958
APPENDIX VII
ORIGIN
a
1
1
■ *
+
!
■
1
i
■ A
!
i
i
I
(Hartem)
1
^ B S G (H y b rtd )
H
k
HB
(Woolwich)
I
j
^H
(Baltimore)
I
N
^■ (S eattle)
| Al
1
■(B abin ga)
I
1
Bart's
1 «
1
1
jsphakki
j
l B>
H A '
Richmond
BB
hybrid
FIGURE V II-1
Relative mobilities of some hemoglobins on cellulose acetate (pH 8.4).
molecular biological techniques employed in the evalua-
tion of hemoglobin disorders are discussed in Chapters 23
and 28.
HbS can be differentiated from most other hemoglobins
by its
insolubility upon deoxygenation.
The test is carried
out in the presence of a reducing agent (sodium dithion-
ite or sodium metabisulfite, which consumes dissolved
T A B L E V II-I
Amino Acid Substitutions and Net Charge Alterations
in Hemoglobins Whose Relative Mobilities Are Shown
in Figure VII-1
Hemoglobin
Amino Acid
Change*
Charge
Alteration
in Tetramer*
C
Glu—>Lys
+ 4
E
Glu—>Lys
+4
O-Arab
Glu—>Lys
CGlu6—> Val "I
+ 4
C-Harlem
[_ Asp—>Asn
J
+ 4
S
Glu—»Val
+ 2
G-San Jose
Glu-»Gly
+ 2
D-Los Angeles
Glu—>Gln
+ 2
Sydney
Val—»Ala
0
I-Philadelphia
Lys—»Glu
- 4
*As compared to HbA. Hemoglobins A2, F, Bart’s and H have multiple
amino acid differences from HbA, and their electrophoretic mobilities
depend on the net effect of all these differences on their net charge at
pH 8.4-8.6.
I
ORIGIN
I
*
|i'
I
|
kcrle
eu
J C hARLEM
I
■
MONTGOMERY
j l
■
Q jr a b *
!l°
I I
e
! |
g
I H
MOST FAST
I
■
MOVING
m
■HB
BOW DEN
!
1
F IG U R E V II-2
Relative mobilities of some hemoglobins on citrate agar (pH 6.0). The
differences between this gel and cellulose acetate are the differential
interactions of the hemoglobins with the gel and the changes in net charge
caused by the lower pH.
oxygen) in an appropriate buffer. When HbS is added, the
solution becomes turbid owing to precipitation of deoxy-
HbS. The turbidity can be quantitated spectrophotometri-
cally and related to the amount of HbS added. By using the
same reagents with intact erythrocytes, HbS can be precip-
itated within the cells, causing them to sickle. The extent
of sickling can be assessed by microscopic examination.
Because HbC-Harlem and Hb-Bart’s behave like HbS in
the solubility test, they may give false-positive results in
tests for HbS.
HbF is identified and quantitated by its
resistance to
denaturation by strong alkali.
The hemolysate is mixed
with an alkaline buffer (pH 12.8) and incubated to denature
the nonfetal hemoglobins. The denatured hemoglobin is
Alpha Chain Mutants
Hemoglobin
Structure
Mobility in Citrate Agar pH 6.0
+
:
s
A
J P a ris
Q
1 2 A la " A s p
1
4 O x f o r d
Q
1 5 G ty -* A s p
1
1
a 1 6 L y s * G lu
F o r t W o rth
Q 2 7 G lu — G ly
H a s h a r o n (S e a ly )
a
4 7 A s p -* H is
■
M o n tg o m e r y
Ct
4 8 L e u
A rg
■
R u s s
a
5 1 G ly
A rg
S h im o n o s e k t
0 5 4 G in
A rg
■
G -P h ila d e ) p h ia
0 : 6 8 A sn -» L y s
W in n ip e g
O 7 5 A s p
T y r
I n k s te r
a
8 5 A s p
V al
B r o u s s a is
O 9 0 L y s
A sn
T itu s v ille
O 9 4 A s p
A sn
■
G -G e o rg ia
O 9 5 P r o
L e u
■
R a m p a
O 9 5 P r o
S e r
■
F IG U R E V II-3
Mobilities of «-chain mutants on citrate agar electrophoresis relative to
hemoglobins C, S, A, and F. (Courtesy of Dr. Rose Schnieder. Reproduced
with permission from Helena Laboratories, Beaumont, Texas.)
