THE JOURNAL OF I~OLOGIC~ CIIE~~ISTRY Vol. 241, No. 2, Issue of January 25, 1966

Printed in U.S.A.

Studies on the Yeast Photoreactivating Enzyme

I. A METHOD FOR THE LARGE SCALE PURIFICATIOK AND SOME PROPERTIES OF THE ENZYME*

(Received for publication, August 6, 1965)

AMIR MUHAUMED t From the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

SUMMARY

Large scale preparation of purified photoreactivating en- zyme from commercial bakers’ yeast has been described. The enzyme has been purified approximately 3000-fold over the crude extract with an over-all yield of 5 to 6%. The purified enzyme has a pH optimum at 7.2 and does not re- quire any metal ions for activity. High phosphate concentra- tion inhibits enzyme activity with an optimum activity at 0.02 M phosphate. The purified enzyme shows no absorption peaks between 300 and 400 rnp and has a small amount of typical cytochrome absorption. Analytical ultracentrifuga- tion of the purified enzyme indicates the presence of one large homogeneous component and another small component which does not show a peak in the schlieren pattern, but absorbs light at 546 rnp. The average molecular weight of the large component is ~30,000.

A photoreactivating enzyme was first discovered in Escherichia coli extracts by Goodgal, Rupert, and Herriott (1) and in yeast by Rupert (2). However, no purification procedure has been published on this enzyme, and the nature of the enzyme and the mechanism of photoreactivation in vitro of ultraviolet-irradiated deoxyribonucleic acid is largely unknown. It was postulated that the enzyme might have a prosthetic group with an absorp- tion spectrum similar to the action spectrum of photoreactivation in vitro with yeast enzyme (3). The only known reactions catalyzed by the enzyme are the monomerization of different types of pyrimidine dimers in ultraviolet-irradiated polydeoxy- ribonucleotides (4, 5). The reaction involves rearrangement of the cyclobutane ring (6) to give two double bonds. Identifica- tion of the chromophore would help in elucidation of the mecha- nism of this reaction. Since the enzyme is apparently present in rather small amounts in commercial bakers’ yeast., it was found essential to work with large quantities of raw material.

This paper describes the large scale purification of the enzyme and some of its properties.

* This ilrvestigation was supported by the United States Atomic Energy Commission under contract with the Union Carbide Corporation.

t On leave of absence from Pakistan Atomic Energy Centre, T,:rhore, Pakistan.

MATERIALS AND METHODS

The methods for preparat,ion of competent Hemophilus in- fluenzae cells, extraction of H. in&enxae DNA carrying the streptomycin marker, and the transformation assay were es- sentially those described previously (7, 8). DNA at a concentra- tion of 10 pg per ml in 0.15 M NaCl was irradiated from a ger- micidal lamp at an incident dose rate of approximately 7.0 ergs per mm2 per set (incident dose, 2450 ergs per mm2). Photoreac- tivating conditions were as previously described (9) except that the illumination was supplied by five GE 15-watt black light bulbs and the incident intensity to the samples was 5000 ergs per mm2 per min.

Materials for Chromatography-DEAE-cellulose (Matheson, Coleman, and Bell) was processed as follows. Dry DEAE- cellulose (100 g) was allowed to sink into 2 liters of 0.5 N NaOH containing 50% ethanol, stirred for 1 hour, and filtered with a Buchner funnel. The cake was washed on the funnel with large volumes of distilled water, suspended in 2 liters of 0.5 N

HCl, and stirred for 20 min. The slurry was again filtered and washed with distilled water. The adsorbent was then suspended in a large volume of distilled water (10 to 15 liters), stirred, and decanted after 5 min to remove the fine particles. The washed DEAE-cellulose was suspended in 2 liters of 0.5 N NaOH, stirred, and filtered. The adsorbent was again washed with distilled water and its pH was adjusted to 8.0 with dilute HCl. Finally, the adsorbent was washed with 0.1 M phosphate buffer (only potassium salts have been used for preparation of phosphate buffers), pH 7.5, containing 1 mM EDTA, and suspended in the same buffer.

