and the Pathogenesis of Hemoglobin Instability - Blood Journal

and the Pathogenesis of Hemoglobin Instability - Blood Journal

From www.bloodjournal.org by guest on February 8, 2018. For personal use only. Hemoglobin K#{246}Inin a Black Splenectomy and Red the Cell 17-...

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Hemoglobin

K#{246}Inin a Black

Splenectomy

and

Red

the

Cell

17-yr-old

and

black

R. Pedersen,

male

pigmenturia

to

Lave

but

Paul

hemoglobin

EFG5I). kinetic

studies

cell

but

the

with

4

t

51

red

Cr

was

cell

days).

The

H

EMOGLOBIN

Since making variant

structural that time hemoglobin associated

the

red

Supported

Texas;

47.2

site

methods.

and

type

at

in part

was

first

R. N.

pariment

of Cell

and

Ph.D.: Professor D.Sc.:

by

Gmune

42.

and

the

substitution

in

51Cr

and

1962,l

but

the

and

and

German K#{246}lndis-

evidence

con-

R.

McCurdi’.

of

M.D..

HL

College Cell

of

and

Reed

of and

Georgia.

Molecular

Medical

Biologt’ Ga. Biologt’

30902. and

Medical Medicine,

Washington. Technology.

Research

Instructor,

De-

Ga.

30902.

L. L.

and

Research

Augusta.

Molecular Augusta.

of

Hospital, of

M .S.:

General

A mmv

Department

Department

and

of Georgia.

C.

Division of Cell

Beaumont

Walter

D. C. General

J. B. Wilson,

D.

the

and ht’ the LI. S. A mit.

William

Professor,

Professor,

Augusta.

and Department

12958

Hematology. M.D.:

of Cell

the

4. 1973.

and

Officer.

of

Hospital. and

.

Hematology.

Medical

Medical

ofGeorgia.

Mat’

05/68.

Chief

.

30902.

Department

College

HL

Assistant

College

D. C.

Ga.

McCurdy,

and

Department

Professor, Paul

R.

General

accepted

Division

Ga.

Medical

.

LISA

Biology.

Professor,

Washington.

14/3/

M .5.: Augusta.

Medical

Augusta.

Fellow, Paul

D. C.

Washington, 1973,

HE

of Medicine

Molecular

Regents’

.

from

its instability.

described

DF32P

Division,

MC.

former/v.

of Medicine.

requests:

S. E.

Major.

Georgia.

Associate

of Medicine,

with

Center.

April30.

Grants-

Wrightstone,

of

sociate

hemo-

results

acid

salt

indicated formation

normal

This

for

various

pH’s dimer

where

of amino

of the

of hemoglobin

and various tendency to

conditions

disparity

Study

derivative

is a tetramer.

accounts

ofGeorgia.

revised

School

College

Smith,

Medical

D. C. 20012.

(Jniversitt

Medical

Blood,Vol

ultracentrifugation

under

studies

Medical

College

M . D.:

Paso,

D. C. 201XJ3.

A tint’

by USPHS

R . Pedersen, El

survival

8, 1973;

Washington.

/973

K#{246}lnby

DISEASE

University

Medical

in part

Georgetown

C

survival

normal

the

of instability.

Reed

Biolog3’.

Reprint

cell

Georgetown

Submittedfanuarv

A venue.

the

globin

red

for

cyanferri

concentrations an increased

5.6%/day. was

it

accounting

isolated

was the

KOLN

Walter

Molecular

Ph. D.,

from

was

surgery

abnormality of the variant was not determined until 1966.2 over 20 cases from several families have been reported,3 Kdln probably the most common unstable hemoglobin with hemolytic disease and red blood cell inclusion bodies.

chemistry

Hematolog,y,

ment

Cr

-

after

between

doubled,

(DF32P) (30.6

of

with

the

Center,

than

improved

along

Hospital.

platelets

5iCr)

with this disorder have thus far been mostly of English It is our purpose to report the first black with hemoglobin

cerning

Paul

of

after surgery normal. The

span

‘elution”

Individuals ancestry.

and to

more

life

modestly

From

because

tagged

presplenectomy

(1 .9%/day).

