l-Asparaginase from Erwinia carotovora

The physicochemical properties of the antileukemic Lasparaginase from Erwinia carotovora have been studied and compared with those of the corresponding clinically effective enzyme from Escherichia coli B. Although both enzymes are tetramers with similar molecular weight, the amino acid compositions are distinctly different and the Erwinia enzyme is more basic as judged by polyacrylamide electrophoresis. Of the 48 to 50 tyrosyl residues in native Erwinia asparaginase, only about 10% ionize with a normal pK, while 30% of these residues in the E. coli enzyme titrate normally. The native tetramer (s~(,,~ = 7.2) is considerably more stable than the corresponding tetramer from E. coli. Asparaginase from Erwinia is only partially dissociated in 8 M urea, whereas the E. coli enzyme is completely converted to the 1.7 S monomer under the same conditions. Guanidinium chloride at concentrations of 3.5 M completely dissociates Erwinia asparaginase. Dissociation is accompanied by the appearance of an ultraviolet difference spectrum with a maximum at 288 nm. The rate of dissociation is markedly increased by the addition of alcohols to the denaturant. Dissociation in the presence of alcohols occurs in two distinct steps whose rates increase as the length of the alkyl side chain of the alcohol increases. Succinylation of the lysyl residues results in marked increases in mobility of the modified enzyme on polyacrylamide gels, but does not affect the state of aggregation of the tetramer or its catalytic activity. In addition succinylation does not affect either the rate of dissociation in guanidinium chloride or the extent of reconstitution to the active enzyme. The presence of five bands on polyacrylamide gel electrophoresis of hybrids prepared from the native and succinylated enzyme indicates that Erwinia carotovora asparaginase is composed of four identical subunits.

Of the 48 to 50 tyrosyl residues in native Erwinia asparaginase, only about 10% ionize with a normal pK, while 30% of these residues in the E. coli enzyme titrate normally.
The native tetramer (s~(,,~ = 7.2) is considerably more stable than the corresponding tetramer from E. coli. Asparaginase from Erwinia is only partially dissociated in 8 M urea, whereas the E. coli enzyme is completely converted to the 1.7 S monomer under the same conditions. Guanidinium chloride at concentrations of 3.5 M completely dissociates Erwinia asparaginase. Dissociation is accompanied by the appearance of an ultraviolet difference spectrum with a maximum at 288 nm. The rate of dissociation is markedly increased by the addition of alcohols to the denaturant. Dissociation in the presence of alcohols occurs in two distinct steps whose rates increase as the length of the alkyl side chain of the alcohol increases.
Succinylation of the lysyl residues results in marked increases in mobility of the modified enzyme on polyacrylamide gels, but does not affect the state of aggregation of the tetramer or its catalytic activity.
In addition succinylation does not affect either the rate of dissociation in guanidinium chloride or the extent of reconstitution to the active enzyme. The presence of five bands on polyacrylamide gel electrophoresis of hybrids prepared from the native and succinylated enzyme indicates that Erwinia carotovora asparaginase is composed of four identical subunits.
-1 larger supply of the enzyme that would furnish sufhcicnt material for clinical testing leas provided by E'scherichia coli 1< (3). Many bacterial aaljaraginases \verc subsequentl> screened to determine their effectiveness as antilymphoma agents (4). The enzyme isolated from 1+winia carotovora is pharmacologically active (5) and is as effective as the enzyme from E. coli 13 in the treatment of acute lymphocgtic leukemia.' In an effort to dett,rmine the molecular bases of clinical effectiveness, the l)rcsCllt study \vas undcrtakcn to compare the physicochemical propertics of the asparaginasc from &winia carotovora xith those of the c~~zyme from I<scherichia co2i I< (6-9). M.\TISRI.\LS ASI) METHOI)S L-iZsl)aragitl:~str from Krwinia carofovora was generously supplied by thcl 1 Jrug Evaluation Isranch of the National Cancer Institute.
The 1yol)hilizrd po\vdcr nas dissolved in 0.05 RI phosphate buffer, pH 7.5, to girt a stock solution containing 15 mg per ml. This solution was dialyzed against several changes of the same buffer in order to remove salts. The enzyme x-as homogeneous as determined from the monodisperse boundary obtained in the analytical ultracelltrifuge.