Whatman phosphocellulose, Grade P-l, was obtained from H. Reeve Angel Company and treated initially as for DEAE-cellu- lose. The adsorbent was finally washed with 0.1 M phos- phate buffer (pH 7.0) containing 1.0 mM EDTA and suspended in the same buffer.

Hydroxylapatite was prepared from brushite as described by Levin (10). Exactly 5 min of settling time were allowed during the last heating steps, and the hydroxglapatite prepared in this way had very good flow rates. Calcium phosphate gel was prepared according to the method of Keilin and Hartree (11). The gel was stored in 0.05 M phosphate buffer (pH 6.8) after t)he fine particles were removed by decantation; it was used after 2 to 4 weeks of storage.

Chromatographic runs were carried out in a Technicon fraction collector with an Instrumentation Specialities Company, Inc., ultraviolet absorption recorder.

516

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0 0.05 0.10 0.15 0.20 0.25 PROTEIN CONCN. (mg/ml )

517

FIG. 1. Effect of increasing enzyme concentration on photoreactivating activity. Hydroxylapatite-purified concentrated enzyme was diluted in 0.04 M phosphate buffer (pH 7.0), containing 1.0 mM EDTA, to give the protein concentratjions as indicated. Enzyme activity was measured as usual.

Protein and DNA Estimations-Protein was estimated rou- tinely from the ultraviolet absorption at 280 and 260 rnp (12). For turbid solutions and where the nucleic acid content was high, the method of Lowry et al. (13) was used. Concentration of DNA solutions was determined from the absorption at 260 rnp

(14). Enzyme Assay and Unit-In the standard assay, 0.20 ml

containing 0.25 pg per ml of ultraviolet-irradiated DNA (I’% survival) was added to 0.2 ml of enzyme solution suitably diluted in 0.04 M phosphate buffer (pH 7.0) containing 1 mM EDTA, and the mixture was left at 37” in the dark for 5 min. After warming, the mixture was illuminated under the black light for 5 min at 37” and assayed for ability to transform H. inJluenzae cells to streptomycin resistance.

Since the exact nature of the substrates (i.e. the ultraviolet lesions) for the enzyme is not known, the enzyme unit could not be defined in the usual manner. However, the enzyme unit was arbitrarily fixed as follows. The increase in streptomycin transformants (ratio of the number of cells transformed by ultraviolet-irradiated DNA after a 5-min photoreactivating period to the number at zero time) obtained when various dilu- tions of a purified enzyme preparation were used was plotted against protein concentration (Fig. 1). The initial straight line part of the graph was divided into units in such a way that the factor increase in streptomycin transformation, minus 1, is equal to the number of enzyme units, the factor increase in streptomycin transformation at zero enzyme concentration being 1.

All operations were carried out in the cold room at O-4” or on ice, unless otherwise stated. Centrifugations were done at O-4.”

EXPERIMENTAL PROCEDURE AND RESULTS

Purijkation of Enzyme

Preparation of Yeast Extract-Fifty pounds of fresh bakers’ yeast (Federal Yeast Corporation, Baltimore) were dried in 17 large trays (45 x 60 cm) at 37” with frequent turning. The yeast was thoroughly dried in 30 to 40 hours. A total of 7060 g of dry yeast were obtained from 50 pounds of fresh yeast.

Seven kilograms of dry yeast were stirred into 28 liters of 0.066 M

K2HP04 and the slurry was stirred mechanically at 37” for 6 hours. The viscous extract was then centrifuged in a refrigerated Sharples No. 16 centrifuge at top speed (13,600 x g) for 20 min. The residue was discarded and the supernatant solution was stored in the cold room if it was not used immediately. Ap- proximately 22 liters of extract were obtained by this procedure.

