Vol. 78, No. 4, 1977

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FACTORS AFFECTING TERMINALDEOXYMICLEOTIDYL TRANSFERASE ACTIVITY IN CACODYLATE BUFFER ThomasP. Chirpich Department of Chemistry Memphis State University Memphis, Tennessee 38152 Received

September

2,1977

SLMY: For reproducible terminal deoxynucleotidyl transferase activity in cacodylate buffer, assay tubes need scrupulous cleaning, and for maximal activity with the commercial enzyme, zinc ion is required when dATP is polymerized. Potassium ion is also required for maximal activity, though ammonium ion may be used in somesituations. The enzyme is readily inhibited by a variety of negative ions, and inhibition by the monovalent anions follows the chaotropic series. This result suggests that cacodylate ion belongs at the beginning of the chaotropic series and so may be generally useful in assaying enzymes with labile activity.

One assay for terminal deoxynucleotidyl transferase uses cacodylate buffer 2+ and either dATP and Mg or dCTP and Co’+ (1). More recently, a number of laboratories

have begun using Tris-buffered

cases, the activity

systems (2-5);

in the Tris system was compared to the activity

cacodylate system (3) and found to be l/26 the activity cacodylate system. The Tris system was still been unable to reproduce the high literature system.

Difficulties

number of factors system.

in one of these

reported for the

used because the workers had values given for the cacodylate

had also been encountered in this laboratory were found to affect

in the

the activity

and a

measured in the cacodylate

These are reported here. MATERIALSANDMETHODS

Purified terminal transferase was purchased from PL-Biochemicals. The pI-1 of stock solutions of buffers had to be greater than the pH required under assay conditions. For instance, a 1 M potassium cacodylate buffer at pH 7.56 was needed to obtain a pH of 7.2 at 0.25 M. Enzyme assay: Two assays for terminal deoxynucleotidyl transferase were used depending upon whether dATP or dCTPwas the deoxynucleoside triphosphate substrate (1). In both cases, the total volume of an assay solution was 25 ~1. WhendATP was the substrate, each assay contained 0.79 mM[3H]dATP (0.0047 mCi/

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8 1977 by Academic of reproducfion in any

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nmole dATP), 8.0 I&I Mg(OAc)2, 4 r&l 2-mercaptoethanol, 0.84 mg albumin/ml, 254 JI$! potassium cacodylate pH 7.2, 0.14 mMZnSO4,13 $4 d@T)3, and terminal transferase in the amount indicated. Assays with dCI’P as the substrate contained 0.97 I&I [3H]dCFP (0.0038 mCi/nmole dCTP), 1.2 n&l CoC12, 2.0 r&l Z-mercaptoethanol, 0.84 mg albumin/ml, 254 n&l potassium cacodylate pH 7.2, 0.14 mMZnSO4, 13 J&! d(pT)3, and terminal transferase. The mercaptoethanol was added only after both the dCTP and Co2’ were added; addition of the mercaptoethanol before the Co2+ had complexed with dCTP resulted in a red-brown precipitate that did not readily dissolve. For both of these assays, the stock solution of enzyme was first diluted, and 3 ~1 of the diluted enzyme added to the remaining components. The enzyme solution contained 0.29 mg albumin/ml, 118 a&Jpotassium cacodylate pH 7.2, 1.2 a&JZnSO4, 109 $I d@T)3, and terminal transferase; when the initiator concentration was varied in a series of assays, it was omitted from the diluted enzyme solution and added directly to the assay solutions. Other appropriate changes in assay conditions are noted in the text or in figure legends. The assay solution was incubated at 34’ in 15 ml Corex tubes. After one hour, the reaction was stopped by the addition of 0.4 ml of 0.5 mg carrier roe DNA/ml, 0.025 M sodium EDNApH 7.4, and 0.1 M Na4P207. Five ml of cold 10% trichloroacetic acid was added and the tubes chilled in ice for 10’. The precipitated material was collected on a glass fiber filter which had been prewashedwith 5 ml of 8 mMsodium pyrophosphate in 10% trichloroacetic acid to reduce adsorption of labelled deoxynucleoside triphosphates. The precipitate was washed 3 times with cold trichloroacetic acid, once with 95% ethanol, and twice with diethylether. The dried filter was placed in a scintillation vial; 0.5 ml of hyamine hydroxide added; and the loosely capped vial incubated in a 70° oven for 15’. Ten ml of scintillation fluid (Orrmifluor in toluene) was then added and the sample counted. The total cpm in assays with [3H]dATP were 58,900 and in assays with [3H]dCTP, 44,000. RESULTS Reproducible activity--Initially, assays varying reproducible IJltrasonic alcoholic

