Purification of leaf nucleotides and nucleosides on insoluble polyvinylpyrrolidone

ANALYTICAL

54, 276288

BIOCHEMISTRY

Purification

of

Leaf

on Insoluble

(1973)

Nucleotides

and

Nucleosides

Polyvinylpyrrolidonel

Determination of plant nucleotides is often hampered by the presence of other ultraviolet-absorbing materials, especially phenolic compounds (1,2). Stewart and Guinn (2) found it necessary to purify nucleotides from cotton leaves by chromatography on Sephadex” G-10 even after separation by anion exchange chromatography. The interfering materials resulted in high background absorbance that, in some cases, prevented identification and quantitation of nucleotides without further purification. Activated carbon did not separate interfering phenolics from nucleotides. An insoluble form of polyvinylpyrrolidone, Polyclar AT”, sorbs phenolics (3,4) and has been used to purify plant extracts that contained enzymes (4)) hormones (5)) guanosine diphosphate derivatives of various sugars (6)) and ATP (7). Although ATP is not retained by Polyclar AT (7)) purines and pyrimidines are retained to some extent (8). This paper reports (a) elution profiles and recoveries of known nucleotides and nucleosides, alone and in the presence of cotton leaf extract, from columns of Polyclar AT; (b) the amount of purification obtained by passing leaf extracts from alfalfa, cotton, grape, and orange through Polyclar AT at different pH levels; and (c) compares extraction with isoamyl alcohol and ether with purification on Polyclar AT for removal of uv-absorbing impurities. MATERIALS

AND

METHODS

Elution and Recovery of Nucleotides .and Nucleosides. Elution profiles were determined by eluting known nucleotides and nucleosides (Sigma Chemical CO.~) from columns of Polyclar AT. The Polyclar AT was purified and fines removed as reported previously (7). The nucleotides and nucleosides were dissolved separately in 1 mM HCl to give 0.1 mg/ml. Five-milliliter portions were loaded onto 2 X 6-cm columns (18.8 cm3) of Polyclar AT and eluted with 1 mM HCl at room tempera’ Journal Paper No. 1967 of the Arizona Agricultural Experiment Station. ‘Mention of a trade name or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. 276 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

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COMYIUNICATIONS

277

ture. Flow rate was about 2.5 ml/min at a head of 1.3 m. Elution was monitored with an ISCO~ Model UA-4 absorbance monitor at 254 nm until 60 ml of effluent were collected in a graduated cylinder. Recoveries were calculated by comparing absorbancies at 260 nm of samples eluted from the columns with equal dilutions of samples not passed through columns of Polyclar AT. (By this method, of course, indicated recovery would be less than 100% if any impurities were present and were sorbed by the Polyclar AT.) In order to determine whether any of the components of leaf extract affected recovery, individual nucleotides and nucleoeides were mixed with cotton leaf extract prior to plirificntion on columns of Polyclar AT. Five-milligram portions of nucleotidcs and nucleosides were dissolved separately in 20 ml of 1 mill HCl. Five-gram portions of lyophilized cotton leaf tissue, ground to 1~~s :I 4%mesh screen, were extracted with 100 ml of ice-cold 0.5 X HClO, for 5 min at slow speed in a Virtis? homogenizer. After centrifugation, the pH of the supernatant fraction was adjusted to 3.0 with 2 A’ KOH ancl the extract was centrifuged again to remove KClO,. Five-milliliter portions of leaf extract were mixed with (al 5 ml of nuclcotide or ntlclcoside or (h) 5 ml of I mM HCl. Five-milliliter portions of the rc.Qulting misturcs were passed through 2 X B-cm columns of Polpclnr AT and eluted n-it’ll 1 mM HCI until 60 ml of effluent were rollectcd. Net’ absorbance due to the known nucleotide or nucleositle, mixed I\-ith leaf extract,, was determined by subtracting absorbance at 260 nm of purifietl lcnf extract (b) from that given by the mixture of leaf extract and known nucleoticle or nucleoside (a). Recovery was calculated by comparing this value with absorbance given by an equal dilution of unpl~rifierl reagent nucleoticle or nucleosicle. Attempts were made to detcrminc cllltion profiles of nuclrotides and nucleosides in the presence of cot,ton leaf extract, but this was not possible because the abs0rbanc.e ljrofilcs were influenced by components in the leaf extract. Elution volumes to absorbance peaks were recorded, however, and give some indication of the effect of leaf extract on elution of the known nucleotides and nnclcosidcs. pH and PWificntion. Leaves of alfalfa (Me&cnf~o safizin cv. LYMoapa”), cotton (Gossypiuw hirsutwu cv. “DPL 16”) , grape (Vitis vinifera w. “Thompson seedless”), and orange (Citrus sinensic cv. “Arizona sweet”) were washed with deionized water, lpophilized, and ground to pass a 40-mesh screen. Samples of 900 me were est.racted with 15 ml of ice-cold 0.5 N HClO, for 5 min at slow speed with a VirTis” homogenizer. Insoluble mat’erial was removed by ccntrifugation and suction filtration. The supernatant fraction was adjusted to ihe appropriate pH with 1 N KOH while cooling in ice water and the extract was crnt,rifuged again