Thrombo-

Cr

and

Instability

R. N. Wrightstone.

whereas

val-.met

Post-

T. H. J. Huisman

cells

found

$

done

hypersplenism. count returned

more

ease,

was

thrombocytopenia.

suggested the platelet red

was

and

hemolysis

anemia K#{246}In (a2

Splenectomy

complicating

(DF”2P

R. McCurdy.

L. L. Smith,

with

no

and

of Hemoglobin

J. B. Wilson,

A

Survival

Pathogenesis By Paul

: Pre..

T.

As-

H . J. Huisman,

Professor,

Depart-

Ga. 30902. General

Hospital.

19th

and

Massachusetts

D. C’. 20003.

& Stmatton,

No. 5 (November).

Inc.

1973

771

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

PEDERSEN

772

Table

1 . Clinical

Lab oratory

Data.

Presplenectomy 1966

Hemoglobin

(gm/mI)

Hematocrit

ml)

46.0

Red cell count

(x 1O6/cu

mm)

Platelet

(x 1O3/cu

mm)

count

White cell count (x 103/cu Reticulocyte count (%) Bilirubin(direct/total; Plasma

mg%)

hemoglobin

Serum

(mg/i

haptoglobin

Heinz

bodies

(fresh

00 ml)

(U/gm

Hb)’4

Routine

1 .5 0

0

0

4+

istics

of

done

the

studies

by

red

cells

DF32

solution,

sent

Ga. Red cell lysed

with

carbon

gel

the

modification

-

4.5 6.2

-

7.7

method

hemoglobin

hemoglobin

CM-Sephadex analyses

was

ofits

540 and crease used

Drabkin’s ratios

the

280 nm and in

at

NaOH

heme

content

approximately (pH

range,

11.2-12.0).

appropriate

buffer. patterns

(9) ofthe

hemoglobins

The and

280

the

the

and

with to

washed with

ACD

Augusta,

cells

0.2

were

volume

of

at 4’C.

9.019

The

and

alakali-resistant

Schlicht.20

hemolysate

was

on

The

The

heat

determined

pH

8.5.

using

The

the

columns

fraction

of CM-Sephadex,

by

pH

eluate

of

a

DEAE-

containing

was

the

Elution

6.5.24

dialyzed

acid-acetone

nm

the

was assumed

The

in

of

abfrom

against

method.25

dis-

Structural

was

hemoglobin

were

the

same.

cyanferri citric

(pH

coefficients

were

in water

in the

to be 0.749

(pH

ml/g

at 20’C

from

manner. and

0.751

the

distilled KOln

at de-

buffers

4.9-5.9): and

against

because

a significant

follwing

range,

8.9-10.7):

calculated

against

hemoglobin

excludes

K#{246}In. The

range,

usual

and

used

DEAE-Sephadex

by dialysis A

for 24 hr at 4’C

a

using

a temperature

was

from

observation

hemoglobin

and

derivative

removed

acid-Na2HPO4

studies,21’25

rpm,

isolated

isolated

This

previous

60,000

cyanferri

fractions

ferricyanide

was dialyzed

to 20’C

speed

the

NaCI-glycine-NaOH

sedimentation

described

a rotor

of

strength:

Each sample corrected

column

treating

excess

isolated

ionic

6.9-7.9):

(pH range. schlieren

of 0.1

pH Marti,

8.0.2223

0.5 g/ 100 ml.

by

densities

at 415 and

at

chromatography

procedures

were The

optical

out

by

at

prepared

solution.

of

g, and

pH

followed

concentrations was

saline

the

pre-

Dacie.21

globin

ultracentrifuge

It

The

both

51Cr

mixed

at 10,000

in

with

D.C.

by shaking

Betke,

metabolic character-

previously.21’26

analyses

stability.

with The

outlined

arrival.

cell with

kinetics

removed

of

buffer,

into

3.1

flow

autosurvival

Washington,

buffer,

0.2 M Tris-HC1

Red

to splenectomy,

isolated

on a small

converted

E analytical

Hemoglobin

column

with and

velocity

greater

water.

