Hemoglobin GCoushatta: A beta variant with a delta-like substitution

Vol.

26, No.

4, 1967

BlOCHEMiCAi

HEMOGLOBiN

GCoushat+a

Barbara

BlOPHYSlCAt

: A BETA

VARIANT

Bowman’,

Don

H.

Genetics

AND

Foundation,

RESEARCH

WITH

A DELTA-LIKE

R. Barnett

University

COMMUNlCATfONS

and

SUBSTITUTION’

Rodney

of Texas,

Hite’

Austin,

Texas

Received January 16, 1967

HemoglobinGCoushattais which

was

who

live

found

to be inherited

on a reservation

analysis

revealed by alanine

alteration

in this

in tryptic

hemoglobin

ponent,

Hb A2,

and

in rare

chains,

i.e.,

the

globins

occurs

appears

Lepore

to be the

result

peptides

previously purified

pH 6.5,

3.5 solvent

1958;

by high and

voltage

1.8.

which

two

glutamic

are

related

N-terminus

normally of junction

appears 22nd

This

acid

acid

residues

had

been

that

site

of

the

products

to be the

amino

amino

occurring

1965).

tribe

of the

to chromosomal

substitution the

acid

the

(Baglioni,

events

and

indicates

in the

composed

hemoglobins

Indian

here

from

chain

hemoglobin

Fingerprints

described

S

out

part

The authors investigated

327869).

no.

address,

The

1962).

in varsol

chromatography

8 chain. minor

com-

of both

8

latter

class

crossing

over.

result

of the

of hemoIn

of a single

8 chain.

long

for

1205

and

high

was added

containing

University,

New

466

York,

with

New

acid

the

(Spinco

tubing

acid

following

at-

part

leading

of

: butasol:

of 6.6

the hemoglobin and HD-01963 York

B-V

Amino

cuvettes

card

and buffers

of @-II.

sensitivity

to Dr. Rose G. Schneider for providing work was supported by grants AM-08331

Rockefeller

: acetic

analyser

to the

@-Ill volatile

purification

range

described

B-II,

a pyridine

automatic

an expanded

extension

Peptides

the

Integrator,

by techniques

tanks

with

necessary

Model

3530034),

compared

Hagopian,

lnfotronics

A 3 meter

are indebted here. This

were

electrophoresis

on a Spinco

(Spinco

and

and

v / v ) was

path

length

GCoushatta

Descending

CRS-12A5

Present

of the

of human

Alabama-Coushatta

1964).

residue

specifying

Naughton

a model

2

study

acid

triplet

tachments:

1

The

of genetic

the amino

(103:30:150:120

was carried

837662

one

of the

type

of Hbs A and

(Ingram,

analysis

B-111

variant

Methods

Tryptic

water

residue

of the

et al .,

is the 22nd

hemoglobins

however, Coushatta’ transversion in the nucleotide

were

peptide

in the 22nd

Hb G

Experimental

(Schneider,

variant

also

slow

members

in Hb GCoushatta.

Alanine

%

in several

in Texas

that

replaced

and

an electrophoretically

mm

nos. from

the

preparations from the N .I .H

Vol. 26, No. 4, 1967

reaction

bath

After

high

they

the

Instrument

amino

with

Co.,

acid

T-S

These

Worthington) M &Cl, 0.005

violet

were

compared

peptides

were

@hoenix

hydrolysates

were

flash

at pH 3.28

eluted Recision

evopomted

and

acid

dried

after

performed

G.

The

and

3.5

subjected

carried

at pH 60,

and

out

by odding

by the

in 0.1

8.5. 90,

submitted

into

(Hunt

of peptide

30,

and

(Worthington)

was

M Tris

in vacua was

elastase

to

120,

method

0.05

180

acid

solvent

The

pl a t es were

examined

hsitions

of the

Dansyl

amino

acid

derivatives

of peptides

to authentic

Dansyl

amino

acids

on the

same

removed 210

analysis. Horecker

employed

v/v).

mg

and

and

(38:4:3

1959).

were

of Morse

chromatography

frag-

ml of water,

150,

to amino

smaller

Ingram,

Aliquots

acid

of 8-111

established for

had

was

under

a long

8-11

chromatography

the

site

peptidase

ospartic Because

c! chains of the

8

of alanine

(Table

and

wave

p-Ill

plate.