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COMMUNICATIONS

to remove KCIO,. Other extractants, such as trichloroacetic acid plus 8-hydroxyquinoline (9)) might give more nearly quantitative extraction of nucleotides; HClO, was used in this study because it does not absorb light at the wavelengths used in subsequent analyses. Five-milliliter portions of each extract were purified on 2 X 6-cm columns of purified Polyclar AT preconditioned at the appropriate pH. Effluent was collected in a loo-ml graduated cylinder during loading and subsequent elution. Dilute HCl (lCV, 3.2 X 10-3, 10-3, and 3.2 X 1W M) was used as eluent at measured pH values of 2.0, 2.5, 3.0, and 3.5. Deionized water was used to elute cotton leaf extracts loaded at pH 4.5 and 5.5. A total of 60 ml of eluate was collected and absorbancies at 230, 260, 290, 320, and 350 nm were compared with those given by an unpurified portion of the same extract. Comparison of Isoamyl Alcohol, Ether, and Polyclar AT. Samples of cotton leaf extract, adjusted to pH 3.0, were extracted four times with an equal volume of isoamyl alcohol (3-methyl-1-butanol) and then twice with ether, were extracted six times with an equal volume of diethyl ether, or were purified on Polyclar AT as outlined above. Isoamyl alcohol and ether were withdrawn by aspiration after samples were centrifuged. Residual ether was removed in a rotary evaporator after the final extractions. Absorbancies of appropriate dilutions of the resulting extracts were determined and compared with those of unpurified extract. RESULTS

AND

DISCUSSION

Elution and Recovery of Nucleotides and Nucleosides. Although differences in retention were noted, all of the tested nucleotides and nucleosides were eluted in less than 60 ml (Table 1). Guanosine was retained longer than any of the other materials tested, but it was completely eluted after 54 ml had passedthrough the column. Very little tailing was evident at pH 3.0, but some tailing occurred, especially with guanosine, 2’ & 3’-AMP, uridine, and 2’ & 3’-UMP, when samples were loaded at pH 3.5 and eluted with water instead of 1 mM HCl (data not shown). Recoveries were good and were little affected by the presence of leaf extract (Table 1). Recoveries of the two samples that contained glucose, ADPG and UDPG, were apparently decreased slightly by the presence of leaf extract, but were still 89% each. There was little discernible effect of leaf extract on elution of known nucleotides and nucleosides except in the cases of GMP, GTP, and the uridine derivatives. In those cases leaf extract apparently caused more rapid elution, as indicated by lower volumes to the absorbance peaks (Table 1). Even though leaf extract and the known nucleotide or nucleoside contributed about equally

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Elution Profiles Eluted from

TABLE 1 and Recoveries of Known Nucleotides and Nucleosides 2 X 6-cm Columns of Polyclar AT with 1 mM HCl Elution

Sample

Start

volume” Peak

End

ml

ml

(28)b (24) (23) (23) (22) (22) (24) (22) (38) (26) (23) (24) (22) (21) (22) (21) (22) (20) (21)

