Proton-enhanced 13C NMR of normal human erythrocytes: Characterization of motionally restricted molecules

Vol. 106, No. 4, 1982 June 30, 1982

BIOCHEMICAL

Proton-Enhanced Characterization

National

Received

RESEARCH COMMUNICATIONS Pages 1161-1168

13C NMR of Normal Human Erythrocytes: of Motionally Restricted Molecules

K. Ackerman,

Samuel

AND BIOPHYSICAL

Constance Tom Noguchi, and Dennis A. Torchia

Alan

N. Schechter

Laboratory of Arthritis,

of Chemical Biology Digestive and Kidney Diabetes, and National Institute of Dental Research National Institutes of Health Bethesda, Maryland 20205

Institute

Diseases

May 7, 1982

Solid-state natural abundance 13C nuclear magnetic resonance spectroscopy demonstrated the presence of physically immobilized material in intact human The NMR signal was shown to originate in bilayer lipid, with erythrocytes. a minor portion from membrane-associated protein. This technique is potentially useful in studying physical changes of membranes in intact cells under physiological conditions. Introduction Although

few techniques

biophysical

processes

ultimately

require

intracellular

abundance

knowledge

of sickle

13C nuclear

and quantitative

probe

which

this

intact

technique

cells During

spectrum

is

under the

of both

investigation

cells,

Previous cell

resonance

of aggregation

course

of those

fully

oxygenated

this

studies, sickle

QPresent address: Division of Biochemistry 8800 Rockville Pike, Bethesda, MD 20205

on the

natural

(NMR) is S (1,Z).

a sensitive Of particular

enhancement

to quantitate

will

in the

that

to motionally

conditions

function

laboratory

demonstrated

of proton

sensitive

physiologic

from

of hemoglobin

been able

of cell

spectroscopy

technique

selectively we have

have

or

of macromolecules

studies

anemia

state

of biochemical

understanding

of the behavior

magnetic

has been the solid

decoupling, Using

intact

environment.

pathophysiology

value

in

permit

with

restricted hemoglobin

dipolar molecules.

S polymer

in

(3). we found erythrocytes

that

the proton and normal

and Biophysics,

Bureau

enhanced erythrocytes

of Biologics

Vol. 106, No. 4, 1982 displayed

a small

restricted that

BIOCHEMICAL signal

indicative

13C-containing

this

technique

2) membrane

AND BIOPHYSICAL of the

molecules.

was detecting

associated

This

presence

of motionally

unexpected

either:

peripheral

RESEARCH COMMUNICATIONS

result

1) immobilized

or integral

protein

suggested bilayer

lipid,

or 3) membrane-bound

hemoglobin. In the present spectra

of intact

techniques protein

bilayer

lipid

we have further

normal

designed and lipid

from

study

erythroeytes.

to discriminate resonances, with

examined By utilizing

between

we conclude

a component

the proton

spectroscopic

hemoglobin, that

attributable

enhanced

the signal

non-hemoglobin comes primarily

to membrane-associated

protein. Materials

and Methods

Preparation

of Erythrocytes

and Hemoglobin

Solution

To prepare intact erythrocytes for NMR studies (3), normal heparinized venous blood was washed three times in Hepes-buffered Earle's Balanced Salt Solution, pH 7.4 with complete removal of buffy coat. Red cells were resuspended in Earle's Solution and introduced into 8mm diameter NMR tubes (Wilmad #513A-1PP modified to accommodate gas tight rubber gaskets), which Tubes were sealed were centrifuged and decanted to remove supernatant. with rubber serum caps. Oxygenated cells received no further treatment; deoxygenated cells were pre-equilibrated in a tonometer (Instrumentation Model 237) with N2 prior to introduction into the NMR tube. Laboratories, Carbonmonoxy cells were obtained by bubbling carbon monoxide into deoxygenated cells. For preparation of 13C0-labelled erythrocytes, cell suspensions in NMR rubes were evacuated, followed by introduction of 3 ml of 13C0 (Merck, 90 atoms % 13C) and 5 ml of N2 into the sealed NMR tube. After a 2 hour Carbon monoxide was06-fold molar excess over heme. equilibration at 22 C, conversion of the hemoglobin to the carbonmonoxy form was checked by Soret band absorption (4). bx lysis of intact erythrocytes Purified hemogLobin A, was prepared with fifty volumes of distilled water at 4 C, followed by two centrifugations at 10,000 x g for 30 minutes to sediment membrane material. Lysate was concentrated by Amicon filtration (PM-10 Membrane), dialyzed against 0.05M Tris pH 8.3 and applied to a column of DEAE-Sephadex for ion exchange chromatographic purification of hemoglobin according to the standard The hemoglobin A, peak was collected, concentrated and method (5). dialyzed into 0.15 M potassium phosphate buffer, pH 7.4. Preparation

