PURIFICATION AND CHARACTERIZATION OF CHICKEN LIVER XANTHINE DEHYDROGENASE*

BY CHARLES N. REMY, DAN A. RICHERT, RICHARD J. DOISY, IBERT C. WELLS, AND W. W. WESTERFELD

(From the Department of Biochemistry, State University of New York, Medical College at Syracuse, New York)

(Received for publication, April 4, 1955)

Previous studies from this laboratory (1) have demonstrated the exist- ence of several different types of “xanthine oxidase” in nature. The en- zymes in milk and mammalian tissues appear to be quite similar, but can be differentiated by the inhibitor Antabuse, which affects only the tissue enzyme (2). Antabuse can also be used to separate the dehydrogenase and oxidase activities of tissue xanthine oxidase, inasmuch as it has no effect on the dehydrogenation reaction, but blocks the reoxidation of the enzyme by air. Both of these enzymes are fundamentally different from the xanthine dehydrogenase found in bird tissues, since the latter is not autoxidizable to any significant extent (3).

A method was developed for the purification of xanthine dehydrogenase from chicken liver in order to study its properties and the nature of its prosthetic group. It was hoped that a comparison with milk xanthine oxidase would permit an identification of the “dehydrogenase prosthetic group” as separate and distinct from the “oxidase prosthetic group.” Ball (4) originally obtained evidence for the existence of two prosthetic groups in milk xanthine oxidase; one of these was identified as flavin adenine dinucleotide (FAD) and the other was unknown (5). More re- cent studies (6-8) have demonstrated the presence of molybdenum (8-11) and iron (12) in addition to riboflavin in highly purified preparations of the milk enzyme.

The fractionation procedure to be described yielded approximately a 406fold purification of the chicken liver enzyme, and the enzyme so ob- tained was 97 to 98 per cent homogenous electrophoretically. By any criterion, the purified enzyme was more active than analogous prepara- tions of the milk enzyme. Like milk xanthine oxidase, the chicken liver xanthine dehydrogenase contained riboflavin and molybdenum, but in a ratio of 1: 1 instead of 2: 1. The best preparations of liver enzyme also contained iron, which seemed to be associated with the enzyme, in approxi- mately an 8: 1 ratio with MO. Riboflavin was hardly detectable in the

* This study was aided by a grant from the American Cancer Society upon rec- ommendation of the Committee on Growth of the National Research Council.

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294 LIVER XANTHINE DEHYDROGENASE

liver dehydrogenase spectrum, which was almost entirely that of the un- known component of the milk xanthine oxidase spectrum.

EXPERIMENTAL

Determination of Enzyme Activily. Manombic Procedure-The enzyme activity was determined by a method similar to the one previously de- scribed for assaying crude liver homogenates (3), in which methylene blue was used as the hydrogen carrier between xanthine dehydrogenase and molecular oxygen. The rapid loss of activity with the purified enzyme during the determination was reduced and the initial activity increased by the addition of albumin (13). The enzyme solution was prepared for assay in 0.04 M phosphate buffer, pH 7.4. 1.5 ml. of the enzyme, 0.15 ml. of 0.0113 M methylene blue, and 0.2 ml. of albumin1 were added to the body of the Warburg flask; the side arm contained 0.15 ml. of 0.05 M hypoxanthine. Readings were made at 10 minute intervals at 38”, and the enzyme was diluted so that between 10 and 25 c.mm. of oxygen were taken up during the initial 10 minutes. The Qo, (MB) was the c.mm. of 02 consumed in the presence of methylene blue per hour per mg. of dry material.

Enzyme preparations lost 20 to 30 per cent of their activity when dia- lyzed against distilled water at O-2” for 12 hours, but only a slight loss of activity occurred during dialysis against phosphate buffers of pH 6.0 to 7.4. Dry weights were determined by dialysis and drying, or for rapid approximate screening protein concentrations were obtained by the quan- titative biuret procedure (14) and converted to dry weights empirically.

Dye Reduction Method-Although the manometric method was satis- factory for following the purification of the enzyme, an alternative and more convenient dye reduction procedure2 was developed. Approxi- mately 35 mg. of sodium 2,6-dichlorobenzenoneindo-3’-chlorophenol (Eastman) were shaken with 80 ml. of water for 30 to 45 minutes, and the mixture was then filtered; the filtrate was diluted to 100 ml. to give a stock solution which could be kept for 30 days at 04”. An aliquot of the stock solution was diluted approximately 1: 10 each day for use.