(NH&TO4 Fractionation-Twenty liters of the clarified extract were brought to pH 7.0 + 0.2 with dilute (NHI)OH, and then 3.9 Kg of solid (NH&SO4 (Mann) were added slowly with mechanical stirring over a period of 1 hour to bring the (NH&S04 saturation to 33%. The stirring was continued for another hour, after which the mixture was kept at (t4” for 2 hours. The precipitate was then removed by centrifuging in the Sharples centrifuge at top speed for 20 nun and the residue was discarded. The supernatant solution was brought to 50% (NH&S04 satura- tion by adding 107 g of solid (NH&SO4 per liter of the super- natant. After stirring for 1 hour, the mixture was left in the cold room overnight and centrifuged the next day in the Sharples centrifuge at top speed for 20 min. The supernatant solution had very little activity and was discarded. The residue was suspended in 3 liters of 0.05 M phosphate buffer (pH 6.5) contain- ing 1 mM EDTA.

Gel Filtration-The redissolved (NH&SO4 fraction was centrifuged in the Servall refrigerated centrifuge at 10,000 x g for 30 min; 1500 ml of the supernatant solution were passed each time through a Sephadex G-25 column (8 x 100 cm) equili- brated with 0.05 M phosphate buffer (pH 6.5) and eluted with the same buffer. The protein fractions were collected. A con- siderable amount of yellow material and almost all the (NH&SO4 was removed by this procedure, and 4200 ml of the Sephadex- filtered extract were obtained.

Calcium Phosphate Gel Adsorption-Calcium phosphate gel (50 ml; 24 mg per ml, dry weight) was added to 4 liters of the Sephadex filtered extract, and the mixture was stirred gently for 30 min and then allowed to settle for 30 min. The mixture was then centrifuged and the residue discarded. More gel (550 ml) was added to the supernatant solution with continuous stirring.

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518 Studies on Yeast Photoreactivating Enzyme. I

!2

16 0 2b 6b 80 IC TUBE NO.

FIG. 2. Preparative phosphocellulose chromatography I. A column (5 X 45 cm) was equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 1 rn~ EDTA. Five liters of DEAE-cellulose filtrate containing 1.5 mg of protein per ml in 0.1 M potassium phosphate buffer (pH 7.0) were applied to the column at 600 ml per hour. Enzyme was eluted with a linear gradient of 1 liter each of 0.1 and 0.3 M KzHPO, at a flow rate of 150 ml per hour. O-O, A280; O-O, enzyme activity; n---n, A415.

Vol. 241, No. 2

2.0

1.2-

0.8-

0 10 20 30 40 TUBE NO.

,400o

.3200

.a00

0

FIG. 3. Preparative phosphocellulose column chromatography II. A column (2 X 30 cm) was equilibrated with 0.1 M phosphate bufler (pH 7.0) containing 1 mM EDTA. Phosphocellulose I fraction (1500 ml) containing 0.15 mg of protein per ml was applied to the column at the rate of 300 ml per hour and eluted with a linear gradient of 250 ml each of 0.1 and 0.3 M KsHPO( containing 1 m&x EDTA. O-O, Asso; O-O, enzyme activity.

The mixture was stirred for another hour and allowed to stand at O-4” for 1 hour, after which it was centrifuged in an Inter- national refrigerated centrifuge. The supernatant solution was largely inactive and was discarded.

The gel residue was extracted three times with 1 liter each of 0.1 M phosphate buffer (pH 7.0) with the use of a mechanical stirrer, and the supernatant solution was discarded after cen- trifugation at 1000 x g for 10 min in the International refrig- erated centrifuge. The phosphate concentration for extraction was gradually increased to 0.12, 0.14, 0.15, and 0.17 M, and in most runs the enzyme was not extracted up to 0.15 M phosphate buffer (pH 7.0). After three to four extractions with the highest phosphate concentration which did not extract the enzyme (0.15 M phosphate buffer (pH 7.0) in this experiment), the residue was extracted with 500-ml portions of 0.2 M phosphate buffer (pH 7.0) until no more enzyme was extracted (usually five to six times) and the active extracts were combined. In some experiments it was essential to extract the residue with 0.2 M KzHPOl for com- plete recovery of the enzyme; 2700 ml of calcium phosphate extract were obtained. The inactive gel was discarded.

