Purification of flavin-adenine dinucleotide and coenzyme a on p-acetoxymercurianiline-agarose columns

\NZI

YrlC\I

HIOCHtlhllSIKY

Purification Coenzyme

68, 349-357

f 1975)

of Flavin-Adenine

Dinucleotide

and

A on p-AcetoxymercurianilineAgarose

Columns’

I. The adsot-bent p-accto\ymel-curlaniline-S~pharo~e 6B (PARIA-Sephat-ose hB) wah prepared by covalently linking PAMA tc> CNBr-activated Sepharo\e 6B. column wa\ equilibrated with dilute xetic xid fiOnic 2. When the adsorbent strength. I‘/? L U.005. pH j-51 f-AD. FMN. Al-f’ OI- ADP v,u\ f’ound tt) hc oldbarbed to the column. whereas riboflavin. AMP and adcnosine were not. The adxxhed compounds could be eluted with 0. I hf Na(‘l. The xfsorbing c~lpncitle\ tcj the\e compound\ were maximill at pH 3.0. i. When the ~ld\orbcnt column W;L\ tl-eated with I’i 2-mercaptoeth~lnol (2.hlE) and then equilibrated with the buffer t I‘/? 7 0. I-I .O. pH 3-71. reduced (‘0~4. cy\tcine :tnd glutathione we,-e adx)rhed to the column. \h’hcl-e;i\ oxidized <‘<)A, F.-ZD. ATP. ADP and AMP cccl-e not. The\e adsorbed compounds could be eluted with I C’r Z-ME. Ifthe udxxbent column wxs not previou4y treated with I Cc ?-ME. the\e \ulthydryl compound\ were ads,orbed to the column in Hn irreversible manner. 1. By chromatqtxphic puritication employing the ;tdsol-bent column ~llf I’/? = 0.005 and pH 3.0. FAD of’ YXQ purity W;I\ isolated in it yield of Y.Sc; from a partially purified \ample 01‘ 75:; purity. 5. By chrom~~toFt.~~phi~ purification emplc)yinp the xfs>rbent column at I’/2 = 0. I and pH 5.0. I-educed (-‘uA of’ US’3 purit) \h’;l\ ol&ned in 2 yield 01‘ YO? from it cl-udc f’or f-Al> ~;LLI I .7 mg per ml 01‘ the ad\ol-bent under open-sting condition\ of’ I /2 = 0.005 and pH 1.0. and th;it tol- ~-ed~~cecl C <)A N;I\ 7.9 mg per ml of’ the ad\orbcnt ;it l/2 y 0. I xnd pH i.0. Ihe c~~pacitie\ of the XL horbcnt for both compound\ wet-e not changed hy storage at S ‘(‘ in xetone fc>r at Ica\t 2 month\.

Generally. FAD and reduced CoA have been purified from microbial cells or fermentation broth by ion-exchange chromatography ( 1.2). How’ Prr\cnted at the Annual h’feeting uf’ the .lapane\e Biochemical Society. Nq!oya. Japan. Teptemher 30. I Y7i. A Present addrebh: Division of Enrymok>,gy. Institute for- f’rotein Kesearch. OS&I Univer\ity. 53 1 I ynmada-kmi. Suita: Os~lhn 565. J;~pml 3JY Copyright All right,

> 197s hy Academic Pres\. Inc. of reproductwn in any form rcservrd.