The enzyme has a sedimentation coefficient (.Q~,~) of 7.2. The native enzyme showed three distinct bands on polgac~rylamide gels lvhich were isolated and all lvere found to bc catalytically active. Aspartic acid /.-p-nitroanilide n-as a generous gift from Dr. Osamu Takenaka of the Tokyo Institue of Technology. Urea and guanidinium chloride \vere recrystallized from aqueous ethanol using decolorizing charcoal to adsorb impurities. The crystalline reagents mere stored in the cold in dark containers and solutions Fvere prepared immediately before use. Dodecyl sulfate \vas a highly pure grade purchased from Mann. All organic solvents lvere spcctroquality grade and were purchased from Mathcson, Coleman and f<ell.
[W]Succinic anhydride \vas purchased from International ('hemical and Nuclear Corporation and n-as diluted \vith unlabeled succinic anhydride (Eastman) to a specific activity of 1.8 X lo6 cpm per mg.
1GwinG.z asparaginasc \vas succinyfated with [14C]succinic anhgdridc at pIT X.0 according to the method described preriously (7). The extent of reaction with lysyl residues \yas determined from titration of free amino groups with ninhydrin, according to the method of Moore and Stein (lo), and ITith trinitrobenzenc sulfonic acid (1 I).
Enzgmc activity of asparagitlase leas determined with Kessler's reagent by measuring the ammonia released from asparagine (12). Catalytic activity was also determined from the increase in absorbance of aspartic acid P-p-nitroallilidc at 410 nm after hydrolysis (13). The synthetic substrate was used at concentratiolls of 2 mM or less and was prepared in pH 8.0 buffer. 'l'hero was no detectable hydrolysis of t.he substrate in the absencc of enzyme. 1 )isc electrophoresis was carried out using 7 c/C polyacrylamidc gels as dcscribcd previously (6). IXfferencc spectra were obtained with the Cary model 15 spectropllot,oliictcr using tandem double cuvettes such as those described by Herskovits and Laskowski (14). In order to record spectra as far as possible into the ultraviolet region while maintaining a reasonable degree of resolution, it was necessary to incrcasc the voltage 011 the photomultiplier to its maximum level. Fluorescence spectra were recorded with the ~~initlco-l~oTT-mall spcctrophotofluorometcr which was modified according to the specifications described by Chen (15).
Sedimentation velocity studies were carried out using the Spinco model E analytical ultracentrifugc equipped lyith ultra-viol& absorption scanner optics.
When urea and GdnW solutions were used, the observed sedimentation coefficients were corrected to the density and viscosity of water at 20" (s~~,~~), assuming a partial specific volume of 0.73 ml per mg obtained with the enzyme from E. coli J!J (16). In that cast of velocity studies carried out in dodecyl sulfate, that stxdimentation co&icicnts that are reported are observed values sincBe dodecyl sulfate has a tendency to aggregate, and reliable values for its density and viscosity are not available.
Similarly partial specific volumes of the succinylated derivatives were not determined and sedimentation coefficients are reported as observed values. The enzyme was dried to constant weight in a vacuum oven at,55".
The extinction coefficient was determined to be I"" ,219 1,111 = 6.4. This value was used to dcterminr protein concentrations throughout these studies. Amino acid analyses were carried out with the Spinco model 12OC automatic amino acid analyzer.
Asparaginase was hydrolyzed in VCLCUO with constant boiling HCl at 1 IO" in the presence of 10 ~1 of 5% phenol and I ~1 of mercaptocthanol for 24, 48, and 72 hours. Performic acid oxidation was carried out by a modification of the procedure described by ITirs (17).
The rate of dissociation of Er~inia asparaginasc, IT-as followed at 288 nm. A solution of the enzyme (from 1 to 5 mg 1)~ ml) in 0.05 bl phosphate buffer, p1-I 7.5, was placed in the sample compartment of the spectrophotomett~r while the same conren tration of enzyme in the denaturing medium was placed in the solvent compartment.
The incarease in 288 nm absorbance was recsorded within 15 s after mixing enzyme and drnaturant and the change was monitored continuously for 10 min. In a scparate experiment the spectral change from 250 to 330 nm was scanned at 1-min intervals.