DEAE-cellulose Treatment-Calcium phosphate gel extract (2500 ml) was centrifuged at 10,000 x g for 30 min in the Servall centrifuge to remove all of the fine particles. The supernatant solution was diluted twice with 1 InM EDTA and the pH was adjusted to 7.5 with 1 N NaOH. The dilute extract was passed through a DEAE-cellulose column (5 x 33 cm) previously washed with 5 liters of 0.1 M phosphate buffer (pH 7.5) containing 1 mM EDTA. The extract was applied to the column at a rate of 600 ml per hour. The column was washed with 700 ml of 0.1 M phosphate buffer (pH 6.5) until the eluate contained no more enzyme activity, and the washings were added to the DEAE- cellulose filtrate. Approximately 60 to 70% of the original enzyme activity applied to the column was recovered in the

filtrate (DEAE-cellulose filtrate), and the enzyme activity

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remaining on the column was eluted with the use of a linear gradient of 1 liter each of 0.1 and 0.4 M phosphate buffer (pH 6.5). The enzyme was eluted at a phosphate concentration of 0.22 to 0.24 M and stored at -20”. This fraction is relatively unstable as compared with the bulk of the enzyme, which does not adhere to DEAE-cellulose, and has not been investigated further.

Phosphocellulose Chromatography-Five liters of DEAE-cel- lulose filtrate were adjusted to pH 7.0 with dilute HCl and the extract was applied to a phosphocellulose column (5 X 45 cm) equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA by passing 3 liters of the same buffer through the column. The flow rate was 600 ml per hour. The red material in the DEAE-cellulose filtrate, which has an absorption spectrum similar to cytochromes, was bound to the phosphocellulose as a red band at the top of the column. The enzyme was almost quantitatively retained on the column, and the material that passed through was inactive. The column was washed with 500 ml of 0.10 M K2HPOI and the enzyme was eluted with a linear gradient of 1 liter each of 0.10 and 0.30 M KzHPOI containing 1 mM EDTA at a flow of 150 ml per hour. The enzyme was eluted as a distinct peak and was largely contaminated with cytochromes (Fig. 2).

Phosphocellulose II-The active fractions from the above step were combined, adjusted to pH 7.0, and diluted twice with cold distilled water. The extract was applied to a smaller phospho-

T

3

FIG. 4. Preparative hydroxylapatite column chromatography. A column (2 X 10 cm) was equilibrated with0.1 M phosphate buRer (pH 7.5). A phosphocellulose II fraction (200 ml) containing 0.40 mg per ml of protein was passed through the column and eluted with a linear gradient of 100 ml each of 0.15 and 0.30 M

phosphate buffer (pH 7.5). O--O, 4280; O---O, enzyme ac- t.ivity; A---/L, AdIs.

TABLE I Summary of puri$cation procedure

Fraction Total protein Total units RC!COW> Specific

activity

s

Crude extract. 1450 13,000 X g supernatant. 710 (NH&SOa, 35 to 50%

x 10-g %

3060 100 2900 93

units/mg protein

2.1 4.1

saturation. Gel filtration. Calcium phosphate gel. DEAE-cellulose

215 1860 61 8.7 195 1842 60 9.5 13.6 980 32 72

filtrate. 7.2 680 21 95 Phosphocellulose I.. 0.2 512 17 2560 Phosphocellulose II.. 0.09 345 11 3830 Hydroxylapatite. 0.03 186 6 6215

