[55] Immobilized nucleotides for affinity chromatography

[55]

NUCLEOTIDESFOR AFFINITY CHROMATOGRAPHY

475

3 times with 10 m M sodium phosphate at p H 7.8, containing 100 m M NaCl, and 0.5 m M E D T A . The cellulose pellet is then annealed to p o l y ( d C ) by resuspending the pellet in 0.83 ml of this buffer containing 0.11 m M 3H-labeled p o l y ( d C ) '~1 (7.6 X 10 ~ c p m / p m o l e of nucleotide). The mixture is agitated for 4 hours at 21 ° until about 60% of the p o l y ( d C ) has been annealed. The product is washed with the sodium p h o s p h a t e - N a C I - E D T A buffer and stored at 4 ° at a cellulose concentration of 10 m g / m l in this buffer; this is the substrate described in Fig. la. The assay for polynucleotide ligase measures the covalent linking of the "~H-labeled p o l y ( d C ) to the cellulose-dTdC substrate (Fig. 1). 29 31M. J. Chamberlin and D. L. Patterson, I. Mol. Biol. 12, 410 (1965).

[55] Immobilized

Nucleotides for Affinity Chromatography

B y MEIR WILCHEK and RAPHAEL LAMED

Introduction and Principle Nucleosides and nucleotide phosphates occur in many biological systems and act as enzyme substrates, as cofactors, and as coenzymes. Thus, covalently binding these compounds to insoluble matrices provides a useful tool for the study of their interactions with proteins 1,2 as well as media for affinity chromatography. 3,4 A simple and general method for coupling nucleosides, nucleotide phosphate, and coenzymes possessing vicinal free hydroxyl groups to agarose hydrazide columns, has been described. ~,6 (Scheme I ) . These systems are prepared by reacting an excess of a dihydrazide of the structure O O II II NH2N H--C(CH2)~C--N HNH.o to cyanogen bromide-activated polymer, in either sodium bicarbonate or acetic acid solutions. The coupling of periodate-oxidized nucleotides to the agarose hydrazide is carried out under mild conditions; the reac~M. Wilchek, ¥. Salomon, M. Lowe, and Z. Zellinger, Biochem. Biophys. Res. Commnn. 45, 1177 (1971). 2R. Lamed, Y. Levin, and A. Oplatka, Biochim. Biophys. Acta 153, 163 (1973). 3p. O. Larsson and K. Mosbach, Biotechnol. Bioenerg. 8, 393 (1971). 40. Berglund and F. Ekstein, Eur. 1. Biochem. 28, 492 (1972). 5R. Lamed, Y. Levin, and M. Wilchek, Biochim. Biophys. Acta 204, 231 (1973). e D. L. Robberson and N. Davidson, Biochemistry I1, 533 (1972).

476

NUCLEIC ACIDS, NUCLEOTIDES, AND DERIVATIVES

[55] NH 2

I I

NH

[04-

I

I o

HO

C----O

(~--O--H~C\H/O.H..-Base C C

(~)--O--C~/O..~.. Base

(CH2)x I

+

C----O I

NH D

NH

OH

I

C----NH I

O

Sepharose

(~) - - O - - H 2 c - . H I o \ H / B a s e C

C

/C.. IC-. HO H N H OH k

NH l

C-----O I

(CH~)~ I C~O NH i

NH I

C ~NH L

O

Sepharose SCHEME I

tion is fast, and the binding capacity is very high. 5 The same procedure was also used for coupling to polyacrylic hydrazide. The table shows some of the nucleotides coupled by this method. Alternatively, periodate-oxidized compounds can be allowed to react first with the dihydrazide, followed by coupling to cyanogen bromide activated agarose. In some cases it may be of advantage to reduce the hydrazone bond formed between the oxidized nucleotides and the hydrazide with sodium borohydride. This reduction gives a different structural analog, which may be more stable. Preparation of Selective AdsorbentS--Method I