The reaction was carried out by adding the following solutions (main- tained at 30”) to a Klett calorimeter tube: 2.7 ml. of ~/15 phosphate buffer, pH 6.3, minus the volume of enzyme to be added, 2.0 ml. of diluted dye, and 1.0 ml. of 0.05 M hypoxanthine in 0.05 M NaOH. The pH of the final reaction mixture was 6.8 and was so established in order to retard autoxidation of the reduced dye. The enzyme solution, usually 0.01 to

1 Armour’s crystalline bovine plasma albumin, 25 mg. per ml. 2 Based upon the procedure of E. Stotz for succinic dehydrogenase (personal com-

munication).

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REMY, RICHERT, DOISY, WELLS, AND WESTERFELD 295

0.2 ml., was added from a cold bath, and the tube was quickly shaken and inserted into the Klett calorimeter (660 rnl.c filter). The concentration of the dye solution was such that the calorimeter initially read 110 to 120. Arbitrarily the time required for the calorimeter reading to fall from 100 to 60 was measured with a stop-watch. The assay was most dependable when the amount of enzyme was adjusted so that this extent of dye re- duction required between 80 and 150 seconds.

PuriJication Procedure-400 to 1000 gm. of commercial chicken liver were conveniently fractionated at one time, and all solutions were main- tained at O-2” unless otherwise stated. The ground liver was thoroughly homogenized in a Waring blendor with 0.5 volume of iced 0.04 M phosphate buffer, pH 7.4. 2 volumes of the buffer were heated to 75” and added rapidly to the homogenized liver with vigorous stirring. Stirring was continued while the temperature of the diluted homogenate was brought to 56” (15 to 20 minutes) and maintained at 56” for 30 minutes. The viscous preparation was then quickly cooled to O-2” and centrifuged; the precipitate was discarded. Approximately 250 ml. of supernatant fluid and 230,000 manometric units (c.mm. of 02 per hour) of activity were obtained for each 100 gm. of fresh liver used. The yield was twice as large as could be obtained by extraction in the cold. The Qo, (MB) at this point was 50 to 80, as compared with 10 for the original liver.

The supernatant solution was made 0.6 saturated with (NH&SO, by adding 1.5 volumes of saturated (NH&SO4 solution (saturated at room temperature and neutralized with concentrated ammonium hydroxide to pH 7.2 to 7.6). 4 gm. of precipitate (Fraction I) were obtained from each 100 ml. of supernatant fluid by centrifugation. Fraction I was dissolved in 5 volumes of water and made 0.35 saturated with (NH&Sod. After discarding the precipitate, the supernatant solution was brought to 0.55 saturation with ammonium sulfate. The precipitate formed between 0.35 and 0.55 saturation (Fraction II) was about 0.48 gm. from each gm. of Fraction I used, and its QO, (MB) averaged 140; it contained 60 to 70 per cent of the activity present in the original supernatant solution.

Fraction II was dissolved in 6.5 volumes of 0.04 M phosphate buffer, pH 7.4, and any insoluble material was removed by centrifugation. An equal volume of a solution containing 8 mg. per ml. of Difco Pangestin, 1:75, dispersed in 0.2 M Na2HP04, was added, and the mixture was then incubated at 38” for 4 hours. The digestion mixture was cooled to 20” and clarified by adding an optimal amount of CaClz (about 0.032 ml. of 0.5 M CaClz per ml. of incubation solution). As previously reported for the milk enzyme (4), considerable amounts of enzyme may be adsorbed on the calcium phosphate precipitate if an excess of CaClz is used. After 15 to 30 minutes, t.he inactive flocculent precipitate was removed by cen-

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296 LIVER XANTHINE DEHYDROGENASE

trifugation at 0”. The enzyme was precipitated from the reddish brown supernatant solution by making it 0.55 saturated with ammonium sulfate. The centrifuged precipitate was redissolved in 0.04 M phosphate buffer, pH 7.4, and any insoluble material was removed. The Qo, (MB) of this fraction (Fraction III) varied between 1500 and 2.500.