PH

FIG. 5. Effect of pH on photoreactivating enzyme activity. Hydroxylapatite-purified, concentrated enzyme was diluted 200 times in 0.05 M Tris-maleate buffers of different pH values as indicated. Final protein concentration after dilution was ap- proximately 0.017 mg per ml. Enzyme activity was measured as usual.

cellulose column (2 X 30 cm) at a flow rate of 300 ml per hour. The column was washed with 150 ml each of 0.1 M phosphate buffer (pH 7.0) and 0.1 M K2HPOd, which did not elute the en- zyme. The enzyme was finally eluted with the use of a linear gradient of 250 ml each of 0.1 and 0.3 M KzHPOd at a flow rate of 120 ml per hour (Fig. 3). The enzyme was eluted as a sharp peak at 0.20 to 0.21 M K2HP04 concentration and some of the cytochrome was removed. This step also yielded a considerable concentration of the enzyme.

Hydroxylapatite Chromatography-The active enzyme fraction obtained from the second phosphocellulose step was adjusted to

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520 Studies on Yeast Photoreactivating Enzyme. I Vol. 241, No. 2

0’ I I 4 I I 0.02 0.04 0.06 0.08 0.1

PHOSPHATE CONCN. IN REACTION MIXTURE (M)

FIG. 6. Effect of phosphate concentration in reaction mixture on enzyme activity. Purified enzyme was diluted 200 times in phos- phate buffer (pH 7.0). The final concentration of phosphate buffer as shown in the graph is in the reaction mixture and the phosphate concentration in enzyme solution is twice this. The enzyme activity was determined as usual.

TABLE II Effect of metal ions on enzyme activity

Phosphocellulose-purified enzyme diluted 100 times (final pro- tein concentration, 10 pg per ml) with 0.04 M potassium phosphate buffer (pH 7.0) and the salts added yielded the final concentrations given below. The enzyme (0.2 ml) was titrated for activity under the standard assay conditions. Free metal ions were converted to complexes by adding 0.1 ml 1 mM EDTA + 0.1 ml of 1 rnM p- mercaptoethylamine to the 3-ml growth tubes before the photo- reactivated DNA mixture. Controls were run for all the samples, and the number of streptomycin transformants in all nonphoto- reactivated samples were almost identical.

Addition Concentration

MgS04.. CaC12. ZnCh.. MnSOl. cuso4. HgClz AgNO,. None.....

M

.......... 1 x 10-a .......... 1 x 10-a .......... 1 x 10-a .......... 1 x 10-S .......... 5 x 10-4 .......... 1 x 10-E

........... 1 x 10-h ..........

Enzyme activity

Units Per cent

23.6 98 25.7 107 16.6 69 12.2 51

1.6 7 0 0 0 0

24.0 100

pH 7.5 and diluted three times with cold distilled water. It was then applied to a hydroxylapatite column (2 x 10 cm) previously equilibrated with 0.1 M phosphate buffer (pH 7.5) at a flow rate of 50 ml per hour. All the extract was allowed to pass through the column, which then was washed with 50 ml of 0.15 M phos- phate buffer (pH 7.5). The enzyme was finally eluted from the column with the use of a linear gradient of 250 ml each of 0.15 and 0.30 M phosphate buffer (pH 7.5) at a flow rate of 30 ml per hour. The enzyme was eluted as a sharp band at a phosphate concentration of 0.20 to 0.21 M (Fig. 4). The cytochrome-like

TABLE III E$ect of Cu++ and Hg++ addition on enzyme activity after

formation of complex between enzyme and irradiated DNA

Phosphocellulose-purified enzyme was diluted 100 times in 0.04 M phosphate buffer (pH 7.0); protein concentration, 10 rg per ml. Then 0.2 ml of 1.0 pg per ml of DNA was added to 0.2 ml of enzyme and the mixture was left at 37’ in the dark for 15 min. The metal ions were added to the “complex” and the mixture was illuminated with or without formation of metal ion complexes as described below.

