERIVATIVE OF A LYSOZYME

FROM PAPAYA LATEX*

BY EMIL L. SMITH, ST. R. KIMMEL,t DOUGLAS M. BROWN, AND

E. 0. P. THOMPSON

(From the Laboratory for the Study of Hereditary and Metabolic Disorders, and the Departments of Biological Chemistry and Medicine, University

of Utah College of Medicine, Salt Lake City, Utah)

(Received for publication, November 8, 1954)

In his survey of lytic activity in various biological materials, Fleming (1, 2) noted that some plant tissues contain lysozyme. Meyer et al. (3) observed later that crude preparations of the plant proteinases, ficin and papain, are very rich in lysozyme activity. We wish to report in this paper the isolation of a lysozyme obtained as a crystalline mercury derivative from commercial preparations of dried papaya latex. The enzymatic behavior, amino acid composition, and some physical properties will be described and compared with the lysozymes obtained from animal tissues.

Our identification of papaya lysozyme is of interest inasmuch as the protein was crystallized during attempts to isolate other proteinases from commercial latex after the successful preparation of crystalline papain (4). Since the recrystallized protein was devoid of proteinase activity, a survey was made of its physical and chemical properties. A study of the amino acid composition showed a relatively low content of histidine and glutamic acid, and relatively large amounts of arginine and tryptophan. These findings, in conjunction with a.basic isoelectric point (pH 10.5) and a relatively low molecular weight near 25,000, suggested an examination for lysozyme activity with Sarcina lutea as the test organism. The new enzyme proved to have an activity on a molar basis about one-fifth that of crystalline lysozyme from egg white. It would appear that this is one of the rare in- stances in which a protein has been isolated and its enzymatic activity identified from its chemical and physical properties.

Method of Assay

Assays of lysozyme activity were performed essentially by the turbidimetric method of Dickman and Proctor (5). Portions of a frozen suspen-

* This investigation was aided by research grants from the National Institutes of Health, United States Public Health Service, and from the Rockefeller Foundation.

t Postdoctoral Fellow of the American Cancer Society, recommended by the Com- mittee on Growth of the National Research Council.

67

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68 PAPAYA LYSOZYME

sion of S. luted were thawed and diluted with water until 1 ml. of this suspension, mixed with 3 ml. of water and 0.4 ml. of 4 M NaOH, showed an apparent optical density at 440 rnp equal to approximately 0.7. Vigor- ous stirring of this suspension at the time of preparation was necessary to insure the absence of large particles. The 5 ml. assay flasks contained 1.25 ml. of the suspension of the organism, 2.5 ml. of 0.1 M acetate buffer at pH 4.65, and, if necessary, 0.5 ml. of 0.05 M cysteine. This mixture was incu- bated for 10 minutes at 39”, 0.5 ml. of enzyme solution was added, and the mixture was diluted to 5 ml. At appropriate intervals, 1 ml. aliquots of the reaction mixture were transferred to 10 X 75 mm. calorimeter tubes, 0.1 ml. of 4 M NaOH was added, and, after 30 minutes at room temperature, the apparent optical density (At) at 440 rnp was measured in a Cole- man junior spectrophotometer. The absorbancy of a completely lyzed bacterial suspension (Af) was determined in the same manner. Complete lysis was achieved by substituting 0.01 pmole of crystalline egg white lyso- zyme2 for the papaya lysozyme preparation and incubating for 30 minutes at 39”. The absorbancy of the unlyzed bacterial suspension (A,) was determined by omitting the enzyme from the.reaction mixture. In both cases 4 M NaOH was added to aliquots of these mixtures as described and the apparent optical density at 440 rnl.c was measured after 30 minutes.

The lysis of suspensions of S. Zutea by papaya lysozyme is a complex reaction which does not follow first order kinetics as described for egg white lysozyme (5). Reasonably consistent results are obtained if it is assumed that the reaction is second order with respect to substrate concentration in accord with the usual equation

k2 = z a(a - z)t (1)

where a is the initial substrate concentration, x is the decrement of a oc- curring in time t, and kz is the velocity constant. Although this treatment is not completely satisfactory, evidence to be presented below suggests that its empirical use is justified, at least for the initial phase of the lytic reaction.

From the above, the specific activity (C,) of papaya lysozyme has been calculated as follows. The initial substrate concentration, So = A0 - A,. The substrate concentration at time t, St = A, - A t. Then, if So or a is called 100, z = St/So, and,

1 We are indebted to Dr. Sherman R. Dickman for the suspensions of S. lutea used in this work.

2 We are grateful to Dr. Edwin E. Hays of the Armour Laboratories for supply-

ing us with a preparation of crystalline egg white lysozyme.


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SMITH, KIMMEL, BROWN, AND THOMPSON 69

kff = y. lysis

lOO(100 - y0 lysis) (2)

cz = kz

mg. protein N per ml. reaction mixture (3)

The number of lysozyme units is defined as the product of CZ and the mg. of protein N in the fraction.

Protein concentration was estimated by the turbidimetric method of Biicher (6) with crystalline papaya lysozyme as the standard. For some of the physical studies, the quantity of dissolved protein was measured refractometrically by assuming a refractive index increment of 0.00184 for a 1 per cent solution. These estimates were in good agreement with those performed turbidimetrically or calculated from the protein N value of 17.2 per cent for crystalline lysozyme (see below).

Isolation and Crystallization

The crystalline mercury derivative of papaya lysozyme can be prepared from dried papaya latex as a by-product in the preparation of crystalline papain (4). The lysozyme activity follows the proteolytic activity closely during this preparation and is not separated from it until the papain crystallizes. The mother liquor from this crystallization, Fraction 5a (4), contains papaya lysozyme, which is virtually pure on the basis of specific activity. The same crystalline lysozyme can also be obtained from the mother liquor, Fraction 3a (4), after precipitation of papain with 0.4 satu- rated ammonium sulfate. Isolation of the lysozyme from Fraction 3a and Fraction 5a is described.3

Procedure I (from Fraction 5a)-When 180 gm. of dried papaya latex4 are used as the starting material, the volume of Fraction 5a is about 425 ml. Enough solid HgClz is dissolved in this solution at 4” to bring the mercury concentration to 0.01 M. A voluminous white precipitate (Frac- tion 5a-la) appears and is permitted to stand overnight in the cold; this is removed by centrifugation, leaving a clear supernatant solution (Fraction 5a-1).

Fraction 5a-1 is brought to 0.4 saturation with solid ammonium sulfate (250 gm. per liter) and again allowed to stand overnight at 4”. The white precipitate (Fraction 5a-2) formed by this treatment is removed by centrifugation and the supernatant solution (Fraction 5a-2a) is discarded.

3 The numbers of the fractions given in the text and in Table I are the same as previously given for the isolation of crystalline papain.