Preparation of Acid Dihydrazides. One hundred milliliters of diethyl adipate, 200 ml of hydrazine hydrate ( 9 8 % ) , and 200 ml of ethanol are

[55]

NUCLEOTIDESFOR AFFINITY CHROMATOGRAPHY

477

THE BINDINGOF NUCLEOTIDESTO SEPHAROSEHYDRAZIDE Nucleotide

Micromoles added per milliliter of Sepharose

Micromoles bound per milliliter of Sepharose

AMP ADP ATP UMP UTP CMP CTP GMP GTP NAD NADP

4.4 4.8 7 3.9 4.0 4.8 4.2 3.7 4.5 5.0 4.7

4 4.3 3.5 3.6 3.2 3.9 3.5 2.9 3.3 3.75 4.1

refluxed for 3 hours. The resulting adipic acid dihydrazide precipitates and is recrystallized twice from ethanol-water mixture (mp 1 6 9 - 1 7 1 ) . Dihydrazides of longer carbon chains are recrystallized from acetic acid or washed with ethanol.

Preparation o[ Agarose Hydrazide (a) With a Water-Soluble Dihydrazide (Adipate). Sepharose 4B is activated by cyanogen bromide. 7,s The activated Sepharose gel is suspended in 1 volume of a cold saturated solution of adipic acid dihydrazide (approximately 90 g/liter) in 0.1 M sodium carbonate, and the reaction is allowed to proceed overnight at 4 ° with stirring. The Sepharosehydrazide gel is washed thoroughly with water and 0.2 M NaCI until samples of the washings show only minimal color reaction in the 2,4,6trinitrobenzene sulfonate test. Columns containing 6-12/~moles of hydrazide per milliliter of packed agarose are stored at 4 °. The column is washed again before use. (b) With Water-Insoluble Dihydrazide ( Sebacate = NH2NH---C (=O)--(CH~)8--C(--O) NHNH2). The cyanogen bromide-activated Sepharose gel is suspended in 2 volumes of a saturated solution of sebacic acid hydrazide in acetic acid at 20 ° (approximately 50 g/liter). The reaction is allowed to proceed with stirring overnight at r o o m temperature and the product is washed thoroughly with acetic acid:water ( 1 : 1 ) and then with water until samples of the washings show no color on reaction with 2,4,6-trinitrobenzene sulfonide. The sebacate column 7p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968). s R. Ax6n, J. Porath, and S. Ernbiick, Nature (London) 214, 1302 (1967).

478

NUCLEIC ACIDS, NUCLEOTIDES, AND DERIVATIVES

[55]

is more effective for affinity chromatography because of the longer spacer. Preparation of Periodate Oxidized Nucleotides. The method of Gilham 9 is used. Metaperiodate solution is added to cold neutral solutions of nucleotides to a final concentration of 10 mM nucleotide and 9.5 mM periodate although more concentrated solutions may also be used. The oxidation is allowed to proceed for 1 hour in the dark at 0 °. Preparation of the Affinity Adsorbents. To 1 ml of Sepharose hydrazide in 0.1 M sodium acetate at pH 5 are added 4-5 ~moles of periodate-oxidized nucleotide in 2.5 ml of the same buffer. The suspension is stirred for 3 hours at 4 °, and the product is washed with 1 M NaC1 and water. The absorbance of the supernatant liquid and washings are measured to determine the extent of binding (see the table). Preparation of Selective A d s o r b e n t - - M e t h o d II (a) Preparation o] Nucleotide Adipic Hydrazide. A 0.1 M neutral solution of nucleotide is oxidized by addition of solid metaperiodate (0.95 eq). The reaction mixture is stored at 0 ° in the dark for 1 hour and then titrated with HCI to pH 5. An equal volume of 0.1 M solution of adipic acid dihydrazide is added and the reaction is allowed to proceed at 4 ° f o r 3 hours. (b) Coupling of the Nucleotide Hydrazide to Sepharose. One milliliter of cyanogen bromide-activated Sepharose is suspended in 2 ml of the nucleotide-hydrazide solution containing 0.2 M sodium carbonate at pH 8.5-9.0. The mixture is stirred at 4 ° for 16 hours. The resulting conjugate may still contain cyanogen bromide reactive groups, and treatment with ethanolamine or acid is recommended. Reduction with NaBH4 of (l) and (II). The Sepharose-nucleotide derivatives are suspended in 2 volumes of 0.5 M Tricine chloride at pH 8 and stirred at 4 °. Three to four portions of solid N a B H , (10 mg per milliliter of Sepharose) are added at intervals of 1 hour and the resulting derivatives are washed with cold water and stored at 4 ° . Conditions for C h r o m a t o g r a p h y