350

MATUO

ET

AL.

ever, these methods are not advantageous for mass production of FAD and reduced CoA because of the complexity and low yield. Recently. it was found that mercuri-polysaccharide columns are useful for the purification of mononucleotides (3) and for the separation of mercaptopapain and nonmercaptopapain (4). In this paper. PAMA:’ was immobilized by covalently linking it to CNBr-activated agarose for purification of FAD and reduced CoA. The behavior of FAD, FAD-related compounds, and disulfide and sulfhydryl compounds to the PAMA-agarose column was investigated and the adsorbent column was found to be useful for the purification of FAD and reduced CoA. MATERIALS

AND

METHODS

Materials. Sepharose 6B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Florisil (60- 100 mesh) was the product of Floridin Co. (Florida). p-Acetoxymercurianiline was purchased from Katayama Chemical Industries Co., Ltd. (Osaka, Japan). ATP, ADP and AMP were purchased from Sigma Chemical Co. (St. Louis, MO). FAD, reduced CoA and glutathione were the products of Tanabe Seiyaku Co., Ltd. (Osaka. Japan). All other chemicals were of analytical grade. Pwprmtiorl c?f’ partidly plrrified FAD. Production of FAD was performed according to the procedure of Watanabe et [II. (5). The fermentation broth was centrifuged and the resulting supernatant fluid ( 10 liters) containing 8 g of FAD was passed through a Florisil column (bed volumn, 1.5 liters). After the column was washed with 3 liters of 0.5% acetic acid. elution was continued with a mixed solvent of phenol : acetic acid: water ( 1 : 6: 16. by volume). The FAD fraction ( 1 liter) was applied onto a column (bed volume, 150 ml) of Amberlite IRA-401 (Cl type). After the column was washed with 1.5 liters of 0.01 N HCI, elution was performed with 10% NaCl. To the resulting FAD solution (500 ml). 304 g of ammonium sulfate was added. After adding 50 ml of phenol to the solution, the mixture was vigorously shaken. To the separated phenol phase. SO ml of water and 250 ml of ethyl ether were added, and the mixture was shaken. The resulting aqueous phase was collected and used for the experiments as partially purified FAD solution. Its purity was 7S%, and its yield from fermentation broth was 60%. Pwprmtion qf’ c~lrdc r~)ti~rcrd CoA unti osidizeti CoA. Crude reduced CoA and oxidized CoA were prepared from Sar-cYrlrr lutccl according to the method described previously (6). Pwpcrrcltion of’ PA MA-Sepi~urose 6B colurm. PAMA-Sepharose 6B was prepared as follows by the method of Sluyterman and Wijdenes (4) :I Abbreviations

used:

PAMA,

p-acetoxymercurianiline;

Z-ME,

Z mercaptoethanol.

PURIFICATION

OF FAD

AND

CoA

is1

with some modifications. Sepharose 6B (4 liters) was activated by 7 kg of CNBr according to the method of Ax& r’t trl. (7). Cyanogen bromide should be handled with care. The activated Sepharose 6B was suspended in 4 liters of 5% aqueous dimethyl sulfoxide solution. To the suspension, 50 g of PAMA dissolved in 500 ml of dimethyl sulfoxide was added. The mixture was gently stirred for 70 hr in an ice bath, and the mixture was warmed at 3X for I hr and filtered. The resultant PAMA-Sepharose 6B was packed into a column. The column was washed with I00 liters of 20% aqueous dimrthyl sulfoxide solution and IO liters of 0.1 M NaCl. This treatment was repeated until no more me]-= cury compound was detected in the effluent. P,rpcrrrrtio,z 01’ I~/@JIx. Dilute acetic acid solutions (pH 3.0. 4.0 and 5.0). 0.2 M sodium acetate buft;=r (pH 3.0 and 5.0). and 0. I IV sodium phosphate buffer (pH 7.0) were used for equilibration of a column. Dilute acetic acid solutions were prepared by diluting glacial acetic acid with water to the desired pH. The ionic strength of the buffers was adjusted to the appropriate value by adding I M NaCl or by diluting with water. The ionic strength was monitored as described previously (6). Dcto.tlli/lrrtio/z c?f’FAD. Determination of FAD was carried out by the fluorometric method (8). f)etci.rrzi/l~lti0/2 (?f Carl. Determination of C‘oA wax performed by the method described previously (6). Dcto.nzitlLitioti.\ 01’ c,>,stcTinc) lrrltl ,glr/tot/liotrc. Cysteine and glutathione were determined by the ninhydrin method (9) using a Technicon Autoanalyzer. Drtet.nlilltrtion (!I’ rlro~~rlt~~ ~Y)II~~x~~II~~/. Mercury compound in the effluent was detected by extraction of mercuric dithizonate with CCI, at pH 5 according to the method of Irving and Cox ( IO). MotlitotYtr,y 01’ rrhso&trllc~c~ rrt 260 11~1. Absorbance at 260 nm of the effluent was monitored by an LKB K3OOA L!VICORD II (Bromma. Sweden). RESULTS