activity

by more than an order of magnitude. results,

cleaning in a commercial detergent K@Jwas also satisfactory,

Addition --of zinc ion--With

activity

(Tide) was routinely

though more hazardous.

as these two.

in a detergent solution was not sufficient

maximal activity

It was found that,

for

the assay tubes needed scrupulous cleaning before reuse.

methods were not as effective

is clearly

varied widely, with duplicate

In particular,

used, and

Other cleaning thorough brushing

to give reproducible

activity.

commercial enzyme and the assay conditions used,

cannot always be obtained unless zinc ion has been added. This

seen from Table I.

WhendATP is polymerized, most of the enzyme 2+ . is not apparent unless Zn is present. This holds true at various

buffer concentrations;

at the optimal buffer concentration,

enzymatic activity

measured in the absence of zinc ion is less than 3%of that measured with zinc ion present.

This requirement for Zn2+ is not always so stringent , though.

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Vol. 78, No. 4, 1977

Table I:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Effect

of &mGtting Zinc Ion cpm incorporateda [3H]&TP

13H]dCIP

mMpotassium cacodylate 2+

+Zn

-2n2+

+zn2+

-Zn2+

134

7,670

990

4,700

3,330

214

10,730

280

7,320

7,270

294

10,340

100

7,520

6,980

aTenninal transferase activity was measuredwith 13 pM d(pT)3 as initiator. Assays for dATP polymerization and for dCTPpolymerization were performed Lot A2 (0.071 pg) of terminal transferase with and without zinc ion present. was used.

2+

As shown in Table I, omission of Zn

makes little

difference

in the maximal

rate of d&TP polymerization. The effect --shows the effect

of monopositive --ions on terminal transferase

activity--Fig.

1

of potassium, ammonium,tetramethylammoniurn, and sodium ions

on the polymerization

of cLATPonto the initiator,

d(pA)lO.

Maximal activity

is obtained with potassium ion, though ammoniumion gives nearly as much activity,

with tetramethylammonium and sodium ions giving partial

differences

activity.

The

between these four monopositive ions is greater in other cases.

For instance, with d(pT)8 as initiator

and dATP as the polymerized substrate,

ammonium,tetramethylammonium, and sodium ions were relatively they gave 43%, 37%, and 24% of the activity

less effective;

obtainable with potassium ion.

This experiment was performed for all eight combinations of four initiators-either

dbT13, db’08,

dW4,

or d@Al10 --and the two triphosphate

dATP or dcTP. Only potassium ion consistently result

substrates,

gave maximal activity--this

being independent of whether dATP or dCTPwas being polymerized and

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Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6-

I

t

I

I

100

200 mM

1

I

,

300

CACODYLATE

Fig. 1: The effect of monopositive ions on the polymerization of [3H]dATP with d(pA)lo as initiator. Terminal transferase (0.144 ug of Lot 34421) was assayed as described in Methods but with the following modifications: The initiator was 2.8 ti d(pA)lo; of the total cacodylate in each assay, 14 mMwas present with potassium ion as the counterion (carried over from the diluted enzyme solution) and the remainder with the designated monopositive ion as counterion. X--potassium ion; O--ammoniumion; A--sodium ion; O--tetramethylammoniumion.

independent of which of the four initiators almost as effective

was being used. Ammoniumion was

in three of the four cases in which a longer initiator

was

used--d (PA)1o with dATP or dCIP, and d(pT)g with dCTP--but not with the shorter initiators

nor with d(pT)8 when dATP was polymerized.