Metal ion and complexing agent Enzyme activity remaining

% cu++, 2 x 10-S M

EDTA, 1 X 1OW M, before photoreactivation. 89 EDTA, 1 X 1O-3 M, after photoreactivation.. 7

Hg++, 2 X IO-6 M /%Mercaptoethylamine, 1 X 1OW M, before photo-

reactivation b-Mercaptoethylamine, 1 X lo+ M, after photore-

activation.

85

1.2

material present in the phosphocellulose II fraction was largely separated from the enzyme activity as a separate peak which eluted from the column after the enzyme. However, there was a slight overlap of the enzyme activity and 415 rnp absorption peaks, and the hydroxylapatite-purified enzyme fractions had a faint red color. The fractions with a specific activity higher than 5500 units per mg of protein were combined and concentrated by perevaporation in a dialysis tube, which resulted in some loss of enzyme activity.

The purification procedure is summarized in Table I. The over-all yield by this procedure is 5 to 6% and the enzyme is approximately 3000-fold purified over the crude ext’ract.

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Properties of Purijied Enzyme

Stability-The purified enzyme is routinely stored at -20” as a concentrated solution in 0.1 M phosphate buffer (pH 7.5 to 3.0) containing 1 mM EDTA for up to 2 to 3 weeks without ap- preciable loss of activity. At low protein and phosphate con- centration, the enzyme is inactivated rather rapidly. Keeping the purified enzyme at O-4” also results in rapid inactivation. Addition of 1 mM cysteine or 20 y0 glycerol did not help in stabiliz- ing the enzyme from inactivation at 0”. At very low concentra- tions of phosphate buffer (approximately 0.005 M), the enzyme reversibly precipitates, and it is inactivated if stored as such for any length of time. Repeated freezing and thawing result in a gradual loss of enzyme activity.

Effect of pH and Phosphate Concentration on Enzyme Activity- The enzyme has maximum activity at pH 7.2 in 0.05 M Tris- maleate buffer (Fig. 5). The reaction is very sensitive to the presence of a high concentration of phosphate ion in the reaction mixture; maximum activity is obtained at a final phosphate con-

TABLE IV Effect of (NH,)&+04 on enzyme activity

Phosphocellulose-purified enzyme was diluted 100 times (final protein concentration, approximately 10 pg per ml) in 0.04 M

phosphate buffer (pH 7.0), and (NH~)QSOJ was added. Enzyme activity was measured as usual with proper nonphotoreactivated blanks.

Enzyme activity Addition Concentration

Units Per cent

M

(NH&Sod. 1 x 10-Z 24.3 101 (NH&S04. 1 x 10-l 14.1 59 None................ 24.0 100

centration of 0.02 M and very little activity is detectable at 0.1 M

or at a higher concentration (Fig. 6). E$ect of Metal Ions on Enzyme Activity-The purified photo-

reactivating enzyme does not require any metal ions for enzyme activity (Table II). Mg++ and Ca+f do not affect enzyme activity at a concentration of 1 InM. Hg++, Ag+, and Cuff are strongly inhibitory even at very low concentrations. Zn++ and Mn++ also inhibit the enzyme, although to a much lesser degree than Hg++, C&f, and Ag+. Hg+f and Cu++ strongly inhibit the reaction even if added after formation of the complex between the enzyme and irradiated DNA in the dark (Table III). The inhibitory effect of Hg* and Cu++ can, however, be al- most eliminated by adding P-mercaptoethylamine or EDTA, respectively, before photoreact,ivation of the mixture. The presence of Cu++ in the enzyme solution slowly inactivates the enzyme; all the enzyme solutions were therefore kept in 1 mM EDTA. Presence of (NH&SO4 at high concentrations in the reaction mixture inhibits the reaction (Table IV).