To clarify the adsorption specificity of a PAMA-Sepharosc 6B coumn. FAD. FAD-related compounds. and disulfidc and sulfhydryl compounds were separately charged onto the column. When the column wab equilibrated with dilute acetic acid (I’/2 = 0.005. pH 3-S). FAD. FMN, ATP and ADP were adsorbed to the column. whereas riboflavin. AMP and adenosine were not (Table I ). These adsorbed compounds were eluted by increasing the ionic strength to 0.1 with NaCI. The adsorbing capacities of the column for these compounds showed a maximum value at pH 4.0. When the column ~a.4 treated with 1% Z-ME and then

352

MATUO

BEHAVIOR

OF

FAD

AND

ET

TABLE 1 FAD-RELATED

PAMA-SEPHAROSE

capacity

Equilibrated solution

(mgiml

with dilute acetic (T/Z = 0.005) -__ pH

3.0

of adsorbent)

4.0

acid

pH

5.0

0.9 0.3 0

1.2 0.3 0

0.5 0.1 0

ATP ADP AMP

0.6 0.3 0

0.8 0.4 0

0.3 0.3 0

Adenosine

0

0

0

with compound

6B

was

packed

into

a column

(bed

volume.

dilute acetic acid solutions (II/- ? = 0.005) (4 mg) was dissolved in the same dilute

for column equilibration. water ot- addition of with 0.1 for FAD.

ON

FAD FMN Riboflavin

‘I PAMA-Sepharose equilibrated spective

pH

COMPOUNDS

COLUMNS”

Adsorbing

Compound

AL.

and ionic strength I M NaCl after adjusting

M NaCI. Adsorbing FMN and riboflavin

of the solution was the pH. Adsorbed

capacity of the adaorbent and A!,;,, for ATP. ADP.

was AMP

3 ml).

at the acetic

The

column

pH indicated. acid solution

adjusted by compounds

calculated by and adenosine

dilution were

was The reas used with eluted

measuring A,,,, in the eluate.

equilibrated with the buffer (I-/2 = 0.1-I .O, pH 3-7), reduced CoA. cysteine and glutathione were adsorbed, whereas oxidized CoA, FAD, FMN. ATP, ADP and .AMP were not (Table 1). The sulfhydryl comw.:;~:s :lGsorbed were eluted with 1% Z-ME. However. these com+,:.::CS \ve~‘e adsorbed to ihe COICII::: in an irreversibie manner when the column VV’~Snot previously treated wiril 1% Z-ME. These results indicate that PAMA-Sepharose 6B columns treated with 1% Z-ME are useful for the adsorption of FAD under the operating renditions at 1’13 = 0. I--l .O and pH 4.0 with acetic acid and oiY reduced ,::~,4 :‘t “/l = 0. I- 1.O and pH J-7.

When ‘J liters of partially purified FAD solution (amount of FAD -= 0.6 g, 1-13 = 0.005, ?H 4.0) was charged onto a PAMASepharose hB column equi!ibrat-ed with dilute acetic acid solution : l-/2 = 0.005. pH 4.0). the greater pa.? of ?iit‘ ;o;:%minants having an absorbznce at 150 nm was not adsorbed (Fig. I). Adsorbed FAD was eluted by increasing the ionic strength to 0.1 with NaCI. The rssiiiting .fAD fraction (I liter) was evaporated to 500 ml under vacuum. To the 5oiution. 30 g of ammonium sulfate was added and dissolved. After add-