@-Ill

acid

and

was

(Ingmm

and

non-o

chain

that

This

of the

Stretton,

of this

the

substitution

enzyme

liberated whereas

amino 1962},

of Hb GCoushatta

wereiiberated acid

it was

necessary

to determine

was

There

et al .,

1960).

from

whether

were

two

Analyses

with

asparagine,

to examine

analysis

GCoushatia

valine,

when

particularly

22 of

from

the

migra-

in residue

of 8-111

in p-Ill

slower

acid

I),

located

found

the

Amino

(Table

during

differences

This

stain.

was

electrophoretic

mimics

pH.

for glutamic acid 26 and $ (Braunitzer,

by digestion

acid

pepfide

Sakaguchi’s

in Hb GCoushatta glutamic

one

22

an altered

at alkaline with

substantiated 3).

from

22

8

b indicated

Th is was

2).

migration

stained

of substitution,

a and

initially

anodaf

revealed

electrophoresis

been

a substitution

(Table

amino

during

fingerprint

fragments

8 chain

of Hb GCoushatta

Th e s 1ower

hemoglobin

the

possibilities

peptides

(Fig.).

intact

after

sequences

of chromatography

tubes

p mole

ml of 0,5

of Silicagel

of tryptic

obvious

@ and

stains;

Discussion

of the

gestion.

by strip

the

buffer

~-Ill

0.1

acetic

analysis

: acetic

of p-If1

asparagine,

0.025

were

plates

lamp.

and

alonine

and

These

layer

Fingerprints

and

water,

hydrolysis

at pH 6.5

of peptide

ml of glacial

: ethanol

leucine

citrate

with

to approximately

N-terminal

ultra

hydrolyzed

digestion

on thin

chloroform

the

enzymatically

of digestion.

of elostase

sodium

piece

electrophoresis,

The

with

ionogram

on a new

at T TO0 C for 22 hours. in 0.2M

filled

on the

in evacuated

peptidase

Qualitative

tion

final

hydrolyzed

beaker

located sewed

After

amino

with

migration

and

were

by electrophoresis

ml of 0.025

Results

and

sepamted

0.025

0966)

HCI

in a 4 liter

were

were

enzyme

minutes

blade

redissolved

was

of the

mixed

coiled

machine.

23-15)

C and

@-Ill

Leucine

and

was

analysis.

Wptide ments.

a razor

6 N

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

peptides

sewing

with

at 40’

and

electrophoresis,

a domestic paper

quickly

calorimeter

removed

with

from

to the

voltage

were

paper

BIOCHEMICAL

aspartic

Hb A valine,

first

period comparing other

or not

of dithe pertinent

this

variant

acid

Vol.

26, No.

4, 1967

BIOCHEMICAL

AND

Table Amino

Acid

Composition

of Tryptic

3-3

Amino Lysine Histidine Arginine Aspartic Threonine Serine Glutamic Pro1 i ne Glycine Alanine Valine lsoleucine Leucine

Fig.

acids

Acid

Acid

Micromoles

from

x 10

p-3

-2

A and

Micromoles

GCoushatta

from

Hb A

x 10

-2

Residues

0.008 0.011 0.286 0.608 0.010 0.013 0.628 0.023 0.871 0.343 0.859 0.006 0.318

1 .o 1.8

-1.2 2.8

0.344

1 .o

to aT-4.

Hemoglobins

Residues

iti

in relation

COMMUNiCATlONS

p-3

0.919 0.727

of Hb GCoushatta.

from

Coushatta

0.009 0.014 0.307 0.610 0.007 0.008 0.410 0.017 0.980

Fingerprint

RESEARCH

1

Peptides

Hb G

BIOPHYSKAL

Arrows

point

468

to migration

of the

altered

0.9 1.9

2.0 2.7 ifi 1 .o

peptide

@T-3)

Vol.

26, No.

4, 1967

BIOCHEMICAL

Table Amino

Acid

Composition Peptide P-III

AND

RIOPHYSICAL

Acid

Glutamic

of Two Elastase Fragments from Hb GCoushotta

Acid

Liberated

by LAP

-VaI*Asn*Val*Asp*(GI

Digestion

Time

Aspartic Asparagi Glutamic Glycine Alanine Valine Leucine

Acid ne Acid

Gly

Glu)

Digestion

of P-III

5

30

-

.lOO .387

B-lll 60

Composition

Glu

Ala)

of Tryptic

(Micromoles

u or Ala)*Ala

Hb GCoushatta ‘90 120

.220 .359 .38l ,317 - .044 .129 - .027 .133 .323 - .059 .287 .466 .045 1.159 1.1881.3161.357 - .067 .097

.030

Gly

of GCo,,shatta

u or Ala)VaI~Gly*Gly~(Gl

(minutes

Acid

0.132 0.292 0.143 (Gly

3

Table Amino

-2

0.251 0.509

Table Acids

of Tryptic

b

(G&

Amino

x 10

a

Glycine Alanine

COMMUNICATIONS

2

Micromoles Amino

RESEARCH

.405 .317 .235 .531 .553 .309

x 10s2)

- Leu.