4 We are indebted to the American Ferment Company and to the Wallerstein Lab-

oratories for preparations of commercial papain.


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70 PAPAYA LYSOZYME

Fraction 5a-2 is resuspended in water at a protein concentration of about 1 per cent and the small amount of insoluble material is removed by gravity filtration on fluted paper. Solid ammonium sulfate is added to the resulting solution until a faint persistent turbidity develops. This is followed by addition of solid HgClz (2.7 gm. per liter), and the suspension is stirred slowly in the cold.5 Crystallization occurs in 2 to 3 days, as indicated by the marked sheen exhibited by the suspension. These crystals appear as hexagonal plates microscopically (Fig. 1).

Procedure II (from Fraction &)-Solid ammonium sulfate is added to Fraction 3a until 0.8 saturation is reached, when heavy precipitation occurs. This precipitate is removed by gravity filtration on fluted paper and further concentrated by pressing between sheets of Whatman No. 50 filter paper backed by layers of absorbent paper towels. This procedure requires

FIG. 1. A preparation of the mercury derivative of crystalline lysozyme as seen under 440 X magnification.

about 24 hours and is performed in the cold. The resulting product is a thick cream-colored paste. This paste is stuffed into dialysis tubing (no more than two-thirds full), and is dialyzed against changes of distilled water until the dialysate is free of sulfate. The resulting solution (Frac- tion 3a-1) should contain 7 to 9 per cent protein.

Mercuric chloride (2.7 gm. per liter) is added to Fraction 3a-1 and the resulting precipitate (Fraction 3a-la) removed on fluted paper. The fil- trate (Fraction 3a-2) is brought to 0.4 saturation with solid ammonium sulfate (250 gm. per liter) and adjusted to pH 5.5 by addition of 1 M NaOH. After standing overnight at 4”, the precipitate (Fraction 3a-3) is removed by centrifugation and the supernatant solution (Fraction 3a-3a) discarded.

Fraction 3a-3 is redissolved in the minimal amount of water and is treated with HgClz and ammonium sulfate, as described for Fraction 3a-1. Crystallization will take place in the resulting suspension after stirring in the cold for 3 to 4 days.

6 This is conveniently done with a magnetic stirrer.


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Recrystallization-Papaya lysozyme may be recrystallized repeatedly by dissolving it in water and adding solid ammonium sulfate (250 gm. per liter) foll6wed by HgClz (2.7 gm. per liter). The resulting suspension, if stirred gently at 4”, will exhibit a crystalline sheen in 24 to 28 hours, but stirring has generally been continued for 3 to 4 days to insure complete- ness of crystallization.

Yield-Table I summarizes the results for a typical isolation of lysozyme obtained from both Fraction 5a (Procedure I) and Fraction 3a (Procedure II) with an initial lot of 180 gm. of dried papaya latex (commercial papain). It should be noted that the yields of crystalline product are modest, being only 10 units from Fraction 5a and 1 unit from Fraction 3a, or about 5 per cent of the original lysozyme activity.6 However, no attempt was made to develop a procedure which would sacrifice the yield of crystalline papain. The lysozyme may be obtained by the above procedures essentially as a by-product of the papain isolation.

It is noteworthy that the best activity obtained, CZ = 0.10, is only about 3 times the specific lysozyme activity of the initial extract of the latex; for Fraction I, 62 = 0.032.

Inasmuch as the crystalline lysozyme appears to be essentially homo- geneous, a CZ of 0.10 may be tentatively accepted as the activity of the enzyme. On this basis it appears that almost one-third of the soluble protein of the papaya latex is lysozyme. Since papain represents about 7 per cent of papaya extract (4), the two enzymes account for almost 40 per cent of the soluble protein of the commercial dried latex.

Enzymatic Properties

The crystalline lysozyme contains mercury (see below) and does not manifest it,s full lytic activity unless the mercury is removed. As shown in Table II, the full activation of the mercury derivative is achieved by addition of 0.004 M cysteine. Addition of Versene (ethylenediaminetetra- acetate) has no further effect on the activity. The mercury is readily removed by dialysis of the crystalline enzyme against several changes of 0.01 M cysteine, followed by dialysis against water. Table II shows that such a “metal-free” lysozyme is fully active and that further addition of cysteine

6 It is important to note that assays of lysozyme by the use of the second order velocity constant are rather unsatisfactory with crude preparations of the enzyme.

The lysozyme content was frequently higher, when judged by activity measurements, than was to be expected from electrophoretic analysis. For example, Frac- tion 5 (Table I) is crystalline papain of greater than 95 per cent purity, yet the lytic

activity suggests that 35 per cent of the preparation is lysozyme. In view of the crude nature of the lysozyme assay with an organism as the substrate, it is possible that active proteinases such as papain enhance the lytic activity.


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72 PAPAYA LYSOZYME

TABLE I

Isolation of Crystalline Mercury Derivative of Papaya Lysozyme

A representative run giving yields and activities obtained from 180 gm. of dried latex. Proteolytic activity, Cl, was determined at 39” with a-benzoyl-n-argininamide as the substrate in 0.02 M acetate at pH 5.1 containing 0.005 M cysteine and

0.001 M Versene (4). Lysoayme activity, CZ, was measured at 39” in 0.1 M acetate at pH 4.6 with 0.005 M cysteine as the activator and a suspension of S. Zutea as the substrate. Total activity was calculated by multiplying the specific activity, Ci

or CZ, by the amount of protein iS

Fraction No.

1

2

3 3a 4 4a 5

5a 5a-1 5a-la 5a-2

5a-2a 5a-3 5a-4

1 5a-5

3a-1

3a-la 3a-2

3a-3 3a-3a 3a-4 3a-5

3a-6 3a-7 1 3a-8

-

-

Volume

ml.

725 520

775

755

448 575 Insoluble

525 Crystals

Recrystallizations

185

182 192 43

43 43 Crystals

Recrystalliaations

Crystals (3X)

resent in the fraction.

Total protein

gm.

41

40 26 17

7 22

1.8

4.8 2.7

0.21*

0.23 0.29 0.23

0.50 0.19 1.33 0.32

0.87

2.0

0.90 None

17

16 15 0.50 0.07

0.47 0.28

0.067 None

‘roteolytic Cl

-

’ l

-

Total xoteolytic activity

units

1400 1510 1260

625 550 660

382 252

I

.-

,

,

,

/

0.032 0.035 0.045

0.012 0.087 0.030 0.037

0.100 0.070

None

0.080 0.019 0.063

0.07-0.10

0.017

0.019

0.009 0.026 0.061

0.060

None 0.090

Total lysozyme activity

mits 214 230

195 32 95

104 11 79 31

11 4

10

47

1.5

1.6

2.7

1.0

* It may be noted that some of the fractions yielded different values for Ci from those reported earlier (4). Different batches of dried latex exhibit some variation

in yields and specific activities of the fractions. For recrystallized mercuripapain, C1 values as high as 1.7 have been obtained.

has no effect. Such preparations can be lyophilized with no detectable loss of activity.

