Physical chemical studies of reactions of human hemoglobins

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PHYSICAL CBEMICAL STUDIES OF REACTIONS OF HUNAN HE%OGTX>BINS

Thesis by

William Day Hutchinson

I n P a r t i a l F u l f i l l m e n t of t h e Requirements For the Degree o f

Doctor o f Philosophy

C a l i f o r n i a I n s t i t u t e of Technology Pasadena, C a l i f o r n i a

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With a deep sense of g r a t i t u d e

X

acknowledge t h e stimulating

&pidance o f D r , Jerome Vinograd during the course of my graduate study a t the California I n s t i t u t e of Technology. I am proud *a have been a b l e t o work with and l e a r n from h i m .

I wish t o thank the many people who have donated t h e blood used i n this research, Thanks a r e especially due t o my cousin, Nelson C, Jackson, Jr., who donated @oat of t h e s i a k l e e e l 1 hemoglobin used i n t h i s work, Far c l i n i c a l s t u d i e s , I wish t o actknowledge t h e help of D r , P h i l l i p A, Sturgeon and t h e Hematology s t a f f of The Children's Hospital, Us Angeles.

The people o f t h e Ccal Tech~community have made my years here both enlightening and enjoyable,

I wish t o thank D s , Paul E, Lloyd and t h e California I n s t i t u t e of Technology f o r making a v a i l a b l e t o me t h e Paul E, Lloyd Fellowship during my residence here.

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ABSTRACT

The molecular d i s s o c i a t i o n of t h e d e r i v a t i v e s , carbonmonoxy-, oxy-, ferro-, and f e r r i - , o f normal a d u l t hemoglobin has been s t u d i e d a t a pH o f f i v e , Of t h e s e d e r i v a t i v e s , ferrohemoglobin i s l e a s t d i s - s o c i a t e d while ferrihemoglobin is most extensively d i s s o c i a t e d a t a given concentration. The apparent d i s s o c i a t i o n c o n s t a n t s have been c a l c u l a t e d and r e l a t e d t o t h e a c i d d i s s o c i a t i o n o f t h e ffHeme-linkedtt a c i d groups o f t h e s e d e r i v a t i v e s ,

Hemoglobins l a b e l l e d with carbon-14 have been prepared. Using C' l a b e l s on one hemoglobin, t h e exchange of molecular s u b u n i t s h a s been s t u d i e d a f t e r d i s s o c i a t i o n o f mixtures of normal a d u l t and s i c k l e c e l l hemoglobins a t a c i d and a l k a l i n e pH. Hemoglobin '$hybridsw, i n which one type of subunit i s r a d i o a c t i v e and t h e o t h e r n o t , have been formed and i s o l a t e d . These hybrids have been used i n t h e i d e n t i f i c a - t i o n o f t h e polypeptide chain i n which t h e anomaly occurs i n s i c k l e c e l l hemoglobin. This anomaly was found t o occur i n t h e f3 chains.

The k i n e t i c s o f t h e a chain exchange between t h e two hemoglobins has been s t u d i e d a t a l k a l i n e pH. Presumptive evidence o f t h e mode o f d i s - s o c i a t i o n a t t h i s pH has been obtained. Preliminary i n v e s t i g a t i o n s a r e r e p o r t e d on t h e i s o l a t i o n o f hemoglobin hybrids i n which only t h e

c h a i n s a r e r a d i o a c t i v e .

S p e c t r a l d a t a f o r s e v e r a l d e r i v a t i v e s o f hemoglobin have been r e l a t e d t o a r e c e n t l y adopted standard, The use o f t h e f e r r o v e r s e n a t e i o n i n forming ferrohemoglobin from oxyhemoglobin i s reported.

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TABLE OF CONTENTS PART I

DIFFERENCES I N THE ACID INDUCED MOLECULAR DISSOCIATION BEHAVIOR OF SEVERAL DERIVATIVES OF

NORMAL ADULT HEMOGIDBIN

I n t r o d u c t i o n

. . .

¹

. . .

Procedure 2

. . .

P r e p a r a t i o n o f M a t e r i a l s .

3

. . .

Experimental Method

5

R e s u l t s

. . .

⁷

. . .

Discussion of R e s u l t s

18

PART I1

CARBON-14 LABELLEID HYBRIDS OF NORMAL ADULT

AND

SICKLE CELL HEKOGMBIMS

A. PREPARATION AND ISOLATION OF CARBON-14 LAB.E&LEXI HPBRIDS OF

NORMAL ADULT AND SICKLE CELL HEMOGLOBINS

. . .

²²

. . .

^{0 2 2}

. . .

Procedure 25

P r e p a r a t i o n of M a t e r i a l s

. . .

²⁶

Chromatography

. . . O .

30

. . .

R e s u l t s of Hybridization Experiments

32

. . .

S i g n i f i c a n c e of Hybridization o f Radioactive Hemoglobins 39 B

. %m

IDEHTfFICATION OF THE ABERRANT CHAIN I N SICKLE

. . .

CELL WEM0GIX)BIN 4-0

. . .

I n t r o d u c t i o n 40

. . .

Procedure 40

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...

P r e p a r a t i o n o f M a t e r i a l s 40

. . .

R e s u l t s

41

. . .

Discussion 42

THE KINETICS OF SUBUNIT EXCHANGE I N THE ADULT-SICKLF: CELL HEMOGUIBIN SYSTEM RND THE MODE OF DISSOCIATION OF

KmoGmBINATpH11.0

... 44

. . .

⁴⁴

Experimental Procedure

. . . 45

...

R e s u l t s

46

. . .

Conclusion 60

PRELIHINARY INVESTIGATION OF THE ISOLATION OF HEM0GU)BIN

...

HYBRIDS LABEUED I N THE CHAINS 61

. . .

61

...

Procedure

61

. . .

Experimental Procedure 62

...

P r e p a r a t i o n o f M a t e r i a l s 62

. . .

R e s u l t s

63

...

REFERENCES

68

APPENDICES

1. SPECIRC EXTINCTION COEFFICI3ZNTS ) OF

HEM0GU)BIN DERIVATIVES

. . . ~ . . . 70

I T

.

REDUCTION OF OXYHmOGmBIN USING THE FERXIVERSENATE I O N

. .

⁷³

P I 1

.

PREPARATION OF VARIOUSLY WELLED RADIOACTIVE

. . .

SICKLE CELL HEHOGLOBINS 75

I V

.

CHROMATOGRAPHY PXOTOCOL SHEETS ^a

. .

⁷⁷

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PART I

DIFFERENCES I N T H E A C I D INDUCED MOLEUZAR D I S S O C I A T I O N BEHAVIOR O F SEVERAL

DERIVATIVES O F NORMAL ADULT HENOGLOBIN

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I n 1955 F i e l d and OtBrien reported a decrease i n t h e sedimentation c o e f f i c i e n t o f hunnan carbonmonoxyhemoglobin i n s o l u t i o n s of pH below f i v e (1). This decrease i n sedimentation