Absorption Spectrum of Purified Enzyme-The absorption spectrum of the purified enzyme was obtained with a Beckman DU spectrophotometer. The enzyme gave a typical protein absorption spectrum with a maximum at approximately 280 mp (Fig. 7). No other absorption peaks were found in the visible region except for small absorption peaks at 415, 520, and 550 rnp, which are typical of a cytochrome. In view of the separation of cytochrome absorption from enzyme activity on phosphocel- lulose and hydroxylapatite columns, it is assumed that the small cytochrome absorption in the purified enzyme is an impurity and not a part of the enzyme. The question of the possible formation of a chromophore by combination of photoreactivating enzyme and ultraviolet-irradiated DNA in the dark was also investigated. However, under the experimental conditions used, there was no evidence of the formation of such a chromophore (Fig. 7).

Analytical Ultracentrifugation of Puri$ed Enzyme-The schlieren velocity sedimentation pattern of the purified photo-

0.4- ‘\ 0

\ l.-

s .--o-.--y.-.

-2”‘ 0-0-0-0-00-000’ Oh -m-0-@+

-0-o --- 1 I I I I I

260 300 340 380 420 460 500 WAVELENGTH (rnp)

FIG. 7. Absorption spectrum of the purified enzyme. O-O, hydroxylapatite-purified concentrated enzyme; protein concentra- tion, 1.5 mg per ml in 0.05 M phosphate buffer (pH 7.0). O-O, hydroxylapatite-purified concentrated enzyme mixed with ultra- violet-irradiat,ed calf thymus DNA (50 pg of DNA per ml in 0.15 M NaCl, 1 hour under the germicidal lamp), and the mixture was kept at 37” in the dark for 20 min. Final concent,rations in the mixt,ure: prot,ein, 2.0 mg per ml; ultraviolet-irradiated DNA, 30 pg per ml.

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522 Studies on Yeast Photoreactivating Enzyme. I Vol. 241, No. 2

FIG. 8. Ultracentrifugal sedimentation patterns of photoreactivating enzyme (Spine0 model E). The pictures were taken at 20,44, and 132 min, respectively, after reaching 59,780 rpm. The solvent was 0.05 M phosphate buffer (pH 7.0), the temperature was 5”, and the protein concentration was 2.5 mg per ml. The direction of sedimentation was from left to right.

0.5 I 45 46 4; 4’8

r2 (cm*)

FIG. 9. Equilibration sedimentation run on photoreactivating enzyme. The protein concentration in the enzyme solution was 0.5 me ner ml. and the exneriment was carried out at go in 0.05 M

phos;hate buffer (pH 7.Oj. The data were taken 20 hours after sedimenting at 29,500 rpm. The results have been plotted as the log10 of the enzyme concentration measured in microns of fringe displacement (15) against the square of the radius. One fringe corresponds to 268 G. The indicated deviation corresponds to a *5-p error in measurement.

reactivating enzyme is shown in Fig. 8. The ‘enzyme travels as a single peak throughout the run, with some evidence of hetero- geneity. Traces of a more slowly sedimenting red component are seen in the background absorption of the mercury 546-mp light of the schlieren plates.

Preliminary data on the concentration dependence of the sedimentation rate in 0.05 M phosphate buffer (pH 7.0) at 5”

indicate that the enzyme has a sedimentation rate of approxi- mately 3.2X at concentrations of 1 to 3 mg per ml, rising to about 3.78 at concentrations near 0.05 mg per ml, under these condi- tions.

A short column equilibrium molecular weight run by the Yphantis meniscus depletion method (15) gave a weight average molecular weight of 30,000 for most of the material in the cell (Fig. 9) and also showed the presence of small amounts of larger molecular weight material.