hr.

procedures

model

of 25’C.

made

24

Sedimentation

a Spinco

was

cell

from

carried

and

Tris-HCI

was concentrated

for

used

M

15 mm

present

Meisler,

7

0.3-

included

platelet

prior

was

method

fraction

fraction 0.04

in

blood

upon

was

to the

studies

and

delivery

for

hemoglobin

ofGrimes,

procedures.’2”3

patient

Stroma

centrifuging

hemoglobin

equilibrated

water

of

the

3-

0.0

Other

red

special

water.

according

abnormal

abnormal

tilled

and

1.1415

immediately

of distilled 1 mm

standard

Table

splenectomy,17

mail

5.3 ± 0.1 4- 7.2

METHODS

using

from

by air

1 50 0

-

sieves,16

after

was prepared

determined

ofthe

normal

collected

volume for

was of

The

was

electrophoresis

hemoglobin

Sephadex

and

in ice

hemolysate

tetrachloride

stability

polycarbonate

packed

an equal

Starch

through

before

in

0- 2 50-

-

AND

made

referenced

Blood

and

were

methods

P methods

postsplenectomy.18

1 .3

-

-

5.5

0.5/1.5

3.0

hematologic

51Cr and

0.5/2.1-3.4

(%)15

were

10.2 4.6-

-

MATERIALS studies

8.9 12.9

5.9-

0

kinase

4.71

235

-

blood)

Red cell, 2.3 DPG (m moles/L)14 Red cell G-6-PD (U/gm Hb(14 Methemoglobin

14.4 52.5

4.86

0.2/0.9 00 ml)

(mg/i

Red cell pyruvate

14.6

67

10.0 10.0

6.4-

Normal

1972

51.0

-

125

mm)

Postsplenectomy

1970

-

(ml/100

ET AL.

were

KH2PO4-

Na2HPO4-NaOH

three

changes

of the

peak

positions

in

The

partial

specific

volume

the

ml/g

at 25’C.29

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

HEMOGLOBIN

KOLN

IN A BLACK

Table

773

2.

Eryth ro- and Thrombo-Kine

tic Studies.

Presplenectomy 1966

RBC mass (mI/kg) T 1/251Cr(days) Mean

35 7.3

cell life-DF32P

(days)

30 6 (F)

Serumiron(g/1OOmI) Total

iron binding

Plasma

capacity

iron clearance

(pg/100

(T 1.2,

ml)

mm)

Plasma iron turnover (mg/kg/day) Peak RBC iron utilization (%) Platelet recovery (% at 1 hr( Platelet

.

F F?

survival

finite

=

curve

finite

t Ke

=

(days)

-.

36

-

15.5

than

better

rate of elution

exponential

than

of

51

curve

exponential

Cr from

red cells

26-32

(F?)t

100-120

1.9

-

1.30 ±O.53’

225

110

65-185

-

315

440

300-350

--

-

-

-

42

-

-

65

-

13

96

8.2

with

curve

47.2

25-30

-

-

better

curve

Normal

1971-1972

-

5.6

Ke(%)

t

Postsplenectomy 1970

p

with

10

55

8.8

8.1

8

±

0.8

±

0.05.

<

p

70-1 0.46-0.78 70-100

0.05-0.10.

=

in vivo.

RESULTS Case

Report

In 1965, a 17-yr-old black male was admitted to the District of Columbia General Hospital for evaluation of dark urine which dated to early childhood. There was no other history of illness. He had an elevated reticulocyte count, but no anemia (Table I). A peripheral blood smear showed mild red cell anisocytosis and hypochromia with occasional macrocytes, target cells, and spherocytes. Diffuse decreased but cipitable

and had

punctate normal

hemoglobin

fresh

red

cells,

zine. ber

At the 1969,

was

they

time dark

basophilia morphology,

did

present.