40 37 35 34 36 33 32 30 54 50 51 39 41 38 39 45 35 30 31

ml Adenosine 2’ & 3’ AMP 5’ AMP ADP ATP ADP-Glucose Cytidine 5’ CMP Guanosine 5’ GMP GTP Uridine 2’ & 3’ tJMP 5’ UMP UDP UTP UDP-Glucose NAD NADP

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17 16 16 15 18 17 15 14 27 25 27 16 18 18 18 20 18 15 15

26 24 23 22 25 22 22 20 38 35 37 25 26 26 26 32 26 20 21

Recovery % 97 97 96 98 98 98 92 92 99 97 94 99 97 97 98 95 97 98 98

(95)b (97) (93) (99) (94) (89) (98) (99) (99) (97) (95) (94) (95) (102) (100) (91) (89) (98) (96)

a Elution volumes include 5 ml added during loading of samples and were determined by monitoring absorbance at 254 nm as the effluents were collected in graduated cylinders. * Values in parentheses were obtained when cotton leaf extract was mixed with the known nucleotide or nucleoside prior to purification. Recoveries were calculated by subtracting absorbance at 260 nm of purified leaf extract alone from absorbance given by a mixture of leaf extract and a known nucleotide or nucleoside. Net absorbance due to the known nucleotide or nucleoside in the mixture was then compared with that given by an equal dilution of the same nucleotide or nucleoside that was not passed through Polyclar AT.

to absorbance of the effluent at 260 nm, in most cases only a single peak was observed. Volume to the absorbance peak for leaf extract alone varied between 22 and 23 ml. pH and Purification. Removal of uv-absorbing material by Polyclar AT tended to increase with acidity (Table 2). Purification of cotton leaf extract was poor at pH 5.5 and only fair at pH 4.5. The optimum pH for purification appeared to be between 2.0 and 3.0, with only minor differences between pH 2.0, 2.5, and 3.0. Andersen and Sowers (3) reported that bonding between Polyclar AT and plant phenols increased

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TABLE 2 Influence of pH on Purification of 0.5 N JICIOI Extracts from Grape, and Orange Leaves on 2 X G-c:m %trlurnns of Polyclar as Percent of UV-Absorbing Material Kernwed.) Wavelength,

PI-1

230

260


P’ /C

Alfalfa, Cotton, AT. (Expressed

nm

290

350

c/o

%

Alfalfa 2.0 2.5 3.0 3.5

59.1 58.8 58.6 52.1

62.1 60.2 47.0 37.x

94.4 53.5 74.9 65.9

2.0 3.0 3.5 4.5 5.5

67.9 73.3 71.6 55.8 -

70.3 70.6 G7.7 59.8 38.7

90.6 90.3 8S.l 68.2 21.9

2.0 2.5 3.0 3.5

72.2 73.4 73.8 76.5

75.1 75.1 88.8 69.2

92.8 92.5 86.6 85.5

2.0 2.5 3.0 3.5

68.8 68.0 66.5 57.3

69.5 64.6 65.1 58.2

56.5 85.4 86.4 82.5

94.4 94.5 92.9 86.2

96.0 96.3 95.0 90.1

93.4 93.6 92.0 71.4 19.3

95.3 94.5 93.1 81.4 29.6

96.7 96.6 95.4 95.4

97.8 97.8 97.6 96.9

94.2 93.9 93.7 92.2

96.0 95.5 95.2 93.9

Cotston

Grape

Orange

with increasing acidity down to pH 3.5. Below pH 3.5, however, bonding of caffeic acid decreased rapidly so that less was removed at pH 2.5 than at pH 5.5 (52.7 vs 75.7%). They obtained similar results with other plant phenols studied. My results indicate a lower and much less critical optimum pH. Differences in experimental methods may have caused the apparent difference in optimum pH. Andersen and Sowers (3) used solutions of individual phenols in 10% methanol and adjusted the pH to the desired acidity with acetic acid, whereas, my extracts contained a complex mixture of substances in aqueous HClO,. If carboxyl groups form hydrogen bonds with Polyclar AT, as Andersen and Sowers (3) suggested in their discussion, the increasing amounts of acetic acid needed to obtain pH values below 3.5 may have competed with phenols for hydrogen bonding to Polyclar AT. The concentration of undissociated