DISCUSSION

Since the photoreactivating enzyme is present in yeast in very small amounts, a large scale preparation method had to be developed in order to get a reasonable amount of enzyme for further investigation. All the purification steps have been tested with more than 20 large scale runs, and the results have always been satisfactory. The over-all yield from this purifica- tion procedure is rather low (5 to 6%), in spite of the fact that broad fractions have been taken from ammonium sulfate and calcium phosphate gel purification steps in order to have high yields at the expense of purification. The calcium phosphate step was always carried out on an analytical scale with all new gel preparations before attempting a large scale purification run. The phosphocellulose and hydroxylapatite column steps have always given excellent reproducibility. Considerable difficulty was experienced in concentrating the enzyme without appreciable loss of activity. Carbowax 6000 (polyethylene glycol, obtained from Union Carbide) gives very good concentration and little loss of activity. However, the Carbowax also contaminates the concentrated enzyme, and it was difficult to remove the Carbowax completely by dialysis. Carbowax-concentrated enzyme always gave poor results (smearing) on analytical ultracentrifugation. Perevaporation in the cold room and subsequent dialysis against dilute buffer concentrated the enzyme considerably, but this also resulted in some loss of enzyme activity. However, this method has been used to concentrate the purified enzyme for analytical ultracentrifugation studies and the results have been satisfactory.

The division of enzyme activity into two fractions on DEAE- cellulose chromatography has been observed with all prepara- tions. However, the two fractions are not distinguishable by the biological assay used in this work. It is possible that the

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two fractions represent two different enzymes involved in the photoenzymatic repair of ultraviolet-irradiated DNA or the same enzyme with different charge and elutes in two fractions on the ion exchange column.

The enzyme is very sensitive to high concentrations of phos- phate buffer and ammonium sulfate. It is therefore advisable always to dilute the enzyme samples before assaying for activity, so that the phosphate concentration is approximately 0.04 M and the ammonium sulfate concentration is also low.

Ultracentrifugation of the purified enzyme gives a schlieren pattern which shows a single peak after a 2$-hour run. The red component in the enzyme preparation is identified on the schlie- ren picture by its absorption of the 546-rnp light, and is present in amounts that are too small to give a peak in the schlieren optics. The short column equilibrium run gave a weight average molecular weight of about 30,000, with some indication of the presence of small amounts of heavier material. Preliminary analytical electrophoresis of the purified enzyme on acrylamide gel has indicated that most of the protein moves toward the cathode at pH 7.0 as a single band with a faint smear in the other direction. This observation is consistent with the fact that the enzyme is bound to phosphocellulose and thus carries a positive charge. Detailed investigation of the homogeneity of the en- zyme will be carried out when sufficient amounts of concentrated purified enzyme are available.

The present investigation has not shown the presence of a definite chromophore on the purified enzyme. However, under the experimental conditions used in this work, no chromophore was detected when enzyme and ultraviolet-treated DNA were mixed in the dark, although there is no direct evidence that an appreciable amount of the complex between the enzyme and irradiated DNA was formed to give a detectable absorption spectrum for the chromophore. Experiments on the absorption spectrum of the isolated enzyme-substrate complex are in progress and will be reported in another communication. Another possibility is that the small amount of cytochrome absorption in

the purified enzyme may in fact be part of the enzyme and not a contaminant. Further purification is expected to settle this point.

Acknowledgments-Thanks are due to Dr. Jane K. Setlow for help in writing the manuscript and for helpful discussion during this work, to Mr. Maxon E. Boling for occasional technical assistance, to Dr. A. P. Pfuderer and Mr. D. Halliday for per- forming the analytical ultracentrifugation studies, and to Dr. E. F. Phares for help with the large scale centrifugation with the Sharples centrifuge.

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RUPERT,"~. S., J. Gen. Piysiol., 43, 573 [1960). ’ ’ L SETLOW, J. K., AND BOLING, M. E., Photochem. Photobiol., 2,

471 (1963). SETLOW. J.K., BOLING, M.E., AND BOLLUM, F.J., Proc.Natl.

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Amir MuhammedENZYME

LARGE SCALE PURIFICATION AND SOME PROPERTIES OF THE Studies on the Yeast Photoreactivating Enzyme: I. A METHOD FOR THE

1966, 241:516-523.J. Biol. Chem. 

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