Hazards in the use of Cibacron Blue F3GA in studies of proteins

BIOCHEMICAL

Vol. 86, No. 2, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 252-258

January 30, 1979

HAZARDS IN THE USE OF CIBACRON BLUE F3GA IN STUDIES OF PROTEINS Bruce

H. Weber' , Kenneth

1

Department University,

of Biological 92717

Department

Received

John G. Moe*,

of Chemistry and Institute of Molecular Fullerton, California 92634

2Department California 3

Willefordl,

Chemistry,

of Chemistry,

October

20,

University

Duquesne

University,

and Dennis

Piszkiewicz3

Biology,

California

of California,

Irvine,

Pittsburgh,

State

Pennsylvania

15219

1978

SWY : Cibacron Blue F3GA from several commercial sources is heterogeneous. This crude dye inactivates both phosphoglycerate isoleucyl-tRNA synthetase. Purification of Cibacron Blue F3GA results in a dramatic decrease in inactivation of these enzymes. is shown to be due to covalent modification of phosphoglycerate probably isoleucyl-tRNA synthetase by a minor component present Cibacron Blue F3GA.

shown to be kinase and to homogeneity The inactivation kinase and in crude

INTRODUCTION Blue proteins

dextran that

and its

normally

derivatives

bind

nucleotides

reactions

involving

structure

known as the NAD binding

Cibacron

Blue

affinity

chromatographic

(1,*,3.

The parent binding

structure

and function

Our laboratories Blue

isoleucyl-tRNA heterogeneous commercial study,

domain

media

for

dye Cibacron (3),

and spectroscopic

synthetase.

We have

in composition.

and the implications

yeast

found

studies.

252

available

phophoglycerate

these

F3GA, some of their

@ 1979 by Academic Press, Inc. of reproduction in any form resenvd.

tertiary parent

and used as

number

of proteins (4,5,6)

as a probe

of the

proteins.

We now report

of these

catalyze

and its

matrices

of a great

(3,5,6)

of

of their

dextran

to insoluble

studies

0006-291X/79/020252-07$01.00/0 Copyright All rights

Blue

3).

commercially

with

proteins

F3GA has been used in kinetic

of many of these

Blue

(2,

the purification

have been using

use as probes

a common feature

bound

Blue

wide

Many of these

ATP and NAD, and possess

F3GA in experiments

Cibacron

achieved

(1).

F3GA have been covalently

equilibrium

Cibacron

have

commercial

the resolution effects

preparations kinase

of

and -___ E.coli

preparations of components

to be of

on the two enzymes under

dye

Vol.

86,

No.

2, 1979

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Phosphoglycerate kinase was prepared from yeast by the and Weber (7) or obtained as the crystalline suspension from Mannheim Corporation, as were the glyceraldehyde 3-phosphate ATP, 3-phosphoglycerate, and NADH. Its activity was assayed previously (8).

