Vol. 69, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ON THE EFFICACY OF COMMONLY USED RIBONUCLEASE INHIBITORS George H. Jones Department of Cellular and Molecular Biology Division of Biological Sciences University of Michigan Ann Arbor, Michigan 48109

Received

January

27,1976 SUMMARY

The ability of a number of commonly used inhibitors to inhibit pancreatic ribonuclease has been studied. At ribonuclease concentrations of 10 or 100 g/ml, heparin, polyvinylsulfate and proteinase K, at concentrations reported for their use in the literature, were ineffective in inhibiting RNase digestion of 3H-uridine labelled RNA from Streptomyces antibioticus. In contrast, macaloid, diethylpyrocarbonate and sodium dodecyl sulfate were all effective inhibitors, with the degree of effectiveness decreasing in the order stated. Further, at inhibitor concentrations which allowed RNase conversion of only 50% of the labelled RNA to acid soluble products, a larger percentage of the acid insoluble di estion products sedimented in the "high molecular weight" range 9 4-16s) when macaloid was the inhibitor used than when diethylpyrocarbonate was the inhibitor. In recent from

years,

prokaryotic

effective

inhibitors

ding

dodecyl

2, 3),

6, 7), presumed their

thus

effective

reasoned

in

that benefit

terms

(PVS,

of

8, 9),

and it

inhibitor the

is

from

a direct

inhibitory

refs.

0 1976 by Academic Press, oj reproduclim in any form

Inc. reserrjed.

need for

heparin

dealing comparison These

469

K (5,

without

inclu(DEPC, (HEP, refs.

10).

It was

RNA isolation

of these studies

to

to determine

literature.

with

These

any reference

difficult

the available

activity.

A number

laboratories,

4, 5),

extremely

below.

Copy&f Ail rigtlrs

the

of RNA's

11, diethylpyrocarbonate

employed

literature

from

in various

and proteinase

are frequently ability

properties

to use in RNA isolation.

(SDS, ref.

(MAC, refs.

inhibitory

ques would tors

sulfate

in the

has accentuated

have been used

inhibitors

the most

cells

polyvinylsulfate

macaloid

interest

of ribonuclease

inhibitors

sodium

increased

and eukaryotic

of presumed

refs.

the

presumed are

techniinhibi-

described

VoL69,No.

2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS

Materials

- SDS was obtained

(135

Units/mg)

from

NL Industries

prepared with

from

Pancreatic

Methods

PVS from

and extracting

the

for

Tris-HCl,

1 and 2.

trations

was chosen

min at 30°, remaining

antibioticus (11).

experiments.

dried

and examined

by liquid

as percentage

in each assay

two RNase levels

added

above,

(2.5%)

or MAC (0.5

containing

were

(TCA)

collected

incubated

on glass

scintillation

legends

concenused

was added

in pubfor

5

to precipitate fibre

filters

counting. acid

In the absence

used was sufficient

mg/ml).

was extracted

gradient

0.3 ml incubations 10 pg/ml

Results

insoluble

of inhibitor,

to completely

were

RNase, and either

performed

solubi-

combined with

centrifugation

ethanol.

with

10 A260 units RNA thus

as described

470

as des-

no inhibitor,

At the end of the incubation, (ll),

tRNA and precipitated sucrose

of inhibitor

of the original

tube.

in the

concentrations tubes

RNase,

RNA.

cribed

remaining

the

1.93 A260

per minute);

as indicated

acid were

remaining

'H-RNA,

counts

Assay

RNA precipitates

1 are expressed

in 0.1 ml reaction

the range

so as to encompass

In some experiments,

via

RNA was

described

10 umole;

and inhibitors

RNA.

each of the

coli

H-labelled

performed

precipitable

when 10% trichloroacetic

were

the

were

In each experiment,

RNA isolation

lize

3

Macaloid

of Streptomyces

pH 7.6,

to 6000 acid

to Figs.

counts

Biochemicals,

RNA as previously

RNase activity

1 or 10 F.rg (10 or 100 pg/ml)

in Fig.

cultures

DEPC and HEP

RNase was from Calbiochem.

(equivalent

which

General

Ltd.,

K from Merck.