The data in Table III show that lysozyme is not inhibited by iodoacetate or by p-chloromercuribenzoate; these findings indicate that the enzyme


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Aysis, 15 min.


contains no essential sulfhydryl group. It has also been noted that egg white lysozyme contains no sulfhydryl groups (7). Since Versene and

TABLE II

Activation of Mercury Derivative of Lysozyme by Cysteine

The reactions were performed at 39” in 0.1 hr acetate at pH 4.0 with a suspension

of S. Zutea as the substrate. Reactions with the lyophilized “metal-free” lysozyme were run in 0.025 M citrate at pH 4.65.

Preparation Additions Protein N

Hg derivative “ “ ,............... “ “ .,.............. “ “ . . . . . . . . . .

Lysozyme (“metal-free”). “ “

<‘ “

mg. per ml. per cent

None 0.053 56 5 X 10M6 M cysteine 0.053 53 5 x 10-4 (‘ (( 0.053 78 5 x IO-3 (6 (( 0.053 81 None 0.004 34 5 X 10m3 M cysteine 0.004 32 5 x 10-S “ ‘( + 0.004 34

1 X lo+ “ Versene

TABLE III

Efleet of Metal Ions and Inhibitors on Activity of Lysozyme

The enzyme was a “metal-free” preparation made by dialyzing the mercury derivative against 0.01 M cysteine for 24 hours, followed by dialysis against several changes of distilled water for 48 hours. The reactions were run at 39” in 0.1 M ace-

tate at pH 4.65. A suspension of S. Zutea was the substrate.

Addition Lysis, 30 min.

None......................

p-Chloromercuribenzoate Iodoacetamide. . . . NaF. . . . . HgClz.

“ ,,..,..,.........,... coc12..................... AgN03.

ZnCls

di

0.0001

0.0001 0.01 0.0001

0.001 0.001 0.001

0.001

per cent

44

46 43 45 24

19 16 33

50

cysteine do not inhibit the enzyme, it would seem that the enzyme contains no essential metal ion. The lytic activity is inhibited by Hg++, Co++, and Ag+ ions. It is striking that, although the enzyme crystallizes with a stoichiometric amount of Hg++ ( see e ow , even 0.001 M Hg++ produces b 1 ) only a partial inhibition (Table III) and the crystalline mercury derivative manifests a substantial activity without removal of the metal ion. This


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74 PAPAYA LYSOZYME

suggests that H&+ is only loosely bound to the protein and that the complex dissociates readily in aqueous solution.

It has already been mentioned that a saGsfactory assay may be obtained on the basis of second order kinetics for the lytic activity. For a system described by Equation 2, a plot of (lOO/(lOO - per cent lysis)) versus time should yield a straight line. Fig. 2 shows that this relationship obtains over at least the first 50 per cent of lysis (the ordinate = 2.0).

The second order rate constant, kz, is essentially a linear function of protein concentration over a satisfactory range, as indicated by the data in Fig. 3. All of the assays reported were obtained in the region where the linear relationship applies.

IO 20 30 40 50 60 TIME-MINUTES

FIG. 2. Effect of lysozyme concentration on the rate of lysis of a suspension of S. Zutea. The experiments were performed at 39” in 0.1 M acetate buffer at pH 4.65 in

the presence of 6.065 M cysteine. The numbers adjacent to each line give the enzyme

concentration in mg. of protein N X lo3 per ml. The linearity of the initial slopes

is consistent with a second order reaction.

Fig. 4 shows the activity of lysozyme as a function of pH. The enzyme shows a sharp optimum at pH 4.65. Meyer and coworkers (3) have already noted that Ficus lysozyme shows an acidic optimum. In contrast to the plant lysozymes, egg white lysozyme manifests its optimal activity near pH 6 (5). The data in Fig. 4 also demonstrate that there are no specific ion effects such as those observed by Dickman and Proctor (5) for egg white lysozyme.

Comparison of the act,ivity of egg white lysozyme with papaya lysozyme indicates that, under the conditions of our assay at the optimal pH (4.65), 0.023 mg. of protein of papaya lysozyme produces 27 per cent lysis in 10 minutes. Under the same conditions, but at pH 6, it requires only 0.0028 mg. of protein of egg white lysozyme to achieve the same degree of lysis. Thus, the animal lysozyme is 8.2 times more active. Since the two proteins differ in molecular weight (see below), the relative activities may also be compared on a molar basis. Such a computation shows that the egg white lysozyme is 4.8 times more active.


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The twice recrystallized papaya lysozyme is devoid of papain activity, as indicated by tests with benzoyl-L-argininamide as substrate (Table I). The procedure was that already described (4). A spectrophotometric assay by the method of Kunitz (8) showed that the lysozyme preparation has no proteolytic action on horse hemoglobin.

It is interesting to note that twice crystallized papain contains some lysozyme activity. However, crystalline mercuripapain (4) has no detectable lytic activity when tested in the presence of cysteine and Versene.

8-

, I I I I I I I 0 2 4 6 8

Mg.PROTEIN N /ml.x103

O.O”t / -\e

0.06

C2

0.04

I 7

3 4 ,

5 6 7 PH

FIG. 3 FIG. 4 FIG. 3. Effect of lysozyme concentration on the second order rate constant for

lysis of S. lutea. The values are taken in part from the experiments shown in Fig. 2. FIG. 4. Effect of pH on the activity of papaya lysoeyme expressed as the second

order coefficient, CZ. The buffers employed were 0.1 M acetate (0), 0.025 M citrate (a), and0.1 Mphosphate (m).

Electrophoretic Studies

These observations were made in a Tiselius apparatus equipped with a schlieren scanning device. The runs were made at 1.5” in univalent buffers at an ionic strength of 0.12. The pH values were measured at room temperature with a Cambridge Instrument Company glass electrode standard- ized with a buffer at pH 4.00 of 0.05 M potassium acid phthalate. Only descending boundaries were used for the measurement of mobility.