method of Brake Boehringerdehydrogenase, as described

Homogeneous isoleucyl-tRNA synthetase was prepared by the method of Eldred and Schimmel (9). L- [3H] isoleucine was from INC, EL-- coli B tRNA was from Schwarz-Mann, and ATP was purchased from Calbiochem. Synthetase activity was assayed by the esterfication of L- [3H] isoleucine to tRNA in 0.05 E Tris-HCl at 30° C according to the method of Muench and Berg (10). Samples of Cibacron Blue F3GA were obtained from Polysciences, Inc., Pierce Chemical Company and Ciba-Geigy (gift of Dr. A. Yoshida). Ultrapure guanidine-HCl was purchased from Mann Research. Activated silica gel thinlayer chromatography plates were from Eastman. Silica gel was from J.T. Baker Chemical Company. All other chemicals were of the highest quality available. Reactive dyes were analyzed by thin-layer chromatography on activated silica gel developed by tetrahydrofuran:water (48:7, v/v)" Cibacron Blue F3GA was purified by preparative column chromatography on silica gel. In a typical experiment, 600 mg of the crude dye was dissolved in water and mixed with 3 g of silica gel. This mixture was evaporated to dryness at 40' C in a heated vacuum dessicator. The dried mixture was suspended in ethyl acetate: tetrahydrofuran:water(48:48:4, v/v/v), applied to a column (2.0 x 44 cm) of silica gel equilibrated with the same solvent, and then washed with one liter of this solvent. The eluting solvent was then changed to ethyl acetate: tetrahydrofuran:water (20:48:7, v/v/v). Two liters of this mixture were passed through the column while 20 ml fractions were collected. The fractions were assayed by thin-layer chromatography as described above. The fractions containing the major blue component (Rf = 0.57) were pooled. The organic solvents were removed by rotary evaporation and the aqueous dye solution was lyophilized. This procedure resulted in the isolating of 340 mg of pure Cibacron Blue F3GA. Unfortunately this polysulfonated derivative of a chlorotriazine did not lend itself to infrared, proton magnetic resonance, or mass spectral analysis. Therefore, another apporach to its characterization was taken. The purified dye was coupled to dextran by the method of Bzhme et al (31). The blue product and authentic Blue Dextran 2000 were hydrolyzed in 6 N HCl at 30° C for 60 hrs. Aliquots were subjected to thin-layer chromatography under the conditions described above. The mobilities of both hydrolysis products, presumably the hydroxyl derivative of Cibacron Blue F3GA, were identical (Rf = O-28). Phosphoglycerate kinase (0.1 mg/ml) in 0.090 M Tris-nitrate buffer at pH 8.50 was incubated with reactive dyes at 37' C. Arter sixty minutes of incubation an aliquot was removed for thin-layer chromatography, and the remaining reaction mixt was passed through a Sephadex G-25 column (0.9 x 10 cm) equilibrated with 0.10 M Tris-nitrate pH 8.5 buffer, Fractions were assayed by the method of Lowry (12): those containing protein were pooled and assayed for enzyme activity. Isoleucyl-tRNA synthetase (0.1 mg/ml) in 5 mM sodium phosphate buffer at pH 7.00 containing 10 *6-mercaptoethanol was incubated with reactive dyes at 30 minutes aliquots were withdrawn and assayed for activity as 3o" c, After described above.

were

Sedimentation coefficients of native and modified phosphoglycerate kinase determined in a Beckman Model E analytical ultracentrifuge at 60,000 rPm

2.53

BIOCHEMICAL

Vol. 86, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Effects

of Various

Phosphoglycerate

Dyes on the Catalytic

Activities

Kinase and Isoleucyl-tRNA

Phosphoglycerate Kinase % Activitya

Addition

of

Synthetase

Isoleucyl-tRNA Synthetase % Activitya

None Blue Dextran

100 2000 (100 pgjb

Crude Cibacron

80

Blue F3GAC

Purified Cibacron F3GA

Blue

Purified componentd with Rf = 0.77 from crude Cibacron Blue F3GA

(0.5 jJl$

93

(0.4!J@

60

(5.0 uM)

62

(0.6 uE1

48

(50

uti

6

(0.8 I@!)

35

(100

Id)

0

(1.0 la

21

(1000

PM)

97

0.0 ug

91

(0.05 mg/ml) (0.5 mg/ml)

60 0

aActivities indicated are the percent of initial activity measured after 60 minutes incubation for phosphoglycerate kinase followed by gel filtration, and after 30 minutes incubation for isoleucyl-tRNA synthetase. b Calculated concentration of dye assumi;2g th? chromophore has the same extinction coefficient (E610 = 13.6 ticm ) as Cibacron Blue F3GA. 'These concentrations are approxfmate since it was assumed that there was a single component. dThe molecular weight of this component is unknown.

employing double-sector 280 and 625 nm, were

cells. employed.

Both

schlieren

optics

and absorption

optics,

RESULTS In our initial kinase

or isoleucyl-tRNA

F3GA resulted These

we noticed synthetase

in inactivation

observations

enzymes isolated

studies

led

of the crude by silica

to our Cibacron

gel

that with

incubation

commercial

and incorporation systematic Blue

studies

F3GA and its

chromatography.