Although

appear

evaluation previously of

appeared

stores

normal,

1970

iron

studies

(Table

a spleen

weighing

840

congestive capsule. reactive

red

postsplenectomy and had normal

was no cell. The

Reed General now moderately

with normoblastic megakaryocytes 2)

were

mildly

suggested

g was

removed. of

Hospital severe.

in

(WRGH). A bone mar-

hyperplasia (E:M granulopoiesis The

“hypersplenism,” The

found

service in Novemhepatosplenomegaly

erythroid increased,

decreased.

were

acetylphenylhydra-

results and

histopathology

of in

was

51Cr

August that

of

white pulp markings and a thickened and cord congestion with prominent from surgery was uncomplicated and

longer present. peripheral smear

state and morphology

was

were slightly cells. Heat-pre-

bodies with

examination in military scleral icterus, and

splenomegaly with a loss There was marked sinusoidal germinal centers. Recovery

thrombocytopenia nearly every the ber

and

platelet

Platelets blood

Heinz

incubation

at Walter mild, was

row aspirate was hypercellular ratio of 1 : I ). The number tagged

no

following

of a flight physical urine, minimal

prompted further Thrombocytopenia,

were present. as did white

Heinz bodies were showed changes

hemolysis. Platelets appeared except for a few moderately

now found in consistent with

normal in numlarge ones.

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

774

PEDERSEN

Laboratory

Miscellaneous

Urine

studies

stances

for

spectrum Hemosiderin urine,

were

genase, creatinine

and

studies

it was

negative;

alkaline (and

Erythrokinetic

Studies

true

less

than

cell

life

red

Liver

light

sub-

absorption

biopsy

was

normal.

Coombs

lactic

dehydro-

electrophoresis, uric calcium, phosphorus, time,

acid,

partial

urea nitrogen, electrolytes, and thromboplastin

2) 59Fe clearance red blood cell

found.

after

reducing

visible

transaminase,

prothrombin time.

the plasma The peak

normally

as great as suggested The “elution rate”

absent.

as were thrombin

( Table

span

The

and similar to that described by others.6 Although initially bile was present in the

protein clearance),

normal, level, and

Before splenectomy, turnover increased.

porphobilinogen,

negative.

glutamic-oxaloacetic

phosphatase, creatinine all

slightly

were

always

serum

amylase were time, fibrinogen

iron

myoglobin,

protein

for the urine was nonspecific was present in the urine.

in later

tests

AL.

Studies

hemoglobin,

(Clinitest),

ET

Although

splenectomy

was 59Fe

there

(DF32P

by the results of the (Ke), i.e., the difference

rapid, and the plasma utilization was 65, or

was

30.6

some

-#{247}

SiCr studies between

47.2

improvement days),

in

it was

(T 7.3 -#{247} 15.5 loss of circulating

not

days). DF32P

and circulating 51Cr,3#{176} was 5.6,/day presplenectomy but only I 9(i/day after surgery. Before the spleen was removed, there was increased accumulation of 51Cr activity over the spleen as determined by external counting. RBC mass was slightly above normal (35 mI/kg body weight) before splenectomy and was essentially En’th

after

A-f etaholic

roc’te

The cell

unchanged

results

Evaluation

of various

population

surgery.

(Table

studies

are

The

ascorbic

I ),8

deficiency3’ was abnormal despite This test is known to be a valuable lack of’ specificity to the measurement unstable Af’ter

hemoglobin recovery

5-a-pore

size

through

3-M

H einoglohiii

has f’rom

sieve.

(60,

third

screening

to give an abnormal his red cells passed On

the

other

an

S (Fig.

of’ hemoglobin abnormal 1).

The

band

hand,

test

test.32 normally

for

there

red

G-6-PD assay. and an

through

was

reduced

(minor)

fraction

was

was later overcome

equilibrated

with

eluted

minor A2 were

between

identified also on preparative 0.04

in hemolysate from which migrated

DEAE-Sephadex

showed two major and three K#{246}lnzone and hemoglobin

this hemoglobin separation was Sephadex

in a young

a flow

Studies

hemoglobin

hemolysate hemoglobin

acid-cyanide

found

of’ normal).