B-III 150

180

210

.512 .312 ,233 .69a .723 1.525 .235

.563 .322 .345 .999 .861 1.509 .354

.784 .354 .582 1.506 1.208 1.890 .554

Hb A

60

150

330

.055 .178 .019 .023

,140 .193 .085 .094

-130 .128 .078 .136 .042 -408 N.T,

.579 N.>T.

-028 .705 N.T.

4 Psptides

P-II

and

B-V

of HE IG

Coushatta

@-II

P-V

Micromoles

Expected .I3 -II

Residues

Lysine Aspartic Acid Threonine Serine Glutamic Acid Reline Glycine’ Alanine Valine Methioni ne Leucine Fhenylalanine

0.141 0.011 0.151 0.170 0.030

1.0 0.1 1.1 1.2 0.2

0.176 0.227 0.159 0 .ooo 0.160

1.3 1.6 1 .l 0.0

Tryptophane N -Terminal

Positive Serine

1 1 1 0 0 0 1 2 1 0 1 0 1 Threonine

1.1 Ehrlich

G Iye Arg-

Stain

469

if

Expected Micromoles

Residues

6-v

0.563 1.461 0.563 0.903 0.600 1.176 1.188 0.663 0.621 0.518 0.574 1.698

1 .o 2.6 1 .o 1.6 1.1 2.1 2.1 1 .2 1 .l 0.9 1 .o 3.0

1 3 0 3 1 2 2 1 1 1 1 3

Vol.

26,

was

No.

4,

a Lepore

The

characteristic

C-

terminal

Hb

G Coushatta

of

sides, and

6

chain

tryptic

threonine

of

have on

both (A-C)

residue

in

of the

globin

B chain.

variant

the

structural

been

no

genes

be

umino

acid

failed

to

of serine

and

variants

these

amino

of

the

of

are

COMMUNICATIONS

the

located

two

char-acteristic

residues

,

the

o:.id

of

P-II

and

aspartic

non-a

chain

toward

the

demonstrate N-

ond

(Brimacombe, no

result

of

8 and

8

reported

way

If

al .,

1965)

of

ruling

out

the

crossing-over

polypeptide have

a single

second

d -1ik.c.

ibeen

d-like,

the

result

specifying

occuring

to

this

6 -like

Hb of

time,

G

couslt3

a single

the that

Ln

there

since

possibility

Up

the

of

event

chains.

a!,sent.

in

being

9.

was

is present

Therefore, likely

(Table

acid

it had

is most

in

chain

asplrtic

acid

et

a double

which

and

present.

variant

examined

8 polypeptide

present.

threonine this

were

chat-acteristics

were

no 22

peptides

of

show

serine

8

however,

the

specifying

and

Therefore,

also

is,

RESEARCH

peptides

chains,

triplet

been

hemoglobin

(s

to

nucleotide

have

8 or

substitution,

There

could

other

two

the

the

tryptic

.

8-V

of

fifth

N-terminal

residues

sides

and

N-terminal

and

three

8

the

is the

BIOPHYSICAL

22

found

was

threonine

been

alteration

the

Accordingly,

residue

is p-like

both

serine

peptide.

would

either of

were

AND

second

respectively

Coushatta

In HbG

one

BIOCHEMICAL

hemoglobin.

sequences

the

1967

22nd

this

hemo-

between there

have

alteration.

References Bagiioni,

C.,

Braunitzer,

Biochim.

320,

255

J.

R .,

Proc.

Nat.

J.

A.

Ingram,

V.

Ingram,

V.

Acta,

K.

and M. M.

Schneider,

R.

Trupin,

Acad.

Sci

V.

M.,

Naughton, Barnett,

Biaphys.

Rddloff,

Hifse,

97, B.

37

Gbold

(1966). and

R.

Z.

Muller,

Z.

physiol.

Chem.,

(1960).

BrimaGbe, Hunt,

et

G . , V.

M.

and

A. and

G.,

M.

Science,

Nirenberg,

2,

954

Ingram,

Biochim A,

M.. ., et

P.

Biochlm

Biophys.

0.

W.

H.

Hagopian,

E.

Haggard,

143,

697

Leder,

M.

Bernfield

and

T.

Jaouni,

(1965). et Acta,

Biophys. 28,

Stretton,

539

Bio&m.et Anal. C.

W.

Biochem., McNutt,

(1964).

470

Acta, (1958).

49,

Biophys. 3, J.-E.

520

(1961).

Acta, 276

63,

20

(1962).

(19lj2F

Johnson,

B.

H.

Bowman

and

D.

R.