Reconstitution of the tetramer was &ctcld by dialyzing the denatured subunits in GdnCl against gradually decreasing conccntrations of the denaturant.
For example, the enzyme in 6 >I GclnCl was first dialyzed against 3 in GdnCl followed by dialysis against 1.5 RI GdnCl, etc., until all of the denaturant was rcmovcd.
Enzyme concentrations from 1 mg per ml to 10 mg per ml were used.
Circular dichroic spectra were obtained with a Cary model 60 recording spectropolarimeter fitted wit,11 a model 6001 circular 2 The abbreviations used are: GdnCl, guanidinium chloride; SIX, sodium dodecyl sIllfate. dichrosim attachment and set for a half band width of 15 .\. JJeasuremcnts were made at 27". l'hc concentration of c'nzymc was 0.01 y0 and a path length of 1 .O mm was used.
Hybrids of the native and succinylatcd enzyme wcrc prcparcd by first dissociating the proteins in 6 &f GdnCl.
Aliquots of each were withdrawn and were reconstituted. The remaiiitler of the solutions in GdnCl were mixed in the following ratios of succinylated to native: 9:1, 7:3, 6:4, 5:5, and 2:8. These mistures were dialyzed against buffer and the products were analyzed using polyacrylamide gel electrol)hol,c,sis.

Chemical and Physical Properties
Amino Acid Composition-The complete amino acid composition of Bwinia asparaginase is summarized in Table 1. The moles of each amino acid in a monomer were calculated on the basis of a molecular weight of 33,500 for the subunit (18). Two amino acids that were present in the 13. coli enzyme in small amounts and that are completely absent in Erwinia asparaginase 1 (Zefl). Disc gel electrophoresis of L-asparaginase from Erwinia carotoz~ora @ft) compared with the enzyme from Escherichia coli B (right). Electrophoresis was carried out on 7% polyacrylamide gels using 0.1 mg of protein.
The gels were stained with Amido schwarz.  (19). There are also many other marked differences in amino acid composition of the two enzymes. These differences are also reflected by the patterns obtained on polyacrylamide gels which are shown in Fig. 1. L-Asparaginase from Erwinia carotovora (gel on left) shows three distinct bands very near the top of the gel column.
All three bands have the ability to catalyze the hydrolysis of aspartic acid @-p-nitroanilide.
The gel on the right indicates the greater mobility of asparaginase from Escherichia coli B. The major band which has travelled more than three-fourths the length of the polyacrylamide column represents 95% of the total protein and is the 7.2 S tetramer, while the remaining 5q7b of the protein is assigned to the higher molecular weight octamer (20, 21).
Nature of Tyrosyl Residues-Erwinia and E. coli asparaginase have a similar number of tyrosyl residues (44 in E. coli and 48 in Erwinia), but there is a marked difference in the number of residues that titrate with a normal pK. The number of normal tyrosines can be most readily determined from a comparison of the spectrum at pH 6.5 (where all of the tyrosyl residues are protonated) with the spectrum at pH 10.2, where only the normal tyrosines will be ionized.
Ionization is accompanied by a shift in the absorption maximum from 274 nm (nonionized) to 294 nm (ionized) along with a doubling of the extinction coefficient. The dashed curve in Fig. 2A was obtained when the spectrum of Erwinia asparaginase at the lower pH was subtracted from the spectrum of the enzyme at pH 10.2 and should represent the number of "normal" tyrosines.
The presence of two maxima (290 nm and 298 nm) is not the behavior expected of these groups. 280 300 320 WAVELENGTH hn) from ErwiGa carotovora (A) and from Escherichia coli B (B). A 1.0 X 10V5 M solution of Erwinia asparaginase in pH 6.5 buffer was placed in the solvent compartment of the spectrJphotometer and an equimolar solution of the enzyme at the pH specified in the figure was placed in the sample compartment.
The concentration of E. coli in these experiments was 8.2 X 10-e M.
The usual difference spectrum is shown by the dashed curve in Fig. 2B which was obtained with E. coli asparaginase.
The higher concentration of Erwinia asparaginase was used in these samples so that the difference spectrum at pH 10.2 could be more easily seen.