A second digestion step similar to that described above was carried out as follows: Fraction III, dissolved in 9 volumes of phosphate buffer, was incubated for 2 hours with an equal volume of a 0.2 M Na*HPOd solution containing 4 mg. of Pangestin per ml. This digestion mixture was treated as before; the precipitate thus obtained (Fraction IV) was a deep reddish brown in concentrated solution and a golden brown when diluted. Rou- tine preparations of Fraction IV had a Qo, (MB) of about 3000. The over-all yield was at least 40 per cent and usually up to 60 per cent of the activity in the initial supernatant solution. The preparation at this point showed two major peaks in the analytical ultracentrifuge; the more rapidly sedimenting ferritin-like component accounted for up to one-third of the total protein.

The Qo, (MB) and the yield of the enzyme depended primarily on the activity of the original livers (firm red livers were much more active than pale yellow livers) and the speed with which the isolation procedure was conducted. A buffered solution (pH 7.4) of the enzyme sometimes lost as much as 10 to 15 per cent of its activity in 24 hours at 0”. Storage of the enzyme under 55 per cent ammonium sulfate at 0” resulted in a loss of as much as 50 per cent of its activity in 3 weeks. Freeze-drying of the enzyme at any stage in its preparation caused up to 95 per cent loss of activity.

The ferritin-like impurity was removed by differential centrifugation. Approximately 1 million manometric units of the enzyme (Fraction IV), dissolved in 32 ml. of the phosphate buffer, were divided into eight ultra- centrifuge tubes; the tubes were then filled by overlaying with 8 ml. of phosphate buffer. Centrifugation for 9 hours at 40,000 r.p.m. with a No. 40 rotor in the Spinco preparative ultracentrifuge (average 9 = 105,400) deposited reddish brown pellets which contained nearly all of the enzyme and the ferritin. This preliminary centrifugation removed some non- sedimenting riboflavin-containing material which was unrelated to the enzyme activity.

The pellets were dissolved in 48 ml. of 25 per cent sucrose and centri- fuged again for 10 hours at 40,000 r.p.m. to remove most of the ferritin- like impurity. The precipitate contained 94 per cent of the original 12 mg. of iron (as determined by the method of Kitzes et al. (15)) and 16 per cent of the enzyme activity. In one such experiment the dry weight of the precipitate was also determined, and the iron concentration was found to be 10.7 per cent.

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REMY, RICHERT, DOISY, WELLS, AND WESTERFELD 297

The enzyme was precipitated from the sucrose supernatant fluid by the addition of ammonium sulfate to 0.6 saturation and was redissolved in 0.04 M phosphate buffer, pH 7.4 (final volume 72 ml.). Centrifuging this solution for 4 hours at 40,000 r.p.m. and recentrifuging the supernatant solution for 53 hours deposited two fractions of contaminated enzyme. The QoI (MB) was about 3300; the MO concentration was 0.016 to 0.020 per cent, and the riboflavin-Mo molar ratio was 1. The iron-MO ratio for the first fraction was approximately 100 and for the second fraction 14.

The enzyme remaining in the supernatant solution was concentrated by precipitation with 0.6 saturated ammonium sulfate and was redissolved in the phosphate buffer (final volume 10 ml.). 5 ml. were placed in each of two centrifuge tubes, overlaid with 7 ml. of phosphate, and centrifuged for 44 hours at 40,000 r.p.m. The deposited enzyme pellet had a Qc, (MB) of 3900 to 4000, riboflavin and MO concentrations of 0.078 and 0.02 per cent, respectively (for a riboflavin-MO molar ratio of 1: l), and an Fe-MO atomic ratio of 8 or slightly higher. On the basis of these concentrations, the minimal molecular weight would be 480,000. This was the highest degree of purity achieved (Fraction V), and the enzyme so obtained was sufficiently free of impurity iron so that a valid spectrum could be ob- tained. There was no assurance that the iron present in the final prep- aration was actually a part of the enzyme. However, the supernatant fluid from this pellet still contained about 30 per cent of the enzyme ac- tivity, and the Fe-MO ratio in this fraction was identical with that ob- tained for the precipitate; at this stage of purity the remaining iron sedi- mented with the enzyme.