12 hr old

containing:

lished

BDH Chemicals

and proteinase

- Assays

mixtures units

Sigma,

by incubating

3H-uridine

from

obtained previously

DEPC

the RNA of Escherichia was analyzed (12).

Vol.69,No.

2, 1976

BlOCHEMlCAL

INose

w’.’

,.* .. . .. . .. . *.-

al:

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1DO rg/ml10 “g, ml ....’

..!!!!...a

.* . . . .. . .. . .. -W”’

DECC

........... l . . .. . . .. . .. . .. . . .. . .. . .. l

,

I I 1.0 15 CONCfNlfAlION

0.5 SDS or Df?C

I 20 i%vlvt

I

1.0

1.0 MAC or HfP ADDfD~mphll

- Abilities of various inhibitors to prevent solublization of &il!RN: by pancreatic RNase. Assay conditions were as described in Materials and Methods and inhibitors were present at the concentrations indicated. Results are expressed as the percentage of the add acid insoluble RNA remaining after the incubation.

RESULTS The data tested

of Fig.

to inhibit

pancreatic

can be seen that bition

at the

also

observed

assay

effective

the

presence

PVS and HEP were

that

proteinase employed

20% of the added

RNA.

of 43,

The data effective

presented

ineffective

In experiments

In contrast,

of DEPC and MAC prevented

At 10 pg/ml

not

of the RNase inhibitors

suggested tested.

471

shown,

it

inhibitor

was

under

At 100 ug/ml solubilization

the

RNase,

of about of about

SDS, DEPC and MAC prevented

49 and 67% of the added

above

at RNase inhi-

the solubilization

RNase,

It

SDS, DEPC and MAC were

the

SDS prevented

substances

at 10 or 100 ug/ml.

at some concentration.

RNA while

of the

K was an ineffective here.

inhibitors

of five

completely

used.

30% of the added

solublization

the ability RNase present

concentrations

conditions

all

1 depict

that

RNA, respectively.

DEPC and MAC were However,

the

assay

the most used mea-

Vol. 69,No.

2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

1.

‘? 0 x P $ O.!

fPACTlON

NUMBER

Fig. 2. - Sucrose gradient analysis of the RNA remaining after incubation of 3H-RNA with 10 g/ml RNase in the presence or absence of OEPC or MAC. See text for details of the incubation and RNA extraction. Gradients (5-201) were centrifuged for 13.5 hr at 36,000 rpm and were collected and assayed as described previously (12). Radioactivity values have been corrected for background and the direction of sedimentation is from right to left. The positions of 23s, 16s and 4s RNA's were determined using marker RNA prepared from E. coli cells and run on a parallel gradient.

sured

only

the ability

added

RNA to an acid

hibitormight allow

prevent

significant

molecular this

soluble

weight

complete

RNA which three

as described

centrifugation

equal

levels

of higher

the

insoluble

scale

(0.3

ml)

incubation in Fig.

against

added

an inRNA, but still

RNA to lower

10% TCA.

containing

To examine

and 10 pg/ml

were

no inhibitor, RNase.

The

by sucrose

In the

incubations

chosen

so as to produce

RNase, and in each case,

472

of the

RNase assay mixtures

and analyzed 2.

that

weight in

and Methods

of DEPC and MAC were

of protection

of the molecular

was still

conversion

seemed possible

MAC, respectively,

as described

the concentrations

It

in Materials

after

to prevent

solubilization

large

2.5% DEPC or 0.5 mg/ml RNA was extracted

inhibitor

form.

conversion

possibility,

prepared

of each

gradient

described,

about

about 50%

VoL69,No.

2, 1976

of the the

BIOCHEMICAL

RNA was recovered

recovery

quite

as compared

of TCA precipitable

similar

for

the

incubations

to lower

weight

sence

of MAC.

total

RNA recovered

5% of the RNA from

this

region.

the

cular

than

weight

fractions

in tubes

the

MAC in preventing

50% of

in the

pre-

of the

one observes

that

DEPC sediments the

Thus,

conversion

although

percentage

containing

cpm

RNA was converted

of DEPC than

1-13,

1-13.