Preparations of 2 or 3 times crystallized mercurilysozyme were studied over the range, pH 3.9 to 10.9. The buffer solutions contained 0.02 M cysteine and 0.001 M Versene. The enzyme solutions at pH 10.55 and 10.9 were adjusted to the correct pH with 0.1 M IYaOH; the ionic strength was contributed by NaCl. Fig. 5 presents the electrophoretic patterns obtained in representative runs at four pH values. It is evident from the schlieren curves that the lysozyme preparations are essentially monodis-


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76 PAPAYA LYSOZYME

perse, although occasionally a small amount of an impurity is detectable at levels representing 2 per cent or less of the total area. A

LA B r

FIG. 5. Electrophoretic diagrams of crystalline papaya lysozyme at various pH values. These are descending patterns obtained at 1.5”. A, in acetate buffer at pH 3.9 at a concentration of 0.9 per cent after 166 minutes; B, at pH 10.55 in NaCl at 0.6 per cent after 225 minutes; C, in acetate at pH 5.8 at 0.3 per cent after 150 minutes; D, in Verona1 at pH 7.65 at 0.4 per cent after 210 minutes.

a

I I I I I I I I I I 3 4 5 6 7 8 9 10 11

PH FIG. 6. Electrophoretic mobility as a function of pH for papaya lysozyme (0)

and egg white lysozyme (+). The isoelectric point (PI) of papaya lysozyme is shown. The buffers are identified as follows: a, acetate; u, Veronal; g, glycine; and n, NaCI.

Fig. 6 shows the mobility of lysozyme as a function of pH. Some data obtained in this laboratory with egg white lysozyme under identical conditions are given for comparison. It is evident that the two proteins have the same high mobility near pH 4 but that the curves are different.


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Tanford and Wagner (9) have recently published titration curves for egg white lysozyme. Inspection of these curves shows that they differ greatly from the mobility-pH function for this protein in Fig. 6 and those described earlier (10, 11). It is very likely that these differences indicate a high degree of specific binding of ions by lysozyme. This may also be the cause of the pronounced changes in slope shown by the mobility curve of papaya lysozyme. Papaya lysozyme has an apparent isoelectric point at pH 10.53, whereas egg white lysozyme is isoelectric near pH 11.0 (9).

Sedimentation Xtudies

A Spinco model E electrically driven ultracentrifuge was used for these studies. The runs were made at 59,780 r.p.m., and at room temperature. The controls and procedures were essentially the same as those in earlier

FIG. 7. Sedimentation diagrams obtained with papaya lysozyme at 59,780 r.p.m. A, at pH 3.9 in acetate buffer containing cysteine; B, at pH 7.9 in Tris-NaCl containing cysteine; C, at pH 7.9 with 0.0001 M HgC12; D, at pH 7.9 with the mercury derivative as obtained after recrystallization; E, at pH 7.9 with excess HgC12. The individual pictures are representative of all runs in that only one sedimenting boundary is observed. The sedimentation diagrams illustrated are at the highest protein concentrations shown for the five conditions given in Fig. 8; the capital letters in the two figures correspond.

studies from this laboratory (12-14). The sedimentation data are given in Svedberg units corresponding to water at 20” (sZO,,), as calculated by the procedure of Svedberg and Pedersen (15).

Preparations of 2 or 3 times recrystallized lysozyme gave sedimentation patterns which appear to be monodisperse. Some typical results obtained under various conditions are shown in Fig. 7.

Data relating sedimentation constant and protein concentration appear in Fig. 8. Curves A and B were obtained in the presence of 0.02 M cysteine and 0.001 M Versene in order to remove bound Hg++; under these conditions, the enzyme manifests its full activity. The experiments in Curve A were performed in acetate buffer of 0.12 ionic strength at pH 3.9, whereas those in Curve B were obtained in Tris-NaCP buffer at pH 7.9 at the same ionic strength. Both sets of data indicate that at infinite dilution s20,w = 2.57 S as calculated by the method of least squares.

The measurements in Curve D (Fig. 8) were obtained at pH 7.9 in Tris-

7 Tris = tris(hydroxymethyl)aminomethane.


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78 PAPAYA LYSOZYME

NaCl buffer with the crystalline mercury derivative, but no cysteine or Versene was added. It is evident that the data extrapolate to essentially the same value at zero concentration but the slope indicates that, at higher concentrations, some aggregation of the protein occurs.

A preparation of twice crystallized lysozyme was freed of Hg++ by the dialysis procedure already given. In tests in Tris-NaCl buffer containing 0.0001 M HgClz at pH 7.9, a slight aggregation is produced under these conditions (Fig. 8, Curve C). When a similar experiment was performed with 0.005 M HgC12, a considerable quantity of the mercury salt crystallized

3.4

3.2

3.0

f

?- 2.8c

CONC.- PER CENT

FIG. 8. Sedimentation constant as a function of concentration for papaya lyso-

zyme under various conditions. Curve A, in acetate buffer containing 0.02 M cys-

teine and 0.001 M Versene at pH 3.9; Curve B, in Tris-NaCl buffer containing the same

amounts of Versene and cysteine; Curve C, in Tris-NaCl containing 0.0001 M HgCL; Curve D is mercurilysozyme in Tris-NaCl at pH 7.9; Curve E is at pH 7.9 in Tris- NaCl with excess HgC12. The details of the experiments are given in the text.

during the dialysis. However, studies with this preparation, presumably containing a considerable excess of Kg++, showed that extensive aggregation is produced under these conditions (Curve E). Since in the experiments in Curve E as well as those in Curve C the solutions contained a constant amount of Hg*, successive dilutions having been made with the dialysis medium, it is evident that the extent of aggregation is a function of protein concentration as well as of Hg++ concentration.

Diffusion Measurements

These studies were performed in the electrophoresis cell by the method of Longsworth (16) from photographs taken by the schlieren scanning method. The results were calculated by the height-area method from the


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formula D = A2/4?rH2t, where A is the area under the refractive index curve, H is the maximal height, t the time in seconds, and D the diffusion constant in sq. cm. per sec.

The measurements were made at pH 3.9 in acetate buffer containing 0.02

M cysteine and 0.001 M Versene at a total ionic strength of 0.12. Under these conditions the lysozyme exhibits completely monomeric behavior in the ultracentrifuge (Curve A, Fig. 8). The diffusion studies were made at 1.5” and corrected for the difference in viscosity and temperature in the manner given by Longsworth (16) to give values of D20,w. In each of two runs, the results from the two halves of the cell were computed separately, giving duplicate determinations in each case. Six photographs were taken at intervals between 22 and 120 hours after establishing the boundaries. Areas and heights were measured on tracings of projected enlargements of

TABLE IV

Diffusion Constant of Papaya Lysozyme

The measurements were performed at pH 3.9 in acetate buffer of 0.12 ionic strength containing 0.02 M cysteine and 0.001 M Versene.

Run No. Concentration 020.1 x 10’

per cent sp. cn,. fier sec.

1 0.43 9.29, 9.29

2 1.09 9.14, 9.68

Average .._,__,..,.........._............._...._. 9.35 f 0.17

the individual diffusion curves. The slopes obtained from plots of l/H against 4 gave satisfactory straight lines. The diffusion constants are given in Table IV. The average value for Dpo+ is 9.35 f 0.17 X 10-T sq. cm. per sec.