All of the tyrosyl residues (normal and abnormal) are ionized at pH 12.8 as shown by the solid curves in these figures.
These results show that only 10% of the tyrosyl residues of Erwinia asparaginase ionize normally, while 30yo of these residues in the E. coli enzyme have a normal pK.
Fluorescence Studies-When a solution of Erwinia asparaginase in 0.05 M phosphate buffer, pH 7.5, was excited at 280 nm, a fluorescent band appeared with maximum intensity at 303 nm. This is the behavior exhibited by free tyrosine as well as by proteins that contain tyrosyl residues and no tryptophan (22). In contrast, the E. coli enzyme, which contains a single tryptophan residue along with 11 tyrosyl groups per subunit, exhibits an intense fluorescence at 317 run. This emission band is almost exclusively derived from the tryptophan (6). In order to determine whether or not the fluorescence of tyrosyl residues in Erwinia asparaginase was quenched as it is in a large number of proteins (22), we compared the fluorescence intensity of the 303 nm band obtained from a solution of the enzyme with an absorbance of 0.200 at 275 nm with that from a solution of tyrosine with the same absorbance.
The intensity of the 303 nm band was identical in both cases, indicating that fluorescence quenching of tyrosyl residues does not occur in Erwinia asparaginase. Circular Dichroic Xpectm-There have been many attempts to correlate the shape of circular dichroic spectra and the magnitude of cllipticities at particular wave lengths with the folding of the polypeptide chain (23, 24). Fig. 3 shows the circular dichroic spectrum of asparaginase from Erwinia carotovora (dashed curve) compared with the spectrum of the E. coli enzyme (so/id curve). Although the shape of the tTT-o curves is identical, there is a very significant difference in the magnitude of the ellipticities.
If we use the equation suggested by Greenfield and Fasman (23) to give a rough approximation of the helical content of proteins, 13c/, of the polypeptide chain of Erwinia asparaginase is in the a-helix compared with 3Oc/, for the E. coli enzyme.

Dissociation and Reconstitution
The extent of dissociation of the tetrameric aggregate was measured by sedimentation velocity ultracentrifugation in urea and in guanidinium chloride. The greater stability of the oligomeric structure of &winia asparaginase compared with the E. coli enzyme is indicated by its behavior in urea. Exposure of Erwinia asparaginase to 8 M urea for 5 hours results in 50% dissociation of the 7.2 S tetramer to its 1.7 S monomer (Fig. 4~). I  I  I  I  I  I   I  I  I  I  I  I  I  I  210  220  230  240  250 WAVELENGTH (nm) By contrast the E. coli enzyme is completely dissociated in 4 M urea during the same 5-hour time period. Complete dissociation of Erwinia asparaginase can be effected in 3.5 M GdnCl or by the addition of 10% n-propyl alcohol to the solution of 8 M urea (Fig. 4b).
The extent of dissociation as determined by sedimentation methods is correlated with the absorbance of the 288 nm peak in the ultraviolet difference spectrum of Erwinia asparaginase. The completely dissociated Brwinia enzyme in 6 M GdnCl gives the ultraviolet difference spectrum shown in Fig. 5. Yanari and Bovey (25) suggested that ultraviolet difference spectra of proteins arise when the chromophore of the amino acid residue is transferred from a hydrophobic environment in the native enzyme to a hydrophilic one in the denatured state. The two bands in the difference spectrum of Erwin&z asparaginase (281 nm and 288 nm) are both assigned to tyrosyl residues.
Previous studies with E. coli asparaginase (6) and with ribonuclease (26) suggested that each tyrosyl residue that gets transferred from its microenvironment of low dielectric constant in the native enzyme to the aqueous environment has an extinction coefficient AE of 1000 in the 287 nm band of the difference spectrum.
The extinction coefficient of the 288 nm band shown in Fig. 5 is 42,000 which suggests that 42 out of the total of 48 tyrosyl residues are "buried" in native Erwinia asparaginase.