Starch gel electrophoresis to Augusta, Ga., showed than

to those

normal activity of’ that enzyme by screening test comparable in sensitivity of’ autohemolysis. Blood containing

been found splenectomy,

polycarbonate pores

comparable

blood slightly

chromatogram zones (Fig. incompletely

hemoglobin

K#{246}lnand

of

2).

p1-I

8.0.

About

this

The major separated. A

hemoglobin

as hemoglobin K#{246}ln.This columns by using columns

M Tris-HCI

shipped slower

A:

incomplete of DEAE-

lO#{176}--l2#{176} of the

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

HEMOGLOBIN

KOLN

775

IN A BLACK

Subject

N.L.

-Non-

t.

.

Heme

Proiein

-+----Hb

A2

----Hb

K#{246}In

-*--Hb

S

-‘*--Hb

F

-*-----Hb A

Fig. 1 . Starch gel electrophoresis control samples are from normals. Tns-EDTA-boric acid buffer. pH 9.0.

component graphic made

present of

quantitation

The Fig. after

was mobilities heat

in fresh hemolysate; this fraction changed curve

upon

and chromatoaging, which

65CC,

of

hemoglobin

in

this

hemolysate

is presented

in

2O#{176} of the hemoglobin was precipitated within 10 mm as compared with about 3#{176} of the normal control.

Studies

Amino acid analyses of’ 72 hr hydrolysates (values are mean of five separate analyses; the theoretical 3. 1 (3); Asp

the electrophoretic considerably

N. L The patient.

difficult.

stability

3. Approximately incubation at

Structural

of hemoglobin of red cell hemolysate from subject a sickle cell heterozygote, and a sickle cell anemia Stained with Amido black 10 B.

1 3.3

values for ( I 3): Thr

data

of the /3 chain gave between parentheses

these

results represent

the normal /3 chain): Lys 10.9 (1 1); His 8.9 (9): 6.5 (7): Ser 4.6 (5): Glu I I I ( I I ): Pro 6.9 (7); Gly .

4

0

Fig. globin subject (0.9 x A-50. veloped

2. Elution profile of hemoof red cell hemolysate from N. 1. on a column 50 cm) of DEAE-Sephadex The chromatogram was dewith 0.05 M Tris-HCI buffers. The broken line represents the pH of the effluent.

100

200

300

400

500

EFFLUENT

lmil

600

700

Arg I 2.6

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

776

PEDERSEN

30

subject

__

0

2

N.L.

4

6

Tlmi

8

Fig. 3.

I0

of red

(mEnu?.,)

Ala

14.8

(3); Phe 8.2 replacement covered ofT-lO

(15):

Val

(18);

Met

1.6 (1);

peptides is similar of the normal hemoglobin the expected valyl residue

(Composition:

His

0.83; Phe 0.95.) 98 (FG5) of the

f3chain

lIe 0.1

ofthe

0.90,

Arg

to those A except but did

1.00;

Asp

hemoglobin

(0):

2.10:

efficients

in

erential

solvent

Failure

to

sedimentation

NaCI.29

manner

Leu

18.2

does

such

coefficients

not

bound:

such

comparisons

of the

behavior

of

take

1.18;

Pro

Tyr

2.7

probable were rethe purity acid com-

0.86;

of the valyl as has been

of did res-

Met

residue observed

1.01:

in posiin the

i.e.,

binding at are

theoretical

of

lower

salt

or

concentrations

than

the

and

#{149}.

true

by

coof

pref-

protein.29’33 results

value not,

if

in

water

however,

is

affect

hem oglobins. of

K#{246}ln for

cyanferrihemoglobin

value

20

Fig. 4. The dependence of the sedimentation coefficients (S20w ) of the cyanferri derivatives of hemoglobins KOln and A upon NaCI concentration. The analyses were made at pH 7.2 and at 25#{176}. o. hemoglobin A; hemoglobin K#{246}ln.

calcuusing a at 25#{176}C at

eflects

water

do

mutant

the S20,w value lower than the

were obs)

sedimentation the

salt

considerations

of normal

near neutral pH is only slightly

observed

consideration

high

derivatives of the respective

These values at 25#{176}C(525, of hemoglobin

(P)

the

into

interactions which

preferentially

In 0. I M NaCl was 4.38 5, which

(18);

T-l2b; amino

properties of the cyanferri in Fig. 4, which compares

Correction

interactions,

consider

L. on

K#{246}ln.2

of this

N.

concerns the All peptides

Glu

in NaCI solutions of increasing molarity. from the observed sedimentation coefficients of0.751 ml/g for the partial specific volume

concentrations

hemoglobin

.