Molecular Weight

The molecular weight of papaya lysozyme was computed from the sedimentation-diffusion measurements with the aid of the usual formula (15), M = RTs/D(l - VP), where T is the absolute temperature, R is the gas constant, p is the density of water at 20”, and 7 is the partial specific volume.

7 is 0.726 as computed from the amino acid composition given below and the specific volumes of the amino acid residues (17). The amide groups were assigned equally as glutaminyl and sisparaginyl residues.

With S = 2.57 X lo-l3 and D = 9.35 X 10-‘, the molecular weight is 24,300. The frictional ratio (flf ) o computed from these values is 1.19.


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80 PAPAYA LYSOZYME

Elementary Analysis

Samples of papaya lysozyme which had been recrystallized two or three times were prepared for analysis by washing them three times with 70 per cent ethanol; the final wash fluid gave a negative test for sulfate with BaC12. The crystals were then washed successively with 95 per cent ethanol, absolute ethanol, acetone (twice), and ether (twice). After each washing, the crystals were centrifuged, the wash fluid was discarded, and the crystals were thoroughly dispersed in the medium. The dried crystals were then allowed to equilibrate in the air at room temperature for 1 week. The moisture content was 6 to 7 per cent in different batches.

The nitrogen content was determined by the Dumas method and the sulfur content by the procedure of Elek and Hill (18) ; these determinations were performed by Dr. A. Elek of Los Angeles. Duplicate determinations were in close agreement and showed 17.2 per cent nitrogen and 1.88 per cent sulfur.

The analysis indicates a content of 14 atoms of sulfur for 23,900 gm. of protein or 15 atoms for 25,600 gm. However, the studies of amino acid composition given below indicate the presence of 4 moles of methionine and 8 half cystine residues or a total of 12 atoms of sulfur. The presence of more sulfur than expected from the amino acid composition is not surpris- ing in view of the basic character of lysozyme and the method of crystallization of the enzyme from slightly acidic solutions of ammonium sulfate. It is very likely that 2 or 3 moles of inorganic sulfate are present in the crystalline protein.

The mercury content of two samples of lysozyme was determined by the method of Laug and Nelson (19) with dithizone. All the precautions and controls recommended by these investigators were followed. Four separate analyses gave 2.34, 2.36, 2.28, and 2.38 per cent mercury for an average of 2.34 per cent for the anhydrous protein. This indicates that 1 mole of mercury is combined with 8570 gm. of lysozyme. For a content of 3 moles of mercury, the calculated molecular weight is 25,700 in good agreement with t,he estimate from sedimentation-diffusion data of 24,300.

Amino Acid Composition

The lysozyme used for these studies was a 3 times recrystallized preparation of the mercury derivative which was washed and dried in the manner described for the elementary analyses. The hydrolysates were prepared and worked up as already published for carboxypeptidase (20) and papain (21). The amino acid estimations were performed on Dowex 50-X8 by the chromatographic methods of Moore and Stein (22) with the minor changes already given (20). Typical runs with the elution curves with long and


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short columns for a preparation of lysozyme hydrolyzed for 70 hours at 105’ are shown in Figs. 9 and 1O.s

1.0

0.8 z Z 0.6 z g 0.4

0.2

0.6 z v, 0.4 IF! n 0.2

140 160 180 200 220 240 260 i k-+-pH4.25,50°----+pH 4.25,75:

,

PO

40 4

: !8C 4

,a,, 140 160 180 200 k :I:

220 240 260 pH 6.5 CITRATE Y

FIG. 9 FIG. 10

0.8

z 0.6

So.4 Ei 0.2

FIG. 9. Elution curves for a 70 hour hydrolysate of crystalline papaya lysoayme on a 0.9 X 100 cm. column of Dowex 50-X8. The ninhydrin color yields are given for the 1 ml. fractions as optical density and have been corrected for the base-line color but not for the differences in color yield of the various amino acids. The color produced by proline (PRO) was read at 440 rnp; for the other substances at 570 m. ASP = aspartic acid, THR = threonine, SER = serine, GLU = glutamic acid, GLY = glycine, ALA = alanine, CYS = cystine, VAL = valine, MET = methionine, ILEU = isoleucine, LEU = leucine, TYR = tyrosine, and PHE = phenylalanine.

FIG. 10. Elution curve for a 70 hour hydrolysate of lysozyme for the basic amino acids and ammonia on a 0.9 X 15 cm. column of Dowex 50. Base-line color has been subtracted but no correction has been applied for the color yields of the different substances. TRY = tryptophan, HIS = histidine, LYS = lysine, ARC = arginine, TYR = tyrosine, and PHE = phenylalanine.

The data are given in Table V for analyses after 20, 70, and 140 hours of hydrolysis at 105”. As shown in Fig. 9 and Fig. 10, some of the cystine and tryptophan remain after the prolonged hydrolysis, but no attempt was

8 We are indebted to Mrs. Vina Buettner-Janusch for these determinations.


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82 PAPAYA LYSOZYME

made to estimate these. Tryptophan was determined separately by the calorimetric method of Spies (23). Cysteic acid was estimated after per- formic acid oxidation and hydrolysis as described by Schram, Moore, and Bigwood (24). The separation of cysteic acid was performed on a 0.9

TABLE V

Amino Acid Recoveries from Hydrolysates of Lysozyme

The data are given as gm. of amino acid residue per 100 gm. of anhydrous, ash-

free protein. The values for serine, threonine, methionine, and ammonia in the last column were obtained by linear extrapolation to zero time of hydrolysis. Other values omitted from the averages are given in parentheses. Values for the first thirteen amino acids were obtained on a 0.9 X 100 cm. column of Dowex 50, whereas

the basic amino acids and ammonia were separated on a 0.9 X 15 cm. column. Cys- tine was determined separately as cysteic acid (24). Tryptophan was estimated

calorimetrically (23).

Amino acid

Aspartic acid. Threonine.

Serine Glutamic acid. Proline.

Glycine Alanine. Valine.

Methionine. Isoleucine Leucine

Tyrosine Phenylalanine Histidine.

Lysine. Ammonia. Arginine

Half cystine. Tryptophan

-

-

Time of hydrolysis

20 hrs.

10.35 10.44 5.04 4.91 4.90 5.03

6.31 5.26 7.32 6.98 6.25 5.54

6.08 6.57

(3.04) (2.65) 2.04 (1.16)

(5.01) (4.41)

(6.34) 5.42

8.53 8.86

6.47 7.20 1.70 1.50 5.03 5.46

1.49 1.61

8.88 8.22 3.01 3.04

T 70 hrs.