of Erythrocyte

Ghosts

To prepare erythrocyte ghosts in phosphate buffer (6), washed erythrocytes were lysed with 20 volumes of 10 mOsm/liter potassium phosphate buffer, pH S.0, and spun at 10,000 x g for 40 minutes, resuspended in 20 volumes 310 mOsm/liter potassium phosphate pH 7.4., and recentrifuged. Washing with this buffer was repeated 5 times, and the lightest colored counted and introduced into NMR tubes. ghosts were decanted, To prepare Tris-treated 20 volumes of lysis buffer

ghosts, containing

washed erythrocytes were exposed to 1DmM Tris, 1OmM NaCl and 3mM MgC12, 1162

BIOCHEMICAL

Vol. 106, No. 4, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

pH 7.4 (7), centrifuged 10,000 x g for 40 minutes, and the supernatant This was repeated three times, with three subsequent washes decanted. Ghosts of each type were in 310 mOsm/liter potassium phosphate pH 7.4. enumerated in a hemocytometer. NMR Spectroscopy Natural abundance LH/13C magnetic double resonance spectroscopy was performed essentially as previously described (3), using a Nicol:: TT-14 spectrometer modified for experiments in solids at 15.09 Mz for C. The Hartman-Hahn condition for proton enhanced spectra was obtained with adamantane as standard. The probe was tuned to 50 ohms for each sample. Temperature was maintained by flow of N2 gas through the probe; for low the N2 gas was cooled by liquid N2. temperature experiments, Results Figure

L shows

oxygenated (Fig.

(Fig.

lA),

1C) normal

decoupled

ppm, ho-75

this

cross-polarization resonance

peak

indicating By comparison

this

liganding

its

actual

The proton

molecules

and that

hemoglobin. plasma

normal this

The results

membrane,

is

suggest

from either

feature

that

to that

would

which

LA) is a broad

experiments

the magnitude result

that

membrane

hemoglobin

(Fig.

1163

for

motionally

be accounted

or protein.

alone. 1D) shows

an isotropically

demonstrate

the resonance lipid

hemoglobin, spectra

result

of

from

these

contain

cannot

cells,

from

3 to 4% of intracellular from

are

of the

obtained

we calculated

human erythrocytes phenomenon

which

on the spectrum.

previously

the expected

These

at 120-200

of the deoxygenated

has no effect

be determined

1E shows

illustrates

of the oxygenated

of chromatographed

in solution.

and unliganded

(3),

which

which

(Fig.

The spectra

those

in amounts

spectrum

resonance,

protein

with

cannot

Fig.

resonances

The main

to that

approximately

origin

enhanced

no comparable

comparable

erythrocytes

red ceLLs,

red cells

region.

spectra

of protein

For comparison,

(1).

of hemoglobin

corresponds

immobilization

mobile

of oxygenated

sickle

signal

though

spectrum

of

concentration

to CS2) respectively,

technique

of these

deoxygenated

ppm (relative

are

spectra

of a comparable

and carbonyl

coupling

cells

that

1D).

aromatic

in the aliphatic

and carbonmonoxy

polarization)

1B) and carbonmonoxy-treated

of oxygenated

aliphatie,

with

(Fig.

A, (Fig.

spectrum

ppm and 15-25

(cross

and the spectrum

hemoglobin

the characteristic

observed

deoxygenated

erythrocytes

of chromatographed the scalar

the proton-enhanced

for originates

that

liganded

restricted by bulk from

liquid the

Vol. 106, No. 4, 1982 To verify resonance, Fig.