When subjected to an electrical field in a Perkin-Elmer electrophoresis apparatus at pH 7.2 (phosphate buffer, ionic strength 0.2), the enzyme mi- grated as an anion with a mobility of 2.0 X W6 sq. cm. per volt per sec- ond. At the end of 3 hours, 98 per cent of the material was still present as a single peak. A slower moving component (1 to 2 per cent) was ap- parent at the trailing edge of this peak; the other impurity (about 1 to 2 per cent) had a mobility greater than that of the major component.

Auloxidizability and Reaction with Cytochrome c-The aerobic activity of the enzyme was only 1 per cent of what it was in the presence of meth- ylene blue, whether the enzyme was prepared as described or partially purified without use of heat or Pangestin. Stotz2 found that in an aerobic system composed of cytochrome oxidase, cytochrome c, xanthine dehy- drogenase, and hypoxanthine there was a brisk sustained rate of oxygen consumption which did not decrease with time. Cytochrome b wa8 in- active in this system.

Few&n-Like Impurity-Identification of the iron impurity as ferritin- like was based upon the following properties common to both: (1) typical

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298 LIVER XANTHINE DEHYDROGENASE

absorption spectrum, (2) stability to heating at 56”, (3) stability to pro- teolytic enzymes, (4) similar ammonium sulfate solubilities, (5) non-re- duction by dithionite, and (6) high iron concentration. The ferritin-like impurity precipitated from solution with CdSO,, but this could not be used to remove the ferritin-like impurity from the enzyme since much of the enzymatic activity was lost in the process.

Attempts to separate xanthine dehydrogenase from the ferritin-like impurity by means of the electrophoresis-convection apparatus of Cann

-------- DIFFERENCE

--- E a HCI

300 400 500 MU

Fro. 1. The absorption spectrum of chicken liver xanthine dehydrogenase in the presence and absence of hypoxanthine; the difference curve represents the change in spectrum as the enzyme is reduced by hypoxanthine. The spectrum of the enzyme in 0.066 N HCl is also shown.

and Kirkwood (16) were only partially successful and were impractical from a preparative standpoint. From the rates of transport of the en- zyme and iron-containing material from the top to the bottom compart- ment it was estimated that their isoelectric points fell in the ranges pH 4.5 to 4.9 and 4.9 to 5.2 respectively.

Absorption Specfra-Fig. 1 presents the absorption spectrum of the enzyme (Fraction V) before and after its reaction with hypoxanthine, as determined with a Beckman DU spectrophotometer. There was a slight indication in the 450 rnl.c region of the presence of riboflavin, but the out- standing feature of the spectrum was its similarity to the curve attributed to the unidentified component of milk xanthine oxidase. The curve was also somewhat similar to the spectrum of ferritin (17), but the amount of

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REMY, RICHERT, DOISY, WELLS, AND WESTERFELD 299

iron present (as ferritin or inorganic iron) in the final enzyme preparation was too small to make any significant contribution to the spectrum. The superimposed shoulder at 400 to 410 rnp could not be seen until the ferritin had been removed, but was observed consistently thereafter. Since it was reduced by the addition of substrate, it appeared to be a part of the en- zyme. The addition of substrate also decreased slightly the sweeping visible end-absorption. The ‘Ldifference curve” was not characteristic of flavin, but may have been compounded of flavin plus another group show- ing a maximal change on reduction at 400 to 410 rnp. The spectrum of xanthine dehydrogenase was similar to that of aldehyde oxidase (18) and of alcohol-precipitated milk xanthine oxidase (11).

RiboJlavin-Since the presence of riboflavin in the enzyme could not be established spectroscopically with either the original enzyme or the differ- ence curve, the riboflavin was determined microbiologically with Lacto- bacillus casei (19). After the initial purification steps, which increased the &, (MB) to approximately 1000, the flavin concentration paralleled the xanthine dehydrogenase activity. Final preparations of the sedi- mented enzyme consistently contained riboflavin in a 1: 1 ratio with MO.