10.65

4.34 3.29 6.31

(5.93) 6.37 5.82 3.09

1.97 5.24 5.55

7.93 6.96 1.46

5.19 2.10 8.24

10.26 10.14 3.97 (4.53) 2.95 (3.70) 5.72 5.64 7.03 7.43 6.34 6.10 5.70 6.15 3.05 3.03 1.65 1.65 5.36 5.09 5.63 5.45 9.07 8.09 7.30 6.99

I- 140 hrs.

Average or extrapolated

10.37 f 0.14 5.31 f 0.13

5.70 f 0.12 5.85 f 0.37 7.19 f 0.19 6.12 f 0.24

6.06 f 0.24 3.06 f 0.02

2.10 f 0.08 5.23 f 0.09 5.51 f 0.08

8.50 f 0.39 6.98 f 0.22 1.55 f 0.10

5.23 f 0.16 1.33 f 0.06 8.45 f 0.29 3.03 f 0.02

5.11

X 15 cm. column of Dowex 50-X8; this amino acid emergesat the column volume in two or three 1 ml. fractions when a buffer solution at pH 3.4 is used. The values given in Table V are actual recoveries of cysteic acid as determined by the calorimetric ninhydrin method (22).

The data in Table V show, in general, that the recoveries of serine, threonine, and methionine decrease with prolonged hydrolysis, although the values for serine and threonine obtained after 140 hours are higher than


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expected. An estimate of the initial content of these three amino acids in the protein was obtained by linear extrapolation to zero time of hydrolysis as described earlier (20, 21). Values omitted from these calculations are given in parentheses in Table V.

It may be noted that the amount of serine and threonine destroyed during hydrolysis for 70 hours, as calculated from the linear extrapolation, is 4.2 X 1O-4 mole per 100 gm. of protein. The excess ammonia produced, computed in the same manner, is 4.8 X 10m4 mole. The accord bet,ween these values suggests that the amounts of these amino acid residues and the amide content of the protein as calculated from the extrapolation are rea- sonable estimates. Similar results have already been described for other proteins analyzed in this laboratory (20, 21).

The recoveries of valine and isoleucine obtained at 20 hours are low and have been omitted from the averages. It is to be expected that peptide bonds involving the carboxyl groups of these amino acids are slowly hydrolyzed (20, 21, 25, 26).

In general, the determinations are in satisfactory agreement and, for the values used, the average deviations are less than 5 per cent, with the ex- ception of those for glutamic acid and histidine, which are slightly higher.

Composition of Papaya. Lysoxyme

Estimates of the composition of papaya lysozyme are given in Table VI. The average molecular weight has been computed on the basis of the methionine content, which indicates approximately 4 residues of this amino acid in 1 mole of protein of 25,000 molecular weight. The average molecular weight computed for all the residues (except cystine and ammonia) is 24,745, in excellent agreement with the value estimated from sedimentation-diffusion measurements, 24,300, and from a content of 3 moles of mercury, 25,700.

In terms of the measured nitrogen content of 17.2 per cent, the recovery of nitrogen calculated from the amino acid determinations and the residue weight recovery are slightly high, but are in accord with the estimated pre- cision of the determinations. The protein does not appear to contain substances other than the usual amino acids.

In general, the calculated number of those amino acids present in small amount is reasonably satisfactory. It has already been noted that small fluctuations in base-line color cause greater uncertainties in determining the content of these amino acids than those present in larger amounts (20, 21). Nevertheless, there appears to be little doubt concerning the number of residues of methionine, histidine, and tryptophan.

We have given the actual recovery of cystine, estimated as cysteic acid, and this value is undoubtedly low since Schram et al. (24) indicate that


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84 PAPAYA LYSOZYME

their procedure yields a recovery of only about 90 per cent of the theoretical amount of cystine. In our experience, recoveries of 80 to 90 per cent have been obtained with cystine itself. Since sulfhydryl groups appear to be absent from lysozyme, it is likely that 8 half cystine (or 4 cystine)

TABLE VI

Composition and Molecular Weight of Papaya Lysozyme

Amino acid

Aspartic acid. .....

Threonine. ........ Serine ............. Glutamic acid. ....

Proline. ...........

Glycine. .......... Alanine .......... Valine. ..........

Methionine ........ Isoleucine ......... Leucine ........... Tyrosine ..........

Phenylalanine ..... Histidine .........

Lysine ............. Ammonia. .........

Arginine. .......... Half cystine ...... Tryptophan .......

Total. ........

I .cid pel 00 gm. xotein

Amino acid j

residue a Jer 100 1

m. I protein __-

gm

10.37

5.31 5.70 5.85

7.19 6.12 6.06

3.06 2.10 5.23

5.51 8.50 6.98

1.55 5.23 1.33*

8.45 3.03 5.11

--

01.35 1

cm.

11.99

6.26 6.88 6.67

8.52 8.05 7.59

3.62 2.39 6.06

6.39 9.44 7.83

1.66 5.96 1.41

9.42 3.56 5.60

17.89

N N as Minimal mol. wt. “,:ti-

mol. wt.

SF. per cent total N

1.26 7.33

0.74 4.30 0.92 5.35 0.63 3.66 1.04 6.05 1.50 8.72 1.19 6.92 0.43 2.50

0.22 1.28

0.65 3.78 0.68 3.95

0.73 4.24

0.66 3.84 0.45 2.62 1.14 6.63 1.16 6.74 3.03 17.62

0.42 2.44 0.77 4.48

1110 24,420 1904 24,752

1528 24,448 2207 24,277 1350 24,300

933 24,258 1173 24,633 3239 25,912 6248 24,992

2164 23,804 2054 24,648 1920 24,960

2109 25,308 8852 26,556 2451 24,510

1204 25,284 1849 24,037

3373 26,984 3644 25,508

-___

7.621 102.45 124,745

alculate1 no. of

&dues :or M = 24,745

i P

I

Lssumed no. of

,csidues

22.3 22

13.0 13 16.2 16 11.2 11

18.3 18 26.5 26 21.1 21

7.6 8 4.0 4

11.4 11 12.0 12

12.9 13 11.7 12

2.8 3 10.1 10 20.6* 21*

13.4 13 7.3 8 6.8 7

228.6 228

* These values are omit,ted from the totals. t Average for all the values given except for half cystine and ammonia.

residues are present in this protein. The difference between the measured and calculated sulfur content has been mentioned above.

The high content of basic amino acids and of amide groups is in accord with the basic properties of lysozyme. The sum of arginine, histidine, and lysine is 26 residues. The sum of glutamic and aspartic acids, 33 residues, minus the twenty-one amide groups, yields only twelve free carboxyl groups from these residues. Presumably the thirteen phenolic groups of tyrosine would be in the ionic form at pH 10.5, the isoelectric point of the enzyme.