2 that,

weight

one calculates

incubation

was

of about

37% of the RNA from

MAC is recovered

Fig.

solubilization

presence

In contrast,

effective

from

RNA in the

in gradient

gradients

DEPC and MAC (3500

molecular

if

In addition,

the sucrose

high

For example,

only

taining

complete

RESEARCH COMMUNICATIONS

control.

containing

RNA, much more of the molecular

the

It is evident

DEPC and MAC prevented

the added

with

RNA from

and 3700 cpm, respectively). both

AND BIOPHYSICAL

incubation

DEPC is

of higher

in con-

somewhat to lower

less mole-

RNA by RNase.

DISCUSSION In summary, are

all

should

the data

presented

capable

of inhibiting

be noted,

however,

that

of each

bition

than

isolation culties

higher experiments

the (1,

under

inhibitor

This

employed

of SDS might

necessitate

of the

its

and since

with

RNA (13).

acidic

clay,

interfere ble

that

purified of the

with

Macaloid, whose the

MAC could

on the

presence

assay

SDS, DEPC and MAC

other

concentrations. conditions

required

concentrations

removal,

the

concentrations

4, 9).

when SDS and DEPC are

RNA for

that

RNase at appropriate

the concentrations were

indicate

may lead

RNA isolation,

repeated

in isolation

is

a relatively apparently

In addition,

it

be included

as an RNase inhibitor

RNA, and could be removed by centrifugation assays.

Although

473

they

are

high

to react inert

mixtures

of RNA.

to diffi-

precipita-

DEPC has been reported hand,

inhi-

in RNA

since

ethanol

preparation

RNA in biological

maximal

employed

observation for

employed,

to produce commonly

It

does

not

seems possi-

in solutions

of

prior

to the use

soluble

in aqueous

Vol. 69,No.

2, 1976

solutions, sent

BIOCHEMICAL

the

from

with

DEPC in preventing 2) would

RNA isolation

teinase tive

K, it at lower

conclude higher

that than

is

clear

in biological

the observation conversion

their

assays.

that

of higher

seem to make macaloid

the

that

other

presumed

may be argued

being

These

ab-

con-

MAC is more effective to lower

the

molecular

inhibitor

that

inhibitors these

RNase concentrations.

weight

of choice

one would

expect

Indeed,

tested the

are

used

to observe

even at this quite

same relative

tested,

substances

the RNase concentrations

that

inhibitors

conclude

require

in

procedures.

As regards

six

to be used

coupled

RNA (Fig.

it

of SDS and DEPC would

RNA solutions

siderations, than

properties

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

high

would

it

in these

in cellular

activity

It would

have been effec-

seems reasonable

RNase concentration,

effective.

PVS, HEP and pro-

to

experiments extracts.

However,

three

seems reasonable be manifested

were

of the to at lower

RNase concentrations. ACKNOWLEDGEMENT This research the National

was supported Cancer Institute,

by Grant Number 2 ROl-CA12752-04, DHEW.

awarded

REFERENCES 1. 2.

11.

Girard, M. (1967), Methods. Enzymol. 12a, 581-588. Fedorcsak, I., Natarajan, A. T., and arenberg, L. (1969), Eur. J. Biochem. lo, 450-458. Rosen, C.-G. and Fedorcsak, I. (1966), Biochim. Biophys. Acta 130. 401-405. Mx; B., Koblet, H. and Gros, D. (1968), Proc. Nat1 . Acad. Sci. USA 59, 445-452. Cheng, T.-C., Polmar, S. K. and Kazazian, H. H., Jr. (19741, J. Biol. Chem. 249, 1781-1786. McKnight, G. S. andschimke, R. T. (1974), Proc. Nat 1. Acad. Sci. USA 71, 4327-43331. Schechter, I. (1974): Biochemistry 13, 1875-1885. Stanley, W. M. and Bock, R. M. (196v, Biochemistry 4, 1302-1311. Hancher, C. W., Pearson, R. L. and Kelmers, A. D. (1974), Methods Enzymol. 20e,-510-514.. Mach, B., Eust, C. and Vassalli, P. (1973), Proc. Natl. Acad. Sci., USA-70;451-455. J;r$si7;. H. and Weissbach, H. (1970), Arch. Biochem. Biophys. 137,

12. 13.

Jones, Oberg,

3. 4. 5. 6. i: 9. 10.

G: H. (1975) Biochem. Biophys. Res. Commun. 63, B. (1970), Biochim. Biophys. Acta 204, 430-44E

474

469-475.

by