254

of phosphoglycerate samples

of blue

of Cibacron

dye into

of the effects major

component

Blue

the protein. on these which

was

at

BIOCHEMICAL

Vol. 86, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

chromatography of dyes on silica gel (see Figure 1. Thin-layer A through C are crude Cibacron Blue F3GA from Pierce details). Polysciences, Inc., and Ciba-Geigy, respectively. D is purified Cibacron Blue F3GA.

The effects phosphoglycerate The crude

of crude kinase

dye was found

had no significant Samples thin-layer a major The major

effect

of Cibacron chromatography

blue

component

component

and purified

Cibacron

and isoleucyl-tRNA to inactivate

both

(Rf = 0.57)

was purified

1).

enzymes;

are

from

three

Each of these

and at least in quantity

255

summarized

however,

of either

F3GA obtained

(Figure

F3GA on the activities

synthetase

on the activities Blue

Blue

text for Chemical, Pierce

eight

by column

of

in Table

the purified

1.

dye

enzyme. sources

were

samples

were

other

colored

chromatography

examined resolved

by into

components. on silica

Vol. 86, No. 2, 1979

BIOCHEMICAL

gel,

and upon reaction

that

derived

from

with

phosphoglycerate apparent

in the major

relative

to the control.

fast

component

Blue

followed

by thin-layer but

The most

employing

of inactivation

bound

experimental

binding

kinase

was made 8 -M in gaunidine-HCl

gels

and correspond

velocity

that

both

enzyme showed component

in the

gel

scanned

S20,w

The product

of

Blue

F3GA was also

place.

The blue

s.

kinase

Taken

and

Cibacron

to covalent Attempts

(3),

to

dialyzed

of

produced

Blue-colored

against

nm region

(13)

reaction

not be separated (1.5

if

indicated

x 23 cm) in the presence

6 g guanidine-HCl.

256

that

some

kinase. with

bond formation

the protein

F3GA

s; the unmodified

synthetase

covalent from

Blue

phosphoglycerate

of isoleucyl-tRNA

to determine

G-25

modified

data

In

Cibacron

had S2O'w = 3.7 these

coincided

kinase.

by crude

8 g

was observed.

to SDS elctrophoresis

phosphoglycerate

no

phosphoglycerate

exhaustively

in the 500-700

together,

This

up to concentrations

and Stellwagen

enzyme modified

the

on Sephadex

F3GA.

by crude

employed.

was subjected

F3GA covalently

dye could

Blue

as opposed

were

and then

of control

was

of

chromatography

modified

nm region.

Blue

examined

loss

Cibacron column

no change

at 200 nm and 625 nm; the absorbances

experiments,

= 3.4

one mg of

1).

the 625 and 280 nm absorbances

of Cibacron

chromatography

silica

spectrum

kinase

to the position

sedimentation showed

in the 500-700

phosphoglycerate

and the resultant

crude

of Thompson

in the spectrum

no change

from

disappeared

was the

of ATP or Mg-ATP

change

Dye-reacted

of the

approaches

the procedure

guanidine-HCl;

change

spots

dye molecule(s)

of dye by addition

100 me, following

minor

(see Table

tightly

modification,several

several

phosphoglycerate

with

chromatography,

obvious

was purified

F3GA represented

indistinguishable

F3GA (ml$ was reacted

components,

whether

a product

Dextran.

(Rf = 0.77)

To determine

reverse

yielded

component

was shown capable

Blue

Blue

Cibacron kinase

the fast-moving

dextran

authentic

When unpurified

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cibacron had taken

by column of 6 -M urea

or

BIOCHEMICAL

Vol. 86, No. 2, 1979

After examined

storage

for

several

by thin-layer

corresponded

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

months

the purified

chromatography

to those

observed

showed

for

Cibacron

several

the crude

Blue

F3GA when

components,

some of which

dyes.