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Amino acid analyses of the lysozymes of egg white (7) and spleen (27)

show that these enzymes of animal origin are very similar. The papaya lysozyme resembles these in its relatively low content of histidine and glutamic acid and in its relatively high content of arginine, tryptophan, and aspartic acid; these findings aided in the identification of the plant lysozyme. However, the egg white enzyme is much richer in arginine, tryptophan, aspartic acid, and cystine. The plant enzyme has a much higher content of phenylalanine, tyrosine, and proline.

A comparison of the two crystalline enzymes from papaya latex, papain and lysozyme, whose composition is known reveals that they may be readily distinguished. Papain (21) lacks methionine, contains only 1 resi-

TABLE VII

Recovery of DNP-Amino Acids after Hydrolysis of DNP-Lysozyme

Hydrolysis of the DNP protein was performed at 105” in 6 N HCl. The data are given in moles of DNP derivative per mole of protein. Method I was that of Levy and Chung (30); Method II was that described by Thompson (29).

Time of hydrolysis DNP-glycine by Method I

I

DNP-glycine by Method II

I

e-DNP-lysine by Method II

hrs.

4

8 12 16

20

0.14

0.20 0.26 0.17

0.11

0.34 0.25

8.0

7.3

due of histidine, and has much less proline and phenylalanine, to cite only the most noteworthy differences.

Free Amino Groups

Some preliminary studies of the free amino groups of papaya lysozyme were performed by the methods of Sanger (28). The general procedures followed for the preparation of the dinitrophenylprotein (DNP protein), hydrolysis, and the separation and identification of the DNP-amino acids were those described earlier (29).

The only DNP-amino acids which could be identified by separation on columns (29) or by paper chromatography (30) were DNP-glycine and E- DNP-lysine, and trace amounts of DKP-aspartic acid. Table VII shows the recoveries of DNP-glycine obtained by the method of Levy and Chung (30) after different times of hydrolysis. Separate experiments in which DNP-glycine and c-DNP-lysine were separated by chromatography on Ce- lite columns (29) and estimated spectrophotometrically are also given for two different times of hydrolysis. It is apparent from the results that low


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86 PAPAYA LYSOZYME

recoveries of E-DNP-lysine were obtained in comparison with the estimate of 10 lysine residues found by resolution of hydrolysates on Dowex 50. It is well known that destruction of DNP derivatives is greatly increased in proteins which are rich in tryptophan (31), as is the case for papaya lysozyme.

The recovery of DNP-glycine poses an additional problem inasmuch as this DNP derivative is more labile than most DNP-amino acids to acid hydrolysis. Sanger (28) has found that hydrolysis of DNP-glycine in the presence of insulin, which does not contain tryptophan, results in only a 25 per cent recovery after 12 hours hydrolysis in 6 N HCI. On this basis, our results would indicate that papaya lysozyme contains one N-terminal gly- tine per mole of protein. There is reason to believe, however, that the destruction factors in our experiments were larger because of the lower than expected recovery of c-DNP-lysine, approximately 80 per cent rather than the 95 per cent expected from the studies of Porter and Sanger (32). Fur- thermore, an experiment in which DNP-glycine was added to DNP-lysozyme, and hydrolysis was performed for 12 hours in 6 N HCl, gave a recovery of only 22 per cent of the theoretical amount of added glycine. When this correction factor is applied, a yield of 1.55 moles of N-terminal glycine is obtained.

From these data it appears that papaya lysozyme may consist of two peptide chains each with N-terminal glycine; however, the extensive destruction of DNP-glycine necessitates some caution in accepting this value. Other methods will have to be studied in order to determine conclusively the number of iv-terminal glycine residues. In any case, it is striking that the plant enzyme is different from the animal lysozymes which possess a single N-terminal lysine per mole of protein (31, 33).

DISCUSSION

In his description of lysozyme and his study of its biological distribution, Fleming (1, 2) emphasized the important. r81e which this enzyme probably plays in the defense of the animal organism against many air-borne bacteria. He also noted that many plants contain lysozyme, but that the activities are much weaker than in the rich animal sources. The existence of rich plant sources of this enzyme suggests a similar important r81e for lysozyme in the defense of plants. Presumably in species like papaya and Ficus, which grow in a tropical environment, the lysozymes aid in prevent- ing invasion of the unripe fruits by saprophytic organisms. Inasmuch as the proteolytic enzymes of these fruits can also attack many organisms, including some living animals, these plants appear to be equipped with a strong defense against parasitic invaders.

It is certainly noteworthy that enzymes with the ability to lyze certain bacteria are present in both animals and plants. The work of Meyer,


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Hahnel, and Steinberg (3) indicates that both types of enzymes depoly- merize the same mucopolysaccharide and hydrolyze this fraction with the liberation of acetyl hexosamine.

The high content of lysozyme in egg white (7) and in spleen (27) has been noted. On the basis of admittedly not very precise assays of activity, lysozyme appears to comprise about one-third of the soluble protein of papaya latex. The electrophoretic Component 3 given by Kimmel and Smith ((4) Table II) is the fraction in which lysozyme is found; this represents about 65 per cent of the total protein. However, this fraction contains chymopapain and probably other proteins also, so that it is not possible from the electrophoretic analysis alone to estimate the lysozyme content of papaya latex with any certainty. Nevertheless, both types of estimate, lytic activity and electrophoretic analysis, are consistent with a high content of lysozyme in the latex. Meyer et al. (3) have estimated that the lysozyme content of crude ficin is about 20 per cent.

The properties of the papaya lysozyme reveal many interesting features, perhaps the most noteworthy being that which led to this study; namely, its ability to form a crystalline mercury derivative, and the fact that, as yet, we have been unable to prepare crystals of this enzyme in any other form. *Other crystalline mercury derivatives of proteins have been prepared; enolase (34), mercaptalbumin (35), and papain (4), but in the last two cases there is evidence which suggests that the mercury binds with sulfhydryl, whereas for papaya lysozyme the evidence indicates that such groupings are not involved. This is suggested by the findings that sulfhy- dry1 agents do not inhibit the enzyme, and that the combination of mercury with the protein is much more labile than might be expected for a mercury-sulfhydryl binding, as shown by the fact that the protein-metal complex dissociates on dilution or simple dialysis against water. Rather than a sulfhydryl binding, it is more likely that Hg++ is bound to carboxyl or imidazole groups. The analytical findings of 3 moles each of histidine residues, mercury atoms, and sulfur, presumably as inorganic sulfate, are suggestive. A type of weak binding in which each atom of mercury is bound both to imidazole and to inorganic sulfate would be in accord with all of the known properties of these substances.