520,W values

all

the subject

of the corresponding peptides that of /3 T-l I This peptide contain instead a methionyl

This indicates a replacement f3 chain by a methionyl residue

The diflerences in the sedimentation hemoglobins A and K#{246}lnare shown lated value

of from

at 65#{176}.

that the abnormality a methionyl residue.

(8).

of all other

the /3 chain not contain idue.

instability

hemolysate

from the tryptic digest ofthe AE-f3-K#{246}ln chain except did not allow an accurate analysis ofthis peptide. The

position

Leu tion

16.8

These data indicate of a valyl residue by

The

cell

warming

(13):

ET AL.

of 4.48

5 observed

ln

2.0

.

0

.

I Concentration

2 of

NoCi

(N)

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

HEMOGLOBIN

KOLN

IN A

777

BLACK

A

s2ov(s 3.0

2X

Fig. 5. coefficients

The dependence (S2ow) of the

hemoglobins globin A; S.

of the

sedimentation cyanferri derivatives of K#{246}ln and A upon pH at 25#{176}.o. hemohemoglobin K#{246}ln.

cyanferrihemoglobin

A (Fig.

4). When

4

2.83

the

concentration

K#{246}ln and A as a function observed for hemoglobin

of pH. Maximum K#{246}lnbetween pH

above

decrease

pH

9, the

marked

K#{246}lnis extensively A is a tetramer.

globin

K#{246}ln is almost

completely

globin

A, which

dissociation

into

dimers

of

dimeric

increased

of 4.2 S to 4.4 5 were 8.5. Below pH 6 and

coefficient

(S2o,w

=

2.95

tetrameric

appears

was

cyanferrihemoglobin

S20, values 7.4 and pH

dissociated into In pH 10. 1 buffer,

predominantly

12

in contrast

S)

(S2o.w

to be complete

for

both

brother,

and

the

indicates

that

dimers under for example, = 4.26

condihemoto

S).

At

hemo-

pH

lO.7

hemoglobins.

Studies

Farnilt’

A paternal studied. Red

is still

0

of NaCI

in sedimentation

liganded hemoglobin tions where hemoglobin

.

to a value of 3.7 1 S. whereas that sedimentation coefficients of’ hemosalt concentration: in 2 M NaCI

S. compared to 3.83 for hemoglobin A. compares the sedimentation properties

5

.

8 p11

to 0.25 M, 520.W of hemoglobin K#{246}lndropped of hemoglobin A remained unchanged. The globin K#{246}ln decreased further with increasing S2o,w was Figure

.

6

uncle,

None cells

the

had

from

propositus’

pigmenturia,

each

formed

37#{176}C, but hemoglobin starch gel electrophoretic

anemia, a few

or elevation

Heinz

K#{246}lncould analyses.

not

bodies be

propositus’

of the after

2448

demonstrated

son

were

reticulocyte

by

count.

hr

incubation

at

heat

stability

and

stains

with

DISCUSSION

Heinz

bodies

supravital

dyes

tions the The

affecting tertiary molecular

recently enzyme ability

are

denatured

such one

of the

structure and

heme

anemia. Some Early laboratory

a history only

in earlier fatigue

patients have investigations

of

Amino sites

or the

may result unstable

which acid sites

substitutions

extends moderately

certain or

of bonds

dele-

controlling

in an unstable hemoglobin. hemoglobin syndromes have

bodies may also form when there G-6-PD deficiency, which adversely oxidative stress.34 rather ofdark

cases to a

globin

blue.