BIOCHEMICAL

the erythrocyte

we observed

using

hemoglobin

for

Soret

from

intact

spectra

of the ghosts

is

persists

largely

does not

the magnitude

cells,

even with

of minor

that

the major

the membrane

cells

at 37'

obtained

differs

to the large

aliphatic

major

of the proton

source

To confirm contribution

rule

these

In of intact out

loss

experiments

cells

This

to the resonances

because

originates

in

seconds

absence

in liquid

the relative dispersions

the immobilized

that

considerably that

the

spectrum

protein less

lipid,

not cells

to dissect

the lipid

from

to those

of molecular crystalline

insensitivity in the range

motion

lipid. of overall of contact 1164

spectrum,

is

with

Cornell

the protein contact

signal times

lipid.

correlation

et al. height

the

(8).

from proteins

from bilayer

with

relative

protein,

of the proton-carbon resonances

of

of intact

prominent

in intact

compared

typical

lysates

resonance

prolonged,

2C) provides

erythrocyte

from

suggests

(Fig.

2D shows a spectrum

2C shows

by variation

are

attenuated of the

cells

Fig.

enhanced

times

Fig.

being

we attempted

this,

disproportionately

phospholipid

regions peak.

When contact

confirmed

with

considerably

and aromatic

10m4 -IO-'

to that

we cannot

of intact

from membrane-free

Comparison

the carbonyl

occurs

two ghost

hemoglobin.

equal

preparation,

of the intact

For comparison,

protein,

at -64'C.

times.

ghost

of the resonance

of the spectrum

information.

immobilized frozen

during

Though

hemoglobin,

in the

of retained

cells,

of the

99% of cellular

peak is nearly removed.

2C shows

intact

feature

magnitude

the amount

ghosts

portion

of over

with

cell

itself.

The lineshape additional

with

components

The major

and its

99.6% of hemoglobin

resonance

suggest

loss

Fig.

ghosts

and

hemoglobin,

respectively.

number.

unchanged,

of the Tris

by phosphateResidual

To compare

despite

correlate

prepared

ghosts.

was 0.9% and 0.4% of intact

to cell that

of erythroeyte

ghosts

and Tris-ghosts,

been normalized

of the observed

in Methods.

erythrocytes.

have

preparations

from

band absorptions,

spectra

signal

spectra

as described

the phosphate-

the resonance

fact,

obtained

RESEARCH COMMUNICATIONS

as the source

the proton-enhanced

respectively,

calculated

the

membrane

2A and 2R show spectra

Tris-treatment

AND BIOPHYSICAL

should This times

(9)

be

of

have

from

.OOl to .OlO seconds.

BIOCHEMICAL

Vol. 106, No. 4, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A C

D

A I

L

loo

0

01

I

1

02

p.p.m.

I

I

loo

0

200

200

p.p.m.

Figure 1. Proton enhanced spectra of normal human erythroeytes. A, oxygenated cells; B, deoxygenated cells; C, carbon monoxide-treated cells; D, chromatographed human hemoglobin A, at 36 g/dL; E, scalar decoupled spectrum of oxygenated cells. Proton enhanced spectra (A-D) obtained from 65,536 acquisitions, 1 second each, LH-L3C contact time = 1 msec, line broadening = 50. Scalar decoupled spectrum (E) obtained from 4096 acquisitions and scaling reduced by a factor of 2. Figure spectra

2. Comparison of proton-enhanced spectra of erythroeytes with from ghosts and spectrum of motionally restricted protein solution. A, spectrum from phosphate ghosts; B, spectrum from Tris ghosts; C, speztrum of intact erythrocytes; D, spectrum of erythrocyte lysate frozen at -64 C. Spectrometer setting as for Figure 1. For A-C, acquisitions were 264,144; acquisitfons for D were 4096).

Results

of

obtained sec.

In

our

experiments

from

intact

erythrocytes

Fig.

3B and

3C,

respectively. progressive

are

As loss

can of

an

this

be

readily

initial

shown at has

in

Figure

37'C

been

Figure

3A is

our

normal

contact

lengthened

seen, "limb"

with

3.

the of

1165

to

major the

peak

effect (see

.003

set of

arrows,

this

the

and

spectrum time

of

,005

set,

change Figure

.OOl

is 3),

with

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

lH-13C

Contact

RESEARCH COMMUNICATIONS

Times

,A .m5secf-y-+0

loo p.p.m.