PURIFICATION

FAD

OF

TABLE BEHAVI~K

OF NLICI

2

EOTIDES AND DIWLFIDE PAMA-S~PHAKOS~ Adsorbing

AND SULFHYDKYL COLL’MNV capacity

(mgiml

Equilibrated I‘/? Compound Nuclevtides AMP ADP ATP FAD SS compound O\idizrd SH

(pH

353

CoA

AND

3.0)

I-P

COMPOWDS

of acl~nrhent~ with

(pH

5.0)

T/Z (pH

0. I

0.5

1.0

0. I

0.5

I .o

0. I

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0 0

0 0

0 0

0

OK

Co.4

7.0)

0.5

I .o

0

0 0

0 0

0 0 0

0 0

0

0

0

compound\ Reduced CoA Cy\tcine Glutathionc

” PAMA-Sepharosr pt-c~iously \b~l\hed

hB \vith

WH\ packed IO ml of I?;

cated. Sodium acetate huffel(pH 3.0 employed. The respective compound for column \\atcr or elutscl

with

equilibration. xldition of 1”;

:-ME.

into

:i column

tbeci

volume.

3 ml).

The

column

~‘35

Z-ME ;Ind then equilibrated with the hl;!‘f?rs indiand 5.0) :~nd wdium phosphctte buffer(pH 7.!)1 \ti (20 mg) v,x\ dissolved with the same hufftx ;I\ u\cci

and ionic strength a.)f the solution I hi NaCl after aciju\tin g the pH. Adwl-hing c:tpacit\; a:~\; calcul~ttrcl

wls The hv

ad.ju$ted xisorbed deter-mination

hy

clil!l!itw

\I ith

comnwnci\ wc‘rc‘ of compound

634

SHORT

COMMUNICATlONS

TABLE RECOVERY

Incubation time

(hr) 0 2 16

OF CYSTEIC

Bovine Moles acid/mole

adrenal

of cysteic protein 5.05 5.01 3.59

ACID

2

FROM FERREDOXIN ZINC INCIJEL~TION

ferredoxin

AFTER

THE

Spin&a

Recovery (%o) 100 99 71

Moles acid/mole

ALKALINE

platensis of cysteic protein 4.99 5.08 4.46

ferredoxin Recovery (%o) 100 102 89

over 2 hr with the alkaline zmc reagent resulted in additional release of labile sulfide and the molar ratio of iron to sulfur approached one. The freshly prepared adrenal apoferredoxin yielded essentially no detectable labile sulfide even after preincubation over 2 hr. A quite similar phenomenon was also observed with Spirulina platensis ferredoxin from the alga which contained two gram atoms of iron per mole of protein. The value of one mole of labile sulfide per mole of protein was obtained under the original assay conditions, while extension of the alkaline zinc incubation over 2 hr resulted in release of two moles of labile sulfide per mole of protein. In contrast to the case of these ferredoxins, all labile sulfide in both spinach and parsley chloroplast ferredoxins was rapidly released when added to the alkaline zinc reagent. It was considered that the additional detectable sulfide liberated from the prolonged incubation might be due to H,S derived directly from cysteine residues by a p-elimination reaction, because the action of strong alkali may produce H,S from cysteine residues in protein (14). If this were the case, the cysteine residue should be consumed in amounts equivalent to the additional detectable sulfide. As is evident from Table 2, when the ferredoxin was incubated with the alkaline zinc reagent for 2 hr, there was no effect on the amount of cysteic acid recovered. However, at the 16 hr incubation the recovery of cysteic acid was slightly decreased. Moreover, hydrolysates of the samples incubated with the alkaline zinc reagent were examined for the presence of pyruvic acid, the hydrolysis product of dehydroalanine (15): the samples at the 2-hr incubation did not contain a significant amount of pyruvic acid.