Not shown are the results obtained with the following positive

ion:

and Bis-tris.

lithium,

imidazole,

In these cases, activity

N-ethylmorpholine, The effect ---

tris,

cyclohexylamine,

N-ethylmorpholine,

was generally quite low, except with

which gave low to moderate activity.

of negative --ions on terminal transferase activity--Terminal

transferase activity

varies not only with the type of monopositive ion present

but also with the type of negative ion present. tory,

species as the mono-

Anions are generally

with the cacodylate ion being the least inhibitory.

1222

The relative

inhibiinhibi-

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Vol. 78, No. 4, 1977

i l3 Iv a

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

I

100

300

200 CONCENTRATION.

mM

Fig. 2: Inhibition of terminal transferase activity by negative ions. Terminal transferase (0.03 vg of Lot AZ) was assayed as described in Methods. The deoxynucleoside triphosphate used was dATP, and the initiator was 3.3 $4 d@T)3. Each assay contained potassium cacodylate buffer alone or 214 mMpotassium cacodylate and sufficient added potassium salt to give the total concentration indicated. X--potassium cacodylate alone; O--added KC2H302;A--added KCl; 0 --added KNO3;e--added KBr; A--added KI; l --added K2S04; o--added KC%.

tion by different

anions is similar whether d4TP or dCl’P is the nucleotide

polymerized and whether the initiator longest one, d(pA)l,,.

Fig. 2 shows the results for the polymerization

with d(pT)3 as the initiator. chloride,

nitrate,

tion of sulfate,

is the shortest one used, d(pT)3, or the

Inhibition

bromide, iodide,

of dATP

increases in the order: acetate,

sulfate,

and thiocyanate.

With the excep-

this order is that of the chaotropic series.

The least inhibitory

anion is the cacodylate ion.

(c&J 2~O-j 9 is hardly a commonphysiological

Since cacodylate,

species, a search was undertaken

for an organic ion to see if any such ion might be as compatible with maximal activity

as cacodylate.

A number of organic ions that could reasonably be

expected to be found in animal tissue ions tested above, were inhibitory.

were tested;

and they,

For the polymerization

1223

like the simpler of dATP with either

Vol. 78, No. 4, 1977

BIOCHEMICAL

d(pT)3 or d(pA)4 as the initiator, phoglycerate,

oxaloacetate,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibition

increased in the order: 3-phos-

fructose-1,6-diphosphate,

phosphoenolpyruvate, phosphoribosylpyrophosphate, At a concentration

of 5 mM, inhibition

2-phosphoglycerate,

and 2,3-diphosphoglycerate.

ranged from S-10%for those compounds

at the beginning of this series to approximately

50%for those at the end.

DISCUSSION To obtain high, reproducible activity, detergent or by immersion in alcoholic

cleaning either by sonication

in

KOHis needed. The addition of Zn”

is also required with cormnercial enzyme assays using dATP and Mg2+. requirement for Zn2+ (at a low concentration,

This

0.14 mM) is probably due to the

removal of enzyme-bound Zn‘+ by the EM’A in the commercial preparation of terminal transferase.

(The EI)TA concentration

was 1 mMand in the final

assay, 1.8 fl).

in the stock solution of enzyme

Previous studies by Chang and Bollum

(6) showedthat Zn2+ would overcome inhibition by chelating agents. With these 2+ two conditions--the addition of Zn and cleaning by sonication--reproducible maximal activity

was obtained; without them, especially

cation,

was either low and/or quite variable.

activity

without the soni-

Although Zn2+ was necessary in the assay using dATP and Mg2+, it was not 2+ necessary in the assay using dCIP and Co . This suggests that Co2+ is able to replace the zinc ion bound to the enzyme as well as the magnesiumion complexed with the deoxynucleoside triphosphate. carboxypeptidase A can be reconstituted

than the zinc carboxypeptidase

Two other factors activity

detected;

cellular

in hydrolyzing

carbobenzoxyglycyl-

(7).

also influence the amount of terminal transferase

these are the monovalent cations and monovalent anions present.

Of the monovalent cations tested, activity.

is known that

with cobalt ion in place of bound zinc

ion and that the cobalt enzyme is more active L-phenylalanine

In this regard, it

Such a finding

only potassium ion consistently

is satisfying

in physiological

milieu contains an abundance of potassium ion.