attachment

been reviewed. Heinz abnormalities, such as of the red cell to withstand

mentioned with mild

denaturing cresyl

of the molecule clinical aspects

Our propositus has hemoglobin, complaining toms tion

or

as brilliant

are

certain affect the

common for a patient with an unstable urine since infancy. The range of sympfrom

pigmenturia alone severe nonspherocytic

also developed complicating suggested increased red

or

in associahemolytic

thrombocytopenia.3 blood cell turnover

in

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

778

PEDERSEN

our

patient.

normal

Our

replaces Recent

studies

is in the

and

structural

b

stable.

Our

the

The

molecule. position

9

/

transition

with ability

is in

derivative

tendency

to

a region

that

oxygenated

gel

velocity (and

the

number

of’ abnormal

rivatives

of’ these

have

been

tendency

to

in the Most

of’the

been

retain

A,

even

a one

an amino

up

K#{246}lnis no exception.

In normal

(FG5)

forms

with

valyl

contact,

or

a contact

/ 98 is in the

position deooxy

region

by the

previously

larger

been

stable

reported

exhibit

increased

proximity

to

other

t4l

position.

in persons

Hemo-

(C6).36’4142

distributed.

Studies

family

to

significant

members

failed

is a northern

reported may be establish

European

white

3 9 In addi-

in both

the

oxy

of’ the only of’ because

of’ northern

who

in that

in position

a132 contacts

those of’ Lie-Injo et al., that this hemoglobinopathy

Potentially

formation positions

of’ the adjacent ones as well. K#{246}lnin a black subject is noteworthy predominantly

to

This to the

structures are present

it is possible that replacement residue may cause disruption not

findings and family,’#{176}suggest of’ three

a

de-

similar

dirner

valine

in position

of’several

of’ a

unliganded deoxygenation)

of’ the

such

oxyhemoglobin,

by

derivatives

structure,

in one

the

increased

obtained equilibrium

the oxy and deoxy salt bonds which

substitution

threonine

methionyl

Our

transmission.

itself’

concentrations.28’39’4#{176} other, is likely due

in a Malay

ancestry.”

that

that

an

data

the upon

tetrameric

close

Thus,

the contact involving 9, but The presence of’ hemoglobin it has

in

conformations.36’37’4’

residue

f’act

is also

has

liganded

contrast,

high salt not in the

which acid

t’orms)

the

remain

moderately form, but

variants

have

such

group body

conl’ormational and

with earlier tetramer-dimer

in

predominantly

at

make

and

the for

which

globin tion,

of’

which exist between effect of’ the six additional molecule.36’37

(I13

heme-

heme Heinz

K#{246}lnindicate

liganded

observed

(those

hemoglobin

the

K#{246}ln groups

to the

the

state,

of’ hemoglobin

in agreement

has

in

form

in

ab-

residue

heme-saturated

related

involved

dillerences

liganded

of’the and

a stable,

deoxygenated

Displacement

to

the stabilizing deoxygenated

to

the

properties

mutants

dissociate

conl’orrnational

is directly

labile

K#{246}lnis also

is likely

hemoglobins;’9262728394#{176}

found

form

heat

hemoglobin with heme

results in loss precipitation,

form,

dimers,

form

deoxyhemoglobin

that when is saturated

to

deoxy

other

into

dimeric

the

a methionyl

hemoglobin

derivative

presumably

filtration.3t

towards

cyanf’erri

to

that

transition.36’37

dissociate

dilI’erential

oxy oFthe

sedimentation

cyanf’erri

the

of’ the

in this

involved

The

al.3 have shown the molecule that

to that

the

from

directly

position et

indicate

in contrast

doubt

no

K#{246}lnin which 98 ol’ the 1 chain.2

the deoxygenated state subsequent instability,

to

3 chain

formation.

at the

analyses

leave

hemoglobin

conformation,

Transition

from

to

Wajcman

oxygenated

saturated.

and

analyses

variant is identical the valyl residue

ET AL.