DISCUSSION Our results Cibacron kinase

Blue

F3GA that

or covalent

color

liver

could

or electrophoresis

that

Cibacron

protein

Blue

kinase Since

caution

of Cibacron

must be taken

in

inhibition

by covalent

modification

physical

two facts, is

occurring.

filtration,

and Roskowski

or chemical,

modify

of the reagent since

might

The non-

also

be explained

Therefore,

and whether the purified

proteins,

studies.

Blue

adsorp-

of CAPE? dependent

F3GA covalently

Cibacron

a blue

have reported

formation.

inactivation.

employing

(14)

subunit

(6)

that

charcoal

bond

by it

and consequent

of Cibacron

all

F3GA should

or not

studies,

ascertain

covalent

modification

dye is unstable,

it

should

be

purified.

The fact preparations

that

Cibacron

of it, such as it

Furthermore,

since

are reactive

with

especially

with

F3GA, or contaminants proteins

should

have been used extensively reactive

proteins, if

Blue

may react

compounds

tion,

Blue

reported

F3GA acquired

the use of the dye in kinetic

Furthermore,

used freshly

gel

of covalent

of RNA polymerase

the purity

Blue

the catalytic

as the result

have also

(5) have reported

to Cibacron

by dialysis,

phosphoglycerate

by preparations

Apps and Gleed

F3GA inactivates

preparations

others

of proteins

Witt

in commercial

and inactivate

Recently,

when exposed

probably

contaminants

modify

on SDS gels.

competitive

either

purity.

not be removed

tion

are

modification

NAD kinase

which

there

synthetase.

F3GA of unspecified

pigeon

that

can covalently

and isoleucyl-tRNA

inactivation Blue

demonstrate

they

chlorotriazine they

dyes,

may be useful

show an affinity

257

for

for

found

in commercial

not be surprising dying

proteins

such as Cibacron

reagents

as

for

the NAD-binding

(15-20). Blue

protein domain.

F3GA

modifica-

Vol. 86, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Stellwagen, E. (1977) -Accts. -Chem. %., lo, 92-98. Thompson, S.T., Cass, K.H. and Stellwagen, E. (1975) --__ Proc. Natl. Acad. Sci. U.S.A., __, 72 669-672. Thompson, S.T. and Stellwagen, E. (1976) -Proc. -Natl. -Acad. -Sci. U.S.A., 73, 361-365. Elson, J.E. (1976) Biochem. Biophys. -*Res Commun ., 72, 816-823. Apps, D.K. and Gleed, C.D. (1976) Biochem. J., 159, 441-443. Kumar, S.A. and Krakow, J.S. (1977) J. Biol. w., -., 252 5724-5728. Brake, A.J. and Weber, B.H. (1974) JT Biol. Chem 249, 5452. -., Markland, F.S., Bacharach, A.D.E., ieber, B.H., O'Grady, T.C., Saunders, G.C. and Umemura, N. (1975) J. Biol. m., -,250 1301. Eldred, E.W. and Schimmel, PTR. (1972) Biochemistry, 11, 17-23. Muench, K.H. and Berg, P. (1966) in: _Procedures in Nucleic Acid Research, -_____ 375-383. Bshme, H.J., Kopperschlzger, G., Schulz, J., and Hofmann, E. (1972) 2. Chromatogr., 9, 209. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. E., -,193 265. fieber, K. and Osborn, M. (1969) J. Biol. Chem., __, 244 4406. Witt, J.J. and Roskowski, Jr., RT (1978) Fed. -;, Proc 3J, 1330. Shore, J. (1968) 2. __ Sot. Dyers and ColourGs, 84, 408-412. Shore, J. (1968) J. Sot. Dyers and Colourists, 84, 413-422. Shore, J. (1968) s. ~Sot. Dyers -and Colourists, 84, 545-555. Shore, J. (1969) 2. Sot. Dyers and Colourists, 85, 14-22. Kay, G. and Lilly, M.D. (1970) Biochem. BiophysT-e., -,198 276-285. Konovalova, Z.B. (1967) Tsitologiya, 2, 1371-1377.

258