200

0

loo p.p.m.

200 _

_^

Figure 3. Comparison of the effect of lengthening IH-"C contact time on spectra of intact erythrocytes vs. motionally restricted protein. A-C, intact erythrocytes; D-F, erxthrocyte lysate, 26 g/dL hemoglobin concentration. frozen at -64 C. For A-C, 65,536 acquisitions were obtained, with the indicated contact times; for D-F, 4096 acquisitions were obtained. Line broadening A-F was 150.

the height the

of the major

progressive

enhanced

(not

shown).

does originate

total

spectra intensity

during

we observed From these

in bilayer probably

from immobilized,

on difference the

experiments

of the entire

containing

lysates)

indicate of the signal

In contrast,

unchanged.

attenuation

sample

erythrocyte

at 37'C

resonance limb)

of the

In other

times.

gels

and dramatic

spectrum

membrane-free

peak remaining

lipid,

lengthening

a similar

change

we infer with

protein.

such protein motionally

accounts

restructed

the proton

(frozen of contact

in

sickle

that

a minor

3D-F show

for

protein

similar

membrane-bound, that

spectrum

immobilized

results

Fig.

most

component

hemoglobin of the (the

initial

Calculations at most molecules

for

based 20% of

in the

erythrocyte. Discussion We have proton-enhanced

found

that

normal

l3 C NMR spectrum

erythrocytes in which 1166

display the major

a reproducible contribution

to the

signal

BIOCHEMICAL

Vol. 106, No. 4, 1982 is

lipid,

with

a minor

results

are

of potential

Erom membrane These

protein. illustrate

the utility

studying

normal

is

long

the major

bulk

state

enhanced

they

"flip-flop"

membrane

are

distinct

present

portion Fig.

they

13C NMR in

oE the erythrocyte

give

molecules

In particular,

domains

of motionally

attempting

rise

spin

with

to efficient

respect

limited

lipid

signal

we

in a liquid

rapidly

one need not

of this

immobilization

In view

(if

proton

about

their

to transbilayer invoke

"boundary

to account

(lo-15),

hemoglobin

could,

in fact,

intracellular

enriched

carbon

apparent,

Although immobilized

for

lipid" the

hemoglobin, integral

source

it

out is

also

membrane and associated

such as capillary

deformability,

membrane-associated

integral

only

to erythrocyte

of the signal.

part

of the

possible

may depend

We

to 13C

no CO peak was However,

experiment.

not

originates likely,

that

or the submembranous

proteins.

immobil ized

hemoglobin.

portion if

red cells

measured,

of

associated

of this

a small

1167

membrane

whether

were

and cytoskeleton

would

to 0.75%

binding

immobilized

proteins

which

equivalent

the

in this

that

that

to determine

cells

of sensitivity rule

roughly

by exposure

of observable

borderline

of spectrin

were

of the protein

3 and the calculations

of hemoglobin

when these

lack

we cannot

in the

be the

hemoglobin

indicating

is

we attempted

monoxide;

at the

hemoglobin

source

of Fig.

in amounts

of the many reports

--in vitro

the

portion

of protein

membranes

labelled

to ascertain

Based on the data

hemoglobin

protein).

complex

abundance

Phospholipids

immobilized

1, the magnitude

intracellular

mainly

though

(9).

presently

from

we are

component

configuration

relatively

of the signal.

result

natural

since

result.

We are

from

associated

significance

phospholipid.

even

since,

motions

or other

lipid

in a bilayer

signals,

axes,

from membrane

general

of proton-enhanced

for

observed

crystalline

component

RESEARCH COMMUNICATIONS

cells.

The source have

AND BIOPHYSICAL

Since

various

in part proteins

in it

originates

cytoskeletal red cell

on the physical and perhaps

functions, state

of

of bilayer

Vol. 106, No. 4, 1982 lipid,

BIOCHEMICAL

proton-enhanced

physiological

AND BIOPHYSICAL

NMR could

mechanisms

in

provide

intact

direct

RESEARCH COMMUNICATIONS

estimates

of important

cells.

Acknowledgements The authors

thank

Ellen

Kirshbaum

for

assisting

in preparation

of this

manuscript.

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