European

its presence more widely direct

genetic

ancestor

generations The most

four in-

volves

removed from the propositus. commonly used technic for measuring red cell auto-survival tagging with ‘Cr.43 As chromate, this ion passes the red cell membrane

where

it is reduced

1

chain.44 f’ollowlng

Recently. splencctoni

to (‘r

and

attaches

significant improvement in a patient with

to hemoglobin, in the hemoglobin

predominantly red

at the

cell ‘Cr T was reported K#{246}lndisease.t The obser-

From www.bloodjournal.org by guest on February 8, 2018. For personal use only.

HEMOGLOBIN

KOLN

IN A BLACK

subject

779

vations

in our

modest

improvement

show was

seen

DF32P.

The

can

be explained

difference

label from red cells when ing the unstable protein Moreover, the than do normal surf’ace scanning

done

l966.

this give

or Heinz review, and the

body note lack

creased affinity ing erythropoiesis.

for

mass.

His

splenic Poor

removal

a more with

of’ the

bodies by the

51Cr

containspleen.45

times more detected by or destruction our

Splenic subject

an

indication

between hemoglobin

readily

5tCr body of’ pitting in the for

hypersplenwith unstable the

apparent rate K#{246}lndisease.”

abnormality

in the

lif’e span,

normal

platelet

in hemoglobin

count

to

to

produce

oxygen

K#{246}ln disease

for

the

mild

by the platelets

I hr recovery

K#{246}ln.3 likely

was

platelet kinetic (l3#{176}’),normal

postsplenectomy

splenectomy

(97)),

confirm

from

the

“poly-

curve.

with hemoglobin in our patient

as implied of tagged

results

stimulatincrease

compensate

dissociation

in patients platelet count

after

as disan in-

to tissues and level and a mild

able

destruction

of platelets, I hr recovery

of the

3-7

considered

less oxygen hemoglobin

was cell

thrombocytopenia depression of the

platelet

not

of’ the disparity in patients with

red

sequestration presplenectomy

normalization

bocytopenia

rapid

because the Heinz from the red cells

it was

marrow in

to the

reported severe

hand, measured-

of’ the hemolytic rate with the 51Cr technic hemoglobin K#{246}lnhas been found to have

bone

secondary

presplenectomy and

a more

other when

anemias.

increase

Others have The moderately due to studies.

the

survival

later because of thrombocytopenic combined tag studies in patients

oxygen,48 thus releasing Our patient had a high

to fourf’old

cythemia”

by

is intact “pitted”

was taken ofanemia

One reason is over estimation cussed above. Additionally,

cell

On

cell

an indication for splenectomy.47 the same picture, as it did for

was done of similar

In a recent of’hemolysis

three-

red

Consequently,

which unaware

hemoglobins

of’ red

the

the spleen are readily

consider would

in

splenectomy, ism. We are

improvement.

in

hemoglobin K#{246}ln /3 chains are said to bind ones.’1 An increase in splenic radioactivity is usually interpreted as splenic sequestration

red cells.46’47 Some of’tagged inclusions studies

a similar

that

throm-

hypersplenism.

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37.

KOLN

BoRon

sional

38. Labie

D:

stable

dimen-

body

horse

deoxy-

697,

2.8

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of A

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a2 95 Arg

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Unstable Heinz

USA

survival

Vertrees,

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Sci

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spleen

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red

Pearson

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Acad

Recommended

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Committee

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Blood 44.

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Hemoglobin

p2).

un-

Hemoglobin

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243:980,

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Chem

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KOIn.

781

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(Lond)

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BLACK

Perutz

at

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A

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W,

fourier

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IN

Cr51

by

tagged

in

body

red

1958

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DR:

KOln

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Oxygen

af-

Blood

39:

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1973 42: 771-781

Hemoglobin Köln in a Black: Preand PostSplenectomy Red Cell Survival (DF 32P and 51Cr) and the Pathogenesis of Hemoglobin Instability Paul R. Pedersen, Major, Paul R. McCurdy, R. N. Wrightstone, J. B. Wilson, L. L. Smith and T. H. J. Huisman

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