The rate of dissociation of the Erwinia tetramer could be monitored by following the rate of appearance of the 288 nm band in the ultraviolet difference spectrum. The most convenient concentration of GdnCl for these studies was found to be 3.5 M. The rate of dissociation of Erwinia asparaginase is shown in the bottom curve of Fig. 6. The addition of alcohols with increasing chain length at concentrations of 5% by volume is also shown in this figure.
In the presence of the alcohols it is apparent that dissociation takes place in two distinct steps, both of which increase as the length of the alkyl chain of the alcohol increases. The results suggest that the alcohols aid in disruption of the hydrophobic forces that participate in subunit interactions. These conclusions are qualitatively in agreement with those found by Herskovits et al. (27) and by Tan and Lovrien (28).
The effect of increasing concentrations of dodecyl sulfate on the dissociation of Erwinia asparaginase was also studied by sedimentation velocity and ultraviolet difference spectroscopy. The sedimentation coefficient of the enzyme decreased from 7. in the sample compartment and the denatured sample was used as the blank. At zero time the enzyme was mixed with the appropriate denaturing solvent and the sample was placed in the solvent compartment,.
The first reading was taken within 15 s after mixing and changes in absorbance were monitored for 20 min. S in 0.05 M phosphate buffer, pH 7.5, to 5.9 S in 0.57$ SIX and to 5.0 S in 57, SDS. This behavior is in sharp contrast with the results obtained with E. coli asparaginasc, which had a sedimentation coefficient of 2.4 S in 0.1% SIX. The ultraviolet absorptiou spectrum of the Erwinia enzyme n-as the same in 0.5% to 50/, SDS as it was in buffer, which also contrasts with the results obtained with the coli protein (6). All of t'hese results suliport the observation that the tetrameric structure of Erwinia asparaginase is considerably more stable than the enzyme isolated from Escherichia coli 13.
Reconstituted Erwinia asparaginase was prepared by rernoval of GdnCl from solutions of the denatured enzyme by dialysis against 0.05 M phosphate buffer, pH 7.5. Concentrations of proteiir greater than 1 mg per ml were avoided in this procedure due to formation of a precipitate.
The reconstituted product had 90% of the original catalytic activity and showed only one band on disc gels. Since the native enzyme from Erwinia showed three bands on disc gels (Fig. l), it appears that dissociation followed by reconstitutioir results in random aggregation of the slightly different subunits.
Intergeneric Hybrids-Although the strength of intersubunit forces is quite different between the Erwinia and E. coli monomers, an attempt was made to prepare a hybrid tetramer composed of subunits from the two different bacterial genera. IMferent ratios of the two enzymes in 6 M GdnCl were mixed and then dialyzed against buffer. Electrophoresis on polyacrylamide gels showed that the E. coli subunits aggregated with each other as was true with the Erwinia enzyme and there was no evidence of the formation of intergeneric hybrids.
Erwinia asparaginase was succinylatcd usirig increasing rnolar concentrations of [i4C]succinic anhydride at pH 8.0. The number of lysyl residues modified by succinylation are summarized in Table II together with the catalytic activity, sedimentation coefficients, and the distance the modified enzyme migrates on polyacrylamide gels. The bands obtained with the 1:2, 1 :l, and 2:l succinylated samples were rather diffuse in contrast to the very sharp bands obtained with the 1:4, 4: 1, and 8 : 1 derivatives. It is apparent that cxtensivc succinylation does not destroy catalytic activity nor does it cause the tetramcr to dissociate. The retention of the tetrameric structure is in sharp contrast to the behavior of the I<. coli enzyme (7) and with t,he behavior of a large number of oligomeric proteins (29)(30)(31)(32)(33)(34)(35).
Kot only is the Erwiniu tetramer retained after succinylation, but the rates of dissociation of the modified enzyme in 3.5 M GdnCl remain unaffected.
The succinglated tetramer could be fully reconstituted from 6 M GdnCl solutions without the formation of a precipitate in solutions where the protein c~olrcentration was as high as 2 mg per ml. The presence of even a few succinyl groups in E. coli asliaraginasc had been found to iriterferc with rrconstitution (7).

Hybridization
All of the succinylated samples obtained with iYr'ru;inia asparaginase fulfilled the criteria described by X&hen and Schachman (29) for a proper chemical derivative that could forrn hybrids with the native subunit.