When the milk enzyme is coagulated in a boiling water bath for 15 minutes, the riboflavin (as FAD) is split off and can be recovered quan- titatively in the supernatant solution (4, 20). Heat coagulation of the chicken liver xanthine dehydrogenase or extraction of the enzyme with 50 per cent ethanol at room temperature, or with 4 per cent trichloroacetic acid, split off approximately 90, 80, and 65 per cent of the flavin, respec- tively, as determined microbiologically. Each of the extracts exhibited shoulders in the absorption spectra at 450 rnp, but otherwise showed a sweeping end-absorption similar to that of the enzyme itself.

UnidentiJied Group-Fig. 2 shows the absorption spectrum of the en- zyme after subtracting the theoretical absorption due to riboflavin and iron. The residual absorption was characterized by a peak at 400 rnp and a generally increasing absorption with decreasing wave-length. A comparable analysis of the spectrum of milk xanthine oxidase did not yield an identical residual curve, but the flavin-free alcohol-precipitated fraction obtained from the milk enzyme by Mackler et’al. (11) had a sim- ilar absorption curve. This residual absorption would seem to be related to the presence of an unidentified group in the enzyme.

Treatment of the enzyme with dilute acid (Fig. 1) diminished the ab- sorption and gave a curve somewhat similar to that of the reduced en- zyme. The effect of acid on the chicken liver enzyme was much less pronounced than on the milk xanthine oxidase (12). The change in spectrum of the milk enzyme with acid treatment was similar to that attributed by Morel1 (21) to a splitting of FAD from the enzyme. If

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300 LIVER XANTHINE DEHYDROGENASE

acid also splits FAD from the chicken liver enzyme, then the large amount of absorption remaining is a clear indication of the presence of another chromogenic group in the molecule. Dialysis of the enzyme against three changes of equal volumes of 0.1 N HCl for a total of 72 hours removed two-thirds of the iron into the dialysate but left two-thirds of the color in the non-dialyzable fraction; the spectrum of the latter was similar to

- ENZYME __ w-.. E - F,.

-- E-Fe

--- - - z E FI. Fe

ii = 0.4 0

a 0 to.2 0

I 400 500 MlJ

Fro. 2

------ OIALYSATE ------ OIALYSATE

- - NON - DIAL. - - NON - DIAL.

0.1 - -. -.

-. -.

300 300 MU 400 SC MU 400 SC

FIG. 3 FIG. 3

FICJ. 2. The absorption spectrum of chicken liver xanthine dehydrogenase contain- ing 2.71 y of Fe and 2.28 ‘y of riboflavin per ml. The theoretical contribution to the spectrum by the iron and riboflavin (as pure substances) was subtracted to obtain the additional curves.

FIG. 3. The absorption spectrum of chicken liver xanthine dehydrogenase before and after dialysis against 0.5 N ammonium hydroxide. The spectrum of the dialy- sate is also shown. All curves were calculated to comparable concentrations.

that of the original enzyme. This partial dissociation of iron from the unidentified chromogenic group argues against the possibility that a unique combination of iron with protein is responsible for the unidentified spectrum.

Similar dialysis of the enzyme against 0.5 N NH,OH gave the spectral changes illustrated in Fig. 3. The shoulder at 400 to 410 rnp in the orig- inal enzyme spectrum disappeared as a result of this treatment, and both the dialysate and non-dialyzable fractions were characterized by sweeping end-absorptions. 90 per cent of the iron remained with the protein frac- tion. The intensity of absorption of the dialyzed pigment was exag-

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REMY, RICHERT, DOISY, WELLS, AND WESTERFELD 301

gerated below 390 rnp, since the sum of the densities of the two fractions exceeded the density of the original enzyme solution. The presence of this pigment in the dialysate is fmther evidence for the existence of an unidentified chromogen in this enzyme.

Substrate SpeciJicity-Milk xanthine oxidase is capable (22-24) of oxidiz- ing a number of oxypurines and a variety of aldehydes, as well as reduced diphosphopyridine nucleotide (DP?;) (25) and xanthopterin slowly (26). The chicken liver xanthine dehydrogenase (Fraction IV) oxidized xanthine and hypoxanthine at the same rate. The activity of the enzyme toward 0.05 M aldehydes is reported in Table I. The manometric procedure was the same as that previously described, except that the buffer had a pII of

TABLE I

Relative Rates of Oxidation of Various Substrates by Purified Chicken Liver Xanthine Dehydrogenase in Comparison with Activity toward

Hypoxanthine -

Xanthine dehydrogcnase Substrate Mi$xd$hine

l

Manometric Dye reduction

Hypoxanthine.. 100 100 100 Xanthine......................... 100 96 100 p-Hydroxybenzaldehyde.. 33 40 130 Acetaldehyde 76 72 Furfural 88 75 Benzaldehyde. 57 80 Butyraldehyde.................... 54 0.6

* Values reported by Booth (24).