Adsorption to and Elution of Glucose-6-phosphate Dehydrogenase from an Agarose-NADP Resin. Agarose-NADP (0.15 ml) is suspended in a total volume of 0.5 ml of 10 mM Tris chloride at pH 8.1. A suspension (10 td) of glucose-6-phosphate dehydrogenase (Sigma, 27 l~g/ml; 70 A.~,0 ..... units per minute) in 2.6 M (NH4)2SO, is added. After stirring for 10 minutes at 25 °, the suspension is centrifuged and samples from the supernatant fluid are checked for glucose-6-phosphate dehydrogenase o p. T. Gilham, this series, Vol. 21, p. 191.

[56]

NUCLEOSIDE PHOSPHATES ATTACHED TO AGAROSE

479

activity according to the method of Goldman and Lenhoff. 1° The immobilized NADP completely adsorbs the enzyme. The agarose pellet is suspended in 0.5 ml of 10 mM Tris chloride at pH 8.1 containing 4 mM NADP and is stirred for 5-10 minutes and centrifuged. The supernatant fluid is checked for dehydrogenase activity. The enzyme is readily recovered by this treatment. Adsorption and Elution of Heavy Meromyosin (HMM) from AgaroseA TP Resin. To a suspension of 3 ml containing 1 ml of Sepharose-ATP and Ca 2÷ (2 mM) in 10 mM imidazole buffer at pH 7, 1.6 mg of heavy meromyosin are added. After 15 minutes of stirring at 0 °, the suspension is centrifuged, and the ATPase activity and protein concentration are determined in the supernatant liquid. The active heavy meromyosin is almost completely adsorbed by the ATP-agarose conjugate. Mixtures containing water or Sepharose hydrazide instead of Sepharose-ATP are used as controls. To elute the HMM, concentrated solutions of ATP or ADP are added to give a final concentration of 5 mM nucleotide. After 15 minutes of stirring at 0 °, the ATPase activity and the protein concentration of the supernatant are measured. Most of the adsorbed HMM is released by this treatment. 1°R. Goldman and H. M. Lenhoff, Biochim. Biophys. Acta 242, 514 (1971).

[56]

Nucleoside Phosphates

Attached

to Agarose

By ROBERT BARKER, IAN P. TRAYER, and ROBERT L. HILL Nucleoside mono-, di-, and triphosphates and many of their derivatives, such as the sugar nucleotides and the vitamin-containing coenzymes, are substrates, inhibitors, or cofactors for a wide variety of enzymes. Thus, nucleotide phosphates attached to agarose should have wide application as affinity adsorbents for the purification of many different enzymes. Derivatives of nucleoside phosphates containing a primary amino group can be coupled readily to cyanogen bromide-activated agarose. The major problem in preparation of the adsorbents is the design and synthesis of the nucleotide ligands to be attached to agarose. At present, four kinds of ligand (I, 2, 3, and 4) have been synthesized containing an alkylor aryl-amino group attached to either the phosphate, the purine base, or the ribosyl ring of the nucleotide. Compound (1), 3'-(4-aminophenylphosphoryl)deoxythymidine 5'phosphate, is a good inhibitor of staphalococcal nuclease, and reacts with agarose through the p-amino group to give a useful adsorbent for the