Bromotrifluoroacetone alkylates hemoglobin at cysteine β93

Vol. 81, No. 3, 1978 April 14,1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

BROMOTRIFLUOROACETONE ALKYLATES

Wray H. Huestis’

+

Chemistry

t

Department,

Stanford

Division of Chemistry Technology, Pasadena,

Received

February

892-899

HEMOGLOBIN AT CYSTEINE 893*

and M. A. Raftery?

University,

and Chemical CA 91125

Stanford,

Engineering,

CA 94305

California

Institute

of

15,1978 SUMMARY

The site of alkylation of determined by peptide mapping, fluoroacetonyl function. This the site of modification to be peptide fragment of the modified In a series 19

F-nmr

events (TFA) Knowles reaction

*-k spectroscopy in a region group (2)

cysteine

described

previously

has been used

to monitor

of the a1R2 interface

introduced

at cysteine

has questioned

and suggested

in the protein. analysis,

of studies

hemoglobin by bromotrifluoroacetone has been using a stable radioactive label for the tristudy and ‘H and “F-nmr experiments confirm the 893 cysteine found in the 8TlO tryptic hemoglobin.

In this

and nmr studies

ligand-induced

893 was the spectroscopic

communication

therein),

conformational

A trifluoroacetonyl probe.

Recently,

of the trifluoroacetonylation

the TFA label

which

and references

of hemoglobin.

the selectivity

that

(1,

occupies

we report

establish

the site

several peptide

nonspecific mapping,

of the TFA group

sites

amino

acid

to be

893. MATERIALS

AND METHODS

HbTFA was prepared from hemoglobin as described (3) and labeled with at the alkylation site by reduction with [%I]-NaBHh (New England Nuclear) Globin was prepared by acid/acetone precipitation (5), taken up in (4). water, and lyophilized. Radioactivity in the heme fraction was determined

*

%

Brimhall and Richard T. Jones, The authors are indebted to Drs. Bernadine University of Oregon Medical Center, for assistance in peptide mapping. This work was supported by NIH Grants HL 18660 and K04 HL 00258 (to WHH), Am 17850 (to RTJ), and GM 16424 (to MR).

*

Abbreviations: fluoroacetonyl; nitrobenzoate)

nmr, nuclear magnetic resonance; pCMB, parachloromercuribenzoate; o

0006-291X/78/0813-0892$01.00/0 Copyright 0 1978 by Academic Press, Inc. AN rights of reproduction in any form reserved.

892

Hb, hemoglobin; TFA, triDTNB, 5,5’-dithiobis(2-

by

BIOCHEMICAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

liquid scintillation counting of the acetone supernatant. The whole globin and subjected to peptide mapping was aminoethylated, digested with trypsin, by column chromatography using a lo-cm column of Aminex A-5 eluted with 0.2 g pyridine-acetate buffer in a gradient from pH 3.1 to 5.0 (6,7). Fractions were collected and analyzed for radioactivity. Those fractions containing activity were rechromatographed to remove contaminants (6) and subjected to amino acid analysis. For comparison, fractions corresponding to the other cysteine-containing peptides also were rechromatographed to purity and subjected to amino acid analysis. The trifluoroacetonyl derivative of mercaptoethanol was prepared as described (3) and the exchange rate of its o-protons was determined by ‘I-I-nmr spectroscopy using a Varian T-60 nmr spectrometer. RESULTS AND DISCUSSION We have blocked of this which

all

shown previously cysteine

product

sulfhydryls TFA

(Hb

was associated

(Table

1).

tion

reduced

) with

r3H]-NaBH4

as much label

as Hb

A peptide

TFA

peptide

species

appeared

label.

Amino which

in

acid

compared

aminoethylcysteine

from heme,

aminoethylated to that

ordinarily fractions

contains 20-21

analyses

of this

favorably

with

was absent

amount

8T10, 2).

that

absent

about

8TlO

1A. (the

(6,7).

A new

that

yielded

a composi-

the normal acid

or cysteine

were

found. Peptides normal. that

cy and 8T12a,

The normal

aminoethylation

been oxidized These fluoroacetone

cleavage

results

indicate

contain

pattern

had been

or otherwise results

which

observed

achieved,

altered that

in introduction

cysteines

hence

for

~~104 and 8112, these

peptides

the cysteines

could

appeared indicated not have

by the TFA modification. the reaction

of hemoglobin

of a borohydride-reducible

893

5%

> 90% of the radioactive

purification

No cysteic

only

HbA

shown in Figure

Cys q93)was

except

filtra-

of heme-labeling.

of HbA, except

peak after

the globin

and gel

incorporated

contained

label

in the globin.