A comparison of papaya lysozyme with those from egg white and spleen reveals many interesting differences and similarities, some of which have already been mentioned. One further point worth noting is that the animal and plant enzymes have similar, although not identical, high isoelectric points, and that the number of arginine and lysine residues in both types of enzyme is very similar. However, in the animal enzymes arginine predominates (7), whereas papaya lysozyme contains almost equivalent amounts of these two basic amino acids. The similarity in biological function of these bases is strikingly revealed by the studies of du Vigneaud and


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88 PAPAYA LYSOZYME

his coworkers (36), who have demonstrated that, in the vasopressins of swine and bovine glands, the hormone of the former contains a single lysine residue, whereas that of the latter contains arginine in the same locus in the peptide chain. A similar biological relationship is seen in the specificity of trypsin which has been found to hydrolyze only peptide bonds involving arginine or lysine (37).

It is to be hoped that the fundamental similarities in the properties of the animal and plant lysozymes shown in their basicity and wealth of basic amino acids, as well as of aspartic acid and tryptophan, may eventually aid in understanding the enzymatic properties of these proteins.

SUMMARY

1. Two methods are described for obtaining a crystalline mercury derivative of lysozyme from dried papaya latex. The enzyme appears to represent about one-third of the soluble protein of the latex.

2. The enzyme is not inhibited by sulfhydryl reagents or poisons of metal ions. Hg* is inhibitory and must be removed from the crystalline material for full activity. Lysis of Xurcina Mea follows second order kinetics for the initial 50 per cent of lysis. The enzyme is opt.imally active at pH 4.65. It is about one-fifth as active as egg white lysozyme on a molar basis.

3. The enzyme is essentially monodisperse on electrophoresis over the pH range from 3.9 to 10.9. The mobility curve of the papaya enzyme dif- fers from that of egg white lysozyme. Papaya lysozyme is isoelectric at pH 10.5.

4. Papaya lysozyme has a sedimentation constant (sZO,,,,) of 2.57 S. In the presence of Hg++ some aggregation occurs. The diffusion constant (DZO,W) is 9.35 X lO-’ sq. cm. per sec. The partial specific volume (8) computed from the amino acid composition is 0.726; the molecular weight 24,300.

5. A study of its composition indicates 3 moles of mercury, 3 moles of inorganic sulfate, and 3 moles of histidine, which suggests a relationship among these components. The amino acid composition indicates a basic protein rich in arginine, lysine, aromatic amino acids, and aspartic acid.

6. The protein contains N-terminal glycine unlike animal lysozymes which possess N-terminal lysine.

7. The possible significance of plant lysozymes is discussed. A comparison of the properties of the animal and plant enzymes is given.

BIBLIOGRAPHY

1. Fleming, A., hoc. Roy. Sot. London, Series B, 93, 306 (1922). 2. Fleming, A., Proc. Roy. Sot. Med., 26, 71 (1932). 3. Meyer, K., Hahnel, E., and Steinberg, A., J. Biol. Chem., 163, 733 (1946).


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4. Kimmel, J. R., and Smith, E. L., J. Biol. Chem., 207,515 (1954).

5. Dickman, S. R., and Proctor, C. M., Arch. Biochem. and Biophys., 40,364 (1952). 6. Biicher, T., Biochim. et biophys. acta, 1, 292 (1947). 7. Fevold, H. L., Advances in Protein Chem., 6, 188 (1951).

8. Kunits, M., J. Gen. Physiol., 30,291 (1947). 9. Tanford, C., and Wagner, M. L., J. Am. Chem. Sot., 76,333l (1954).

10. Longsworth, L. G., Cannan, R. K., and MacInnes, D. A., J. Am. Chem. Sot., 62, 2580 (1940).

11. Alderton, G., Ward, W. H., and Fevold, H. L., J. Biol. Chem., 16’7,43 (1945).

12. Smith, E. L., Brown, D. M., Fishman, J. B., and Wilhelmi, A. E., J. BioZ. Chem., 177, 305 (1949).

13. Smith, E. L., Brown, D. M., and Laskowski, M., J. Biol. Chem., 191, 639 (1951).

14. Smith, E. L., and Brown D. M., J. BioZ. Chem., 196, 525 (1952). 15. Svedberg, T., and Pedersen, K. O., The ultracentrifuge, Oxford (1940). 16. Longsworth, L. G., Ann. New York Acad. SC., 41, 267 (1941). 17. Cohn, E. J., and Edsall, J. T., Proteins, amino acids and peptides, American

Chemical Society monograph series, New York (1943).

18. Elek, A., and Hill, D. W., J. Am. Chem. Sot., 66,3479 (1933).

19. Laug, E. P., and Nelson, K. W., J. Awn. Oficial Agr. Chem., 26,399 (1942). 20. Smith, E. L., and Stockell, A., J. BioZ. Chem., 207,501 (1954). 21. Smith, E. L., Stockell, A., and Kimmel, J. R., J. BioZ. Chem., 207, 551 (1954).

22. Moore, S., and Stein, W. H., J. BioZ. Chem., 176, 367 (1948); 192, 663 (1951). 23. Spies, J. R., Anal. Chem., 20, 30 (1948); 22, 1447 (1950). 24. Schram, E., Moore, S., and Bigwood, E. J., Biochem. J., 67,33 (1954). 25. Harfenist, E. J., and Craig, L. C., J. Am. Chem. Sot., 74, 4216 (1962).

26. Harfenist, E. J., J. Am. Chem. Sot., 76, 5528 (1963). 27. Jolles, G., and Fromageot, C., Biochim. et biophys. acta, 11, 95 (1953); 14, 219

(1954).

28. Sanger, F., Biochem. J., 39, 507 (1945). 29. Thompson, E. 0. P., J. B?‘oZ. Chem., 207, 563 (1954). 30. Levy, A. I,., and Chung, D., Nature, 174, 126 (1954).

31. Thompson, A. R., Nature, 166, 390 (1951). 32. Porter, R. R., and Sanger, F., Biochem. J., 42, 287 (1948).

33. Jolles, P., and Fromageot, C., Biochim. et biophys. acta, 24, 228 (1954). 34. Warburg, O., and Christian, W., Biochem. Z., 310, 384 (1941). 35. Hughes, W. L., Jr., J. Am. Chem. Sot., 69, 1836 (1947).

36. du Vigneaud, V., Lawler, H. C., and Popenoe, E. A., J. Am. Chem. Sot., 76,488O (1953).

37. Bergmann, M., and Fruton, J. S., Advances in Enzymol., 1,63 (1941).


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Brown and E. O. P. ThompsonEmil L. Smith, J. R. Kimmel, Douglas M.

PAPAYA LATEXDERIVATIVE OF A LYSOZYME FROM

CRYSTALLINE MERCURY ISOLATION AND PROPERTIES OF A

1955, 215:67-89.J. Biol. Chem.

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