Vol. 64, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OBSERVATION OF DEUTERIUM-LABELED DIACETYLDEUTEROPORPHYRIN

INCORPORATED IN

CYANOFERRIMYOGLOBIN BY DEUTERIUM NUCLEAR MAGNETIC RESONANCE Oskar

Oster,

George

and Frank Department Indiana Bloomington, Received

December

W. Neireiter

R. N. Gurd of Chemistry University Indiana 47401

30,1974

Summary Sperm whale apomyoglobin was reconstituted with selectively deuterated D,-2,4-diacetyldeuterohemin in which the *H label was confined to the methyl groups of the acetyl moieties. A single resonance was observed in 2H NMR of the cyanoferrimyoglobin derivative, with a chemical shift 0.80 ppm downfield of external D12-TMS at pH 6.7. The corresponding chemical shift of D,-2,4diacetyldeuterohemin-OMe as the cyanide complex in pyridine-water was 0.96 ppm downfield of external D12-TMS. The prominent HOD peak was well separated at 4.4 ppm downfield. The line width of the porphyrin 'H resonances in both the protein and free solvent environments yields evidence of considerable rotational freedom of the -CD, groups about their axes.

It

has been

found

diacetyldeuterohemin

possible selectively

acetyl

moieties

globin

may be reconstituted

with

functional

the present

purpose

Spectra

(2) *

myoglobin

(Fig.

form

over

by *H NMR the

deuterated

with similar

are presented and of the

of 24 hours

free

the

from

groups

2,4-

of the

apomyoglobin.

protein

(1).

of the heme was achieved at room temperature

*H NMR behavior

cyanoferriporphyrin

Myo-

Fe (III)-porphyrins

of the normal

deuteration

showing

sperm whale

artificial

to those

the selective a period

in

certain

signal

in the methyl

1) and incorporated

properties

CH,OD and D,S04

to observe

For in

in the dark

of the cyanoferri-

form

in pyridine-water

solution. Material Dg-2,4-diacetyldeuterohemin-OMe function

in deuterated

solvents

and Methods (2) was hydrolyzed

according

to Fischer

to remove et al.

(3).

the ester Sperm whale

Vol. 64, No. 1,1975

apomyoglobin

BIOCHEMICAL

(4)

was treated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at 4" with

a 1.2 molar

diacetyldeuterohemin

taken

up in

tenfold

After

the pH of the

it

with

water.

was dialyzed

pH 6.2.

first

The artificial

pH 6.2 phosphate checked

buffer,

acetate

on a column for

phosphate Amicon

at 14-16" sample

against

0.1 M.

0.1

(Fisher).

an appropriate

to 5 mM.

time

of 0.211

seconds

was 0.211 The

and water seconds,

ml/O.25

with

porated

equipped

2A shows the

were for

ml),

containing

performed

pulsed

was prein 3 ml of

concentrating

it

32,768

by

The concen-

'H NMR experiment

was done

accumulations.

4 mg KCN.

at 33.77

Fourier

The

in a mixture The recycle

of

time

MHz with

transform

a Varian

operation.

HR 220

External

unresolved for

-CD, groups

(Fig.

the zero

reference.

1).

Dl,-TMS

as the cyan0

'H resonance

The chemical

shifts

occurs in both

represents

resonance 'H nuclei is

was 6.7.

spectrum

of the small

derivative

for

2

overlaid

HOD peak is well

solution

at 0.96 cases

the

resonance

The prominent

2B shows the corresponding

the prominent

The major

'Ihe D 1,-I?%

'Ihe pH of the

diacetyldeuterohemin-OMe

of the D6-diacetyldeuterohemin

derivative.

from external

at 4.4 ppm downfield.

and Discussion

'H NMR spectrum

cyanoferrimyoglobin

ppm downfield

Fig.

with

de-

was used as reference.

in the

spectrum

amount

(4 mg) was dissolved

Results Fig.

sample

was

4096 accumulations.

*H NMR measurements

MHz spectrometer D1,-TMS

(0.75

The

at

with

after

of KCN was added.

was 4.5

buffer

of the product

The protein

proportion

to 10.5

on CM50 Sephadex

The purity

sample

a recycle

adjusted phosphate

M, at pH 6.7 and then

molar

diluted

It was lyophilized

of D6-2,4-diacetyldeuterohemin-OMe

pyridine

0.80

and then

by dissolving

A tenfold

with

had been

electrophoresis.

strength

of the protein

solution

was chromatographed

of Rexyn I-300

ionic

filtration.

water

strength

the NMR experiments buffer

tration

cold

ionic

of the D,-2,4-

1 ml of 0.1 N NaOH and immediately

myoglobin

by cellulose

ionization pared

against

proportion

in pyridine-water,

ppm downfield these

molecule

low-spin

of external Fe (III)

incorpeak at of the

two

in the separated

D,-2,4in which Dlz-TMS. heme com-

Vol. 64, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

CD3

/ / :“o H3C “:Io=c

CH I 3

-N

Hf

\

/

HN

NH

N

\

’

3

/ /

CH3

C-OCOCH3 c -

/

OCH3

d

0

0

Figure 1. Formula of D6-2,4-diacetyldeuteroporphyrin-0Me showing tion of the methyl groups substituted with deuterium in the acetyl The Fe (III) derivative is used for the present study (2).

I

I

1

3

,

the locamoieties.

I

0 8 (mm)

Figure 2. Spectra obtained by pulsed Fourier transform 'H NMR, referred to the resonance position of external D1,-TMS (shown overlaid). A, Da-diacetyldeuterohemin-cyanoferrimyoglobin in 0.1 M ionic strength phosphate buffer at pH 6.7. B, Ds-diacetyldeuterohemin-OMe in a mixture of pyridine and water, 0.75 ml and 0.25 ml, respectively, containing 4 mg KCN.

3

Vol. 64, No. 1,1975

pounds

are determined

the chemical

shift

the contact

solution

The line about

width

any chemical

is

both

to the

line

hyperfine

line

from

coupling

The signal

width

time

for

rotation

of the

deuteron

quadrupolar

Fe (III)

relaxation

given

to (if

l/T,

= l/T1

T, is determined the

Here

the

4 eJs2Tc2