8.2; the dye reduction method was used for the study of volatile aldehydes. Reduced DPK was oxidized by the enzyme with cytochrome c as electron acceptor. Xanthopterin was oxidized too slowly to bc detected by the manometric procedure.

Substrates which were inactive in the manometric test (and in the dye reduction procedure as well when tested) included adenosine-3- and 5-phos- phatc, adenosine triphosphate, adenosine, adenine, guaninc, guanosine, guanylic acid, inosine, inosinic acid, xanthosine, oxidized DPS, uric acid, alloxan, uracil, uridine, uridylic acid, thymine, cytosine, cytidine, cytidylic acid, caffeine, thcophylline, theobromine, paraldehyde, chloral hydrate, and glucose.

Kin&c+--Reaction rates were studied with hypoxanthine substrate and the purified enzyme (Fraction IV) by the manometric procedure described. Readings were taken at 5 minute intervals, and the activities expressed as c.mm. of 02 consumed per 10 minutes per flask.

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302 LIVER XANTHINE DEEYDROGENASE

Fig. 4 illustrates the effect of pH on the activity of a constant amount of enzyme; there was a fairly broad pH optimum around 8.2. The addi- tion of the alkaline substrate and carrying out the oxidative reaction changed the initial pH by less than 0.1 unit, except that initial pH below

I f 524

0” G20

z 16

I

0- BORATE

FIQ. 4. The pH-activity curve for xanthine dehydrogenase in 0.15 M phosphate and 0.1 M acetate or borate buffer.

FIQ. 5. (a), the effect of substrate concentration on the initial rate of activity of xanthine dehydrogenase; (b), the relationship between the amount of enzyme and its activity.

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REMY, RICHERT, DOISY, WELLS, AND WESTERFELD 303

G was increased somewhat more. The remainder of the kinetic studies were carried out in 0.15 M phosphate buffer, pH 8.2.

With a constant amount of enzyme and by varying the amount of substrate from 0.01 to 0.5 ml. of 0.05 M hypoxanthine, the curve in Fig. 5, (z was obtained. The initial rate of oxygen uptake was greater with 0.05 ml. than with the usual 0.15 ml. of substrate, but the rate was not sus- tained as well and the substrate was exhausted too rapidly for routine studies.

The linear relationship between the amount of enzyme and the measured activity is shown in Fig. 5, 6.

Variations from 0.05 to 0.5 ml. of 0.0113 M methylene blue added to the Warburg flask had no effect on the enzyme activity when the amounts of substrate and enzyme were kept constant. Similarly, altering the ratio of the enzyme to methylene blue or substrate to methylene blue had no effect when the activity of the enzyme was compared with the results obtained under the “standard” conditions. Apparently the enzyme was saturated by small amounts of methylene blue, and no evidence of inhibition or competition was detected within the range of concentrations studied.

DISCUSSION

Chicken liver xanthine dehydrogenase is quite similar to milk xanthine oxidase. Both appear to contain iron in an 8: 1 ratio with MO. If the spectral end-absorption is indicative of an additional unidentified compo- nent in the enzyme, then this component is also present in both enzymes in a constant ratio with the Fe and MO. If the optical density at 320 rnti is arbitrarily taken as a quantitative measure of this constituent, it is present in a ratio of 0.220 to 0.240 optical density units per microgram of Fe. If this comparison is made with acid-treated enzymes, then the chicken liver dehydrogenase would have approximately twice the ratio observed for the milk xanthine oxidase. The only observed analytical difference is the presence of 2 molecules of riboflavin in the milk enzyme and 1 riboflavin per atom of MO in the liver dehydrogenase. A concen- tration of 0.03 per cent MO in milk xanthine oxidase (8, 27) and 0.02 per cent MO in the liver dehydrogenase is a reflection of the larger molecular weight of the latter. On any comparative basis, the chicken liver dehy- drogenase is more active than the milk enzyme, and the former is at least 5 times as active as the latter in the aerobic methylene blue assay pro- cedure on the basis of riboflavin content.