The 4: 1 and 8: 1 succinylated samples formed the sharpest bands on disc gels alid migrated farthest from the native sample, making thrrn most suitable for hybridization studies.
The native and succinylated samples were dissociated separately in 6 M GdnCl.
Aliquots from each were withdrawn and dialyzed agaiust pH 7.5 buffer to serve as the controls (Fig. 7). Varying ratios of the dissociated enzymes were mixed and were then dialyzed.
The reconstituted product obtained from a 70 : 30 rnixture of succinylated-native enzyrne gave the disc gel pattern shown by the middle gel in Fig. 7. Five bands can be detected. The slowest migrating band represcrits the unmodified enzyme and the most rapidly migrating band corresponds to the 4:l succinylated derivative.
The three intermediate bands represent hybrid tetramers.
The formation of five bands in the hybridizatioir experiment constitutes evidence that L-asparaginase from Brwinia carotovoru is composed of four identical subunits.
Although the molecular weights of the L-asparaginascs from Escherichia coli (19) and Erwinia carotovoru (18) are identical and both are composed of four identical subunits (7 and the present study), there are some very marked differences in their amino acid composition and the stability of the tetrameric aggregate. Tryptophan and cystine are both present in B. co/i but are completely absent in the Erwinia cnzymc.
The over-all difference in the composition of proteins is also reflected in their migration in an electric field. The E. co/i enzyme travels three-quarters of the distance down a column of polyacrylamide gel, while the Erwinia asparaginase barely penetrates the gel matrix.
The stability of the Erwiniu tetramer is reflected in its resist-+ Unmodified asparaginase >uLL,l l ,y,asparaginase (4: I ) Frc:. 7. Disc gel electrophoresis of hybrids prepared from native and succinylateil aspnragin: se. ('cl (n the /q/'l represents the reconstituted irnmocificd rnzymc, while the gel on the extreme rzghl was the 4:l srlcci1l?rl-2~sl,ar.t~in:lse reconstituted from 6 M CJdnCl. The middle gtl shows the three hybrid bands : s well as the st'irting mat-ri :l obt :inrd from :L 70:30 mi\;turc of succinylnted to native enzyme. ante to dissociation by urea or by dotlccyl sulfate.
Asparaginase from Bscherichia coli is completely dissociated by 4 M urea and by 0.1 y0 SDS, while the Erwinia enzyme is unaffected by either of thrse denaturants.
The addition of 10% n-propyl alcohol to 8 M urea markedly enhaiices the rate of dissociation as is also shown with 3.5 M GdnCl.
The alcohol alone does not affect the state of aggregation of the tetramcr, but probably interferes with the hydrophobic forces that hold the subunits together. The structural changes related to the two distinct steps that occur during the dissociation process are under investigation.
In the absence of tryptophan that served as an indicator of structural changes in the microenvironment of E. coli asparaginase, changes in the ultraviolet absorption spectrum of tyrosine were followed.
Titration of the tyrosyl residues and ultraviolet difference spectroscopy in GdnCl indicated that 900/, of the tyrosyl residues are in a hydrophobic environment in the native enzyme and are not available for titration.
A most dramatic difference in the stability of the Erwinia and E. coli enzymes is their response to succinylation. Succinylation of rnany oligomeric proteins results in dissociation to their subunits.
We previously reported (7) that succinylation of more than 40y0 of the lysyl residues of E. coli asparapinase causes spontaneous dissociation.
Modification of fewer than 40% of the positively charged side chains in the E. coli enzyme also results in a weakening of the intersubunit forces and interferes with reconstitution.
11~ contrast, succinylation of Erwinia asparaginase does not cause spontaneous dissociation, does not weaken intersubunit forces, and does not interfere with reconstitution.
These results might be explained by the greater basicity of the Erwinia enzyme which would neutralize the negatively charged succinyl residue.
Since asparaginase from Erwiniu carotovora is as effective as t.he E. coli enzyme in the treatment of leukemia, it appears that clinical effectiveness of the enzyme'does not depend on the ease with which the tetramer can dissociate nor does it appear to depend on the net charge on the enzyme.
It would be of interest to study physicochemical properties similar to those reported in this paper for n-asparaginase samples that are pharmacologically inactive.