HbTFA is

which

(Table

with

aminoethylation,

a similar

Reduction

radioactive

quantitatively

the radioactivity

, and exhibited

(8).

a stable

the same conditions

identical

which

introduced

of > 95% of

bromotrifluoroacetone

to DTNB reaction

and almost

map of reduced

tryptic

tion

r3H]-NaBH4

under

The map was essentially

of Hb with

accessible

of globin

in retention

with

reaction

irreversibly

Separation

resulted

that

with

bromotrimoiety

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 3, 1978

TABLE 1

Specific

Activity

of [3~]-~a~~4

--Reduced

Hemoglobins

Activity

Species

(xCi/mmole)_

--HbA Whole Globin

hemoglobin (tetramers)

Heme Aminoethyl

HbTFA

0.32

4.44

0.30

4.50

0.032

0.024

m-v

globin

/P

and Fractions

4.48

.T3

I 3j4z30 FRACTION

g&i; -).‘* activity

specifically of other These

NUMBER

(A) Peptide maps of sminoethylated gTl0 is the peptide which normally in peptide fractions.

at Cys 893. amino

acid

findings

No evidence

was found

trypsin-digested RbA(---) contains Cys 693. (B)

for

modification

a report

(2)

or oxidation

residues. are not

in agreement

894

with

that

and "H

bromotri-

Vol. 81, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II Amino Acid Compositions of Cysteine-containing from I& Amino Acid

TFA

8TlO

oT12A theory

@TlZA

theory

found

Lysine

1

1.3

Histidine

1

1.1

1

0.9

Aminoethyl Cysteine*

1

0

1

0.9

Aspartic

1

1.0

Threonine

2

1.6

Serine

1

1.0

Glutamic acid

1

0.8

Glycine

1

0.9

Alanine

1

1.3

acid

1

found theory

Leucine

2

1.9

Phenylalanine

1

1.1

found

1

0.8

1

0.9

1

1.5

2

2.1

3

3.1

1.1

Valine

*

Peptides

2

2.2

TFA-cysteine was found to be unstable to acid hydrolysis

fluoroacetone reacts with hemoglobin in somenonspecific, which results

in reversible

noncovalent manner

labeling of both cy and 8 subunits and possibly of

the heme. Those studies madeuse of bromotrifluoroacetone

radioactively

labeled with tritium

WhenHb was allowed

at the o-carbon (cu-~HbromoTFA (-z>).

to react with CY-'HbromoTFA, normal blocking of exposed cysteines occurred but the tritium

label was found randomly distributed

amongthe globin subunits

and the heme. The discrepancies between these findings and ours are best explained by a study of the exchange rates of TFA cu-hydrogens under neutral aqueous conditions.

The 'H-nmr signal arising

895

from bromotrifluoroacetone

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 3, 1978

(Figure

2) and from the carbonyl

ethanol

(inset,

Figure

a drop of 'H20. diminished

tion it

2) were monitored

with

complete

Under the conditions

(25 minutes

at 25' , pH 7.15),

is not surprising was variable

described

that

fractions

would be expected

protons

before

of the globin

after

Br-CX-CF3 I lH

in less

the protein

label

addition

of

signal

than fifteen

of HbA 3 (or of II) by enoliza-

Rb tetramers)

Thus )

was isolated.

found associated

acid/acetone

with

and that

precipitation

CY-~H little

(2).

label The

to appear at random among the H20-exchangeable and heme.

3 1 H OH I I H2° +Br-C=C-CF3

3HO I II

after

of the o-proton

exchange of these protons

the tritium

mercapto-

for trifluoroacetonylation

(2.4 i 0.9 moles/mole

was found in globin label

in deuteroacetone

exchange observed

should have been complete well

HbTFA

of S-trifluoroacetonyl

For both compounds, the magnitude

rapidly,

minutes.

o-protons

1 HO I II d Br-C-C-CF3 I 5-I

$0 4

+ 23H-O-1H

Hb-lH + 3H-O-1H r? Hb-3H + 'H20 The borohydride

reduction

fluoroisopropyl

moiety

used in the present (s)

which

Rms S CH2 s-! CF 3

Another

disparity

shift

a tritiated

tri-

2

H2°

between our work

19F-nmr properties chemical

produces

in aqueous solutions.

is stable

c3H]-NaBH4

work

and that of Knowles

is found in the

of the two hemoglobin derivatives. We reported (3) a for HbTFA in the range +85.0 to tS5.6 ppm from CFC13

896

I,.,

a’-.

11.0

,

I,

a”-

,

.,

7.0

2.

2 minutes 'H-acetone. carbonyl)

Figure

SWEEP OFFSET IHz).I). SPECTRUM AMPLITUDE.-JD INTEGRAL AMPLITUDE:SPINNING RATE (RPS):-

6.

*

*

,

.

a”.

.

m

I,,

*

,,I,

-1.

,,,I.,

I

- * PPM a

A”TO& (250) (5001 ( 2) I (.05l

6.0

4.0

, Q

,

*

I,

l----J-

.‘a

50

.,

I

BrCH,i!CF, SOLVENT: o,- acetone

’ SAMPLE:

-9..

;

I

,

*

*

,

,

.‘.‘.