A comparison of the analytical values does not establish any reason for the oxidase nature of the milk enzyme in contrast to the dehydrogenase nature of the liver enzyme. Even if it is assumed that the unknown prosthetic group is the dehydrogenating group and the flavin is related to

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304 LIVER XANTHINE DEHYDROGENASE

the aerobic reoxidation of the enzyme, the difference in flavin concentra- tion is only 2-fold, while the difference in autoxidizability is approxi- mately go-fold. The spectra of the two enzymes suggest that the dif- ference in autoxidizability of the two enzymes might be related to the differences in the nature or attachment of the flavin to the enzyme. In the milk oxidase the flavin spectrum is not only obvious but is exag- gerated, while in the chicken liver dehydrogenase a flavin component of the spectrum is hardly detectable. Morel1 (21) emphasized the importance of the attachment between the flavin and the enzyme in relation to the activity of the latter, but how this influence is exerted is still unknown.

SUMMARY

A 400-fold purification of chicken liver xanthine dehydrogenase was achieved by a procedure involving heating at 56”, incubation with Pan- gestin, fractionation with ammonium sulfate, and high speed centrifuga- tion. The last step removed a ferritin-like impurity. The aerobic “oxi- dase” activity of this enzyme was only 1 per cent of its dehydrogenating ability when the latter was measured aerobically in the presence of a hydrogen carrier such as methylene blue. Xanthine dehydrogenase was able to reduce cytochrome c and thereby transfer electrons through cyto- chrome oxidase to oxygen.

Microbiological assays for riboflavin showed that the purification pro- cedure yielded a parallel increase in the concentration of riboflavin and enzyme activity. The enzyme contained Fe, MO, and riboflavin in a ratio of 8: 1: 1. The absorption spectrum of the enzyme was similar to the curve attributed to the unidentified component of milk xanthine oxidase and was characterized by a sweeping increase in absorption with decreas- ing wave-length. The enzyme spectrum had a shoulder around 400 rnp, but no obvious absorption attributable to riboflavin. The reason for the oxidase nature of the milk enzyme in contrast to the predominantly de- hydrogenase activity of the chicken liver enzyme was not established, but may be related to differences in the nature or the mode of attachment of riboflavin to the enzyme.

The enzyme was capable of dehydrogenating xanthine, hypoxanthine, reduced DPN, and aldehydes, but had no effect on other naturally occur- ring purines, pyrimidines, nucleosides, or nucleotides. It had a broad optimal pH around 8.2, and the activity was not affected by wide varia- tions in the concentration of methylene blue.

BIBLIOGRAPHY

1. Richert, D. A., and Westerfeld, W. W., Proc. Sot. Ezp. Biol. and Med., 76, 252 (1951).

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2. Richer-t, D. A., Vanderlinde, R., and Westerfeld, W. W., J. Biol. Chem., 186,261 (1950).

3. Remy, C., Richer-t, D. A., and Westerfeld, W. W., J. BioZ. Chem., 193,649 (1951). 4. Ball, E. G., J. Biol. Chem., 128, 51 (1939). 5. Kalckar, H. M., Kjeldgaard, N. O., and Klenow, H., Biochim. et biophys. acla,

6, 575 (1950). 6. Westerfeld, W. W., and Richer& D. A., J. Biol. Chem., 192, 35 (1951). 7. Richert, D. A., and Westerfeld, W. W., J. Biol. Chem., 192.49 (1951). 8. Richert, D. A., and Westerfeld, W. W., J. Biol. Chem., 203,915 (1953). 9. De Renzo, E. C., Heytler, P. G., and Kaleita, E., Arch. Biochem. and Biophys.,

49, 242 (1954). 10. Totter, J. R., Burnett, W. T., Jr., Monroe, R. A., Whitney, I. B., and Comar, C.

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Doisy, Ibert C. Wells and W. W. WesterfeldCharles N. Remy, Dan A. Richert, Richard J.LIVER XANTHINE DEHYDROGENASECHARACTERIZATION OF CHICKEN

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1955, 217:293-306.J. Biol. Chem. 

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