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TABLE 3 Comparison of Passage t,hrough Polyclar AT at pH 3.0, Extraction with Isoamyl Alcohol then Ether, and Extraction with Ether Alone for Removing UV-Absorbing Substjances from Cotton Leaf Extracts Wavelength, Purification

method

230

360

Polyclar Isoamyl Etherc

ATfl alcohol

a Samples * Samples c Samples

then

et.her*

80 L’1 18

350

removed

C’ / c,

91 2.1 1.7

320

290 Percent

C-’ ic

nm

<” io

0.’ /c

7%

90 29 ‘2 1

94 33 1.7

95 41 1.8

were eluted from 3 X 6-cm columns of Polyclar were extracted four times wit,h isoamyl alcohol were extracted six times with diethyl ether.

AT then

with 1 mM HCl. twice with ether.

acetic acid would he 0.584 M at pH 2.5 in a pure aqueous solution and might be even higher in 10% methanol. Comparison of Isoanzyl Alcohol, Ether, and PolyclaT AT. Isoamyl alcohol apparently removed anthocyanin, as indicated by a bright pink color of the extrsct. The aqueous residue remained amber colored, however, and only 33% of the substances that absorbed at 320 nm were removed by four extractions w-vii11 isoamyl alcohol followed by two extractions with ether (Table 3). Six cstractions with ether removed only 1.7% of t.he material that. absorbed at 320 mn. Polyclar AT, on the other hand, removed 9476 of the substances in cotton leaf extract that absorbed at 320 nm, and did so without appreciable loss of any of the nucleotides or nucleosides tested. Removal of phenolic substances should facilitate subsequent chromatography of nucleotides and nuclcosides and their analyses by ultraviolet absorbance and enzymatic assa,y. SUMMARY

Of the 19 nucleotides and nucleosicles tested, all were eluted by 1 mM HCI in less than 60 ml from 2 X 6-cm columns of Polyclar AT (an insoluble polyvinylpyrroliclone). Recoveries were good and, with the possible exceptions of ADPG and UDPG, the presence of cotton leaf extract did not decrease recovery of known nucleotides and nuclcosides. Passing leaf extracts through Polyclar AT removed most, but not all, of the uv-absorbing impurities that interfere with quantitation of nucleotides and nucleosides. The optimum pH for purification of HClO,

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extracts from leaves of alfalfa, cotton, grape, and orange appeared to be between 2.0 and 3.0. In this pH range Polyclar AT removed from 59 to 91% of the substances in leaf extracts that absorbed at 230 nm and from 93 to 97% of the substances that absorbed at 320 nm. Extraction of leaf extract with isoamyl alcohol was relatively ineffective and extraction with ether was almost completely ineffective in removing uv-absorbing impurities. Because nucleotides and nucleosides quickly pass through a short column of Polyclar AT at pH 3.0 while plant phenols are retained, this procedure provides a simple and rapid method for bulk purification of leaf extracts prior to chromatography and assay of nucleotides and nucleosides. REFERENCES 1. INGLE, J. (1963) Phytochemistry 2, 353. 2. STEW~T, J. McD., AND GUINN, G. (1971) Plant Physiol. 48, 166. 3. ANDERSEN, R. A., AND SOWERS, J. A. (1968) Phytochem@try ‘7, 293. 4. LOOMIS, W. D., AND BATTAILE, J. (1966) Phytochemistry 5, 423. 5. GLENN, J. L., Kuo, C. C., DURLEY, R. C., AND PHARIS, R. P. (1972)

chemistry

11,

Phyto-

345.

6. SELVENDRAN, R. R., AND ISHERWWD, F. A. (1967) Biochem. J. 7. GUINN, G., AND EIDENBOCK, M. P. (1972) Anal. Biochem. 50, 8. LERNER, J., DOUGHERTY, T. M., AND SCHEPARTZ, A. I. (1968)

105, 723. 89. J. Chromatog.

453. 9. ISHERWOOD,

F. A., AND BARRETT, F. C. (1967) Biochem. J.

104,

922.

GENE

Western Cotton Research Lab Agricultural Research Service U. S. Department of Agriculture Phoenix, Arizona 85040 Received August II, 19Yd; accepted March 6, 19YZ

GUINN

3’7,