.,

2.0 REMARKS:

I

I

I

ion

,,.I

.~I-

r.0

I

lH-nmr spectrum of bromotrifluoroacetone in Ds-acetone, before and after addition of DsO. Signals marked "a" arise from residual Inset: Time course of decrease in the proton signal (a, to of trifluoroacetonylated mercaptoethanol, after addition of D,O.

MANUAL SWEEP TIME (SEC): w SWEEP WIDTH IHzl: (=I-I-luldlil FILTER: 1 II~l*I.I.l.I7l~~ RF POWER LEVEL:

I,

+?*O

1 I”“I-“‘l”“l”“I”“I”“I’

I

-

I

*

I

*

,I,

= 0

l i

7

4

0 H*

Vol. 81, No. 3, 1978

(QF

BIOCHEMKAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cooH = 6CFC1 3

19 F chemical ture

(9)

istic

shifts

in

contains

absorbing

It

is

clear

tion

among the protein

of the

distribution

other

mentioned.

for

shift

that

region;

highly

used in our

(Huestis

is not

cedure

interfered

concerning

necessarily

lower,

and the o-chain To prepare

introduce

the TFA label it

Finally, after

oxidation

chain

pure

samples

with

of Geraci

et al --•

(or cu3H-bromoTFA; oxidation

prising; several hemoglobin.

of Hb

removal

were

species

reason

time

25 min).

During

oxidation

can be detected in amino

this

acid

time

with

denaturing (10).

analyses

(10).

We found by our pro-

it

with

hemoglobin

was necessary of the

chains.

of sulfhydryl

to S-alkylation

groups

but

to

by bromoTFA.

This

exposure

explicitly) This

hydrolyses

of Hb to bromoTFA resulted

observation

alkylation quantitative

effectively procedure

no cysteic

of Hb TFA prepared

slowly

surto

denature employs

conversion are minimal,

in quantiis not

and decomposes

some of which

898

to

oxidized that

In addition,

were

in which

were

effects

chains

was invoked

stated

the normal

as is consistent

in a-chains

mechanism

two hours.

fluorinated

be

of separated

the masking

was due not

not

bromoTFA

a reaction

distribu-

to the subunit

introduced

hybrids

that

groups

solution

For this

TFA

radical

of the hemes after

different

general

contaminated

by the observation

conditions

in aqueous

radically

of pGMB and recombination (2)

A free

sulfhydryl

was supported

a TFA group The yields

bronwTFA

of the cysteines.

suggestion

that

has been suggested

the heme and the

(e.g.

of Hb TFA should signals

was seriously

after

reaction

its

of 19 F-nmr

separation.

fraction

tetramers.

differ

relevant

the structure

observation

unpublished)

with

are characterspecies

of &I-TFA Thus,

groups

TFA label.

reports

and Raftery,

observed

exhibited

The litera-

shifts

deshielded

studies.

from cu3H-Hb TFA by the procedure

separated

work

or trifluoroacetonyl

such chemical

and other

subunits

points

Knowles

trifluoroacetyl

the nmr properties

of the normal

Several

detected

+112 to +113 ppm from GFG13.

19 F probe

those

tative

the range

fluorocarbons

from

both

The cu3H-HbTFA used in Knowles’

chemical

of aromatic .

ppm) .

no precedent

in this

Teflon)

- 79.9

3

as short

to Iib TFA (20and no heme

acid

as described.

could

be

8lOCHEMlCAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CONCLUSIONS Peptide site

is at cysteine

the heme were with HbTFA

mapping

tritium

based

of Hb 893.

TFA

Reports

on studies

and which

reduced

exhibited

with

that

NaB3H4 showed

labeling

of a TFA analog 19 F-nmr

occurs which

properties

that

the

labeling

on the cy chains was labeled

radically

or at

reversibly different

from

.

REFERENCES 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11.

Huestis, W. H., and M. A. Raftery (1975) Biochemistry l-4, 1886-1892. Knowles, F. C. (1975) Biochem. Biophys. Res. Commun. 65, 1155-1162. (1972) Biochemistry ll, 1648-1654. Huestis, W. H., and M. A. Raftery and M. A. Raftery (1975) Biochemistry l4, Bode, J., M. Blumenstein, 1153-1160. Winterhalter, K. H. and E. R. Huehns (1964) J. Biol. Chem. 239, 36993705. Jones, R. T. (1970) Meth. Biochem. Anal. l8, 205. Scheider, R. G., R. A. Hettig, M. Bilunos, and Bernadine Brimhall (1976) Hemoglobin 1, 85-96. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-81. Dungan, C. H. and J. R. vanWazer, "Compilation of Reported "F NMR Chemical Shifts'l John Wiley and Sons, New York, 1970. Geraci, G., L. G. Parkhurst, and Q. H. Gibson (1969) J. Biol. Chem. 244, 4664-4667. Tomita, S., Y. Enoki, M. Santa, H. Loshida, and Y. Yosuminisu (1968) J. Nara Med, Assoc. l9, l-12.

899