PURlFlCATlON

” A&orbed pH

5.0):

rcftm

indicate\ precipitate

10 c~uiilircil

the indicates

+ rt‘ciuceii

fraction the

FAD

OF

elutecl fraction

with

AND

I’;

precipitated

Z-ME with

35s

CoA

containing

LiCl

;tcetc)ne-nlrthilli~)1.

(I‘:? Total

= 0.1. Co:4

c‘~,.4.

LiCl-HCI solution (I‘/3 = 0. I. pH S.0). most of the contaminants having an absorbance at 260 nm were not adsorbed (Fig. 2). Reduced CoA adsorbed was eluted from the column with 1% Z-ME containing LiCl (I‘/2 = 0. I. pH 5.0). After the elution of reduced CoA. high absorbance at 260 nm due to 1%’ Z-ME was observed. The resulting reduced CoA fraction (5.6 liters) was lyophilized. Lyophilized powder was dissolved in 500 ml of water and to the solution 5 liters of acetone-methanol f IO: I. by volume) was added. Precipitates produced were collected and dissolved in 500 ml of water. This acetone-methanol treatment was

” The

adwrbent

(100

ml)

wa\

stored

al

5°C

in

acetone

for

the

indicated

period.

The

xiwrbcnt with .~eparately packed into two columns (bed volume, 5 ml in each column). One column for the adwrption of F.4D W;L\ equilibrated with dilute acetic acid (I>/2 = 0.005. pH J.0). Other conditions were the same a4 in Table 1. .Another column for the adsorption of reduced Co.4 waj washed with 10 ml of I? ?-ME and then equilibrated with acetatc hrlffer (1’!? = 0. I pH 5.0). Other condition\ were the \amr a\ in Table 2.

356

MATUO

ET

AL.

A sumrepeated three times to remove Z-ME and LiCl contaminants. mary of this purification procedure of reduced CoA is presented in Table 4. Reduced CoA of 95% purity as reduced CoA was obtained in a yield of 90% (54% yield from fermentation broth) without contamination by mercury compound. Purity of the reduced CoA as total CoA was 100%. indicating that the CoA fraction was highly purified.

The stability of the adsorbent was investigated. The results show that it was stable for at least 2 months, when stored at 5°C in acetone (Table 5). DISCUSSION It was found that PAMA-Sepharose 6B columns could adsorb FAD. FMN. ATP and ADP. when equilibrated with diluted acetic acid of lower ionic strength (I‘/2 = 0.005, pH 3-5). and that the column could specifically adsorb reduced CoA, cysteine and glutathione. when treated with I % Z-ME and then equilibrated with the buffer (I‘12 = 0. I-I .O. pH j-7). It is suggested from these results that the adsorption of FAD to the column is due to an affinity between immobilized Hg and “adenosine + 2 molecules of phosphate” and/o] “isoalloxazine + one molecule of phosphate.” The former affinity is assumed from the result of Simpson (I I) that adenosine associates with methylmercuric ion at pH 2-4. It is also indicated that the adsorption of reduced CoA to the column is due to an affinity between immobilized Hg and SH residues, in good agreement with the results of Sluyterman and Wijdenes (4). Generally. it is very difficult to isolate highly purified FAD and reduced CoA in a high yield by conventional techniques. However, by introducing the adsorption-desorption process with PAMA-Sepharose 6B column to Amberlite IRA-40 I chromatography, highly purified FAD can be isolated in a good yield. We previously reported that purification of reduced CoA by affinity chromatography (6). PAMA-Sepharose 68 is superior to the affinity adsorbent because of higher capacity and higher stability. Therefore, PAMA-Sepharose 6B is very useful for the isolation of highly purified reduced CoA in a good yield. From these results. it was found that PAMA-Sepharose 6B is useful for the isolation of FAD and reduced CoA without contamination by mercury compound. However. industrial utilization of the adsorbent is now prudently under investigation because of pollution by mercury compound. ACKNOWLEDGMENTS

PURIFICATION

OF

FAD

REFERENCES

AND

CoA

357