1224

gave optimal

terms since the intraThis requirement is

Vol. 78, No. 4, 1977

frequently

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less stringent

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with longer initiators,

iators bind to the enzyme more tightly the enzyme sufficiently ment.

than shorter initiators

to dispense partially

did not permit ammonium ion to

for potassium ion was the case in which base pairing

place between the longer initiator deoxyadenylate incorporation

intrastrand

it--maximal

in the presence of ammoniumion was 15.3 dAMP to allow loop formation and sometrans-

base pairing which should diminish binding to the enzyme.

For maximal activity,

then, an appropriate monopositive ion is required

and potassium ion is clearly terminal transferase hibitory,

could have taken

and the product polymerized onto

residues per d@T)g, which is sufficient itory

and so stabilize

with the potassium ion require-

The one case in which a longer initiator

substitute

perhaps because longer init-

the ion of choice.

activity

and the relative

The effect

is somewhatdifferent.

of negative ions on

They are generally

in-

by different ions is independent of the 2+ 2+ used and of whether dATP and Mg or dcTP and Co are used. The

initiator inhibitory

inhibition

power of the ions corresponds to their position

series; the one exception is sulfate,

which is the second most inhibitory

of those tested but which is frequently the series (8-11).

listed

as the least chaotropic

The reason for this behavior of sulfate

it should be noted that sulfate enzyme since the purification

detrimental

minus one ions.

ion in

of the active

procedure uses ammoniumsulfate precipitation.

negative

interaction

ion

is not known, but

is compatible with isolation

Consequently, sulfate produces no irreversible also the only divalent

in the chaotropic

effect

on the enzyme. It is

ion used and as such, might have someadditional

with the active

enzyme which does not occur with the

In this regard, it should be noted that the organic divalent

ions tested were all moderately good inhibitors. The

least

inhibitory

ion is cacodylate.

is equal to, or greater than, that predicted

Since inhibition from their position

by other ions in the chao-

tropic

series,

tropic

ions and may prove generally useful in measuring the activity

it seemslikely

that cacodylate ion is amongthe least chao-

enzymes. For assaying terminal transferase,

1225

of unstable

it is best to eliminate anions

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

other than cacodylate

as much as possible.

in measuring --in vitro

terminal

iological

ion;

and this

cellular

constituent

possibilities

transferase

situation

enzyme is somewhat different

activity,

it

is very useful

is also a very unphys-

suggests that the --in vivo activity

from the --in vitro

that is not included

may be relevant

Though cacodylate

in future

activity

of the

or that there is some

in the --in vitro assay. Both these attempts to determine the physiological

role of this unique enzyme. REFERENCES 1. 2.

Chang, L. M. S., and Bollum, F. J. (1971) J. Biol. Chem. 246, 909-916. Sarin, P. S., and Gallo, R. C. (1975) Biochem. Biophys. Res. Comrnun.

3.

7.

D. (1976) J. Biol. Chem. Kung, P. C., Gottlieb, P. D., and Baltimore, 251, 2399-2404. Marcus, S. L., Smith, S. W., Jarowski, C. I., and Modak, M. J. (1976) Biochem. Biophys. Res. Conumm. 70, 37-44. Monahan, J. J., McReynolds, L. A., and O'Malley, B. W. (1976) J. Biol. Chem. 251, 7355-7362. chang, L. M. s., and Bollum, F. J. (1970) Proc. Nat. Acad. Sci. 65 1041-1048. Coleman, J. E., and Vallee, B. L. (1961) J. Biol. Chem. 236,

8. 9. 10. 11.

von Hippel, P, H,, and Wong, K. -Y. (1963) Biochem. 2, 1387-1398. von Hippel, P. H., and Wong, K. -Y. (1964) Science 145, 577-580. Robinson, D. R., and Grant, M. E. (1966) J. Biol. Chem. 241, 4030-4042. Levison, S. A., Kierszenbaum, F., and Dandliker, W. B. (1970) Biochem.

65, 4. 5. 6.

673-682.

2244-2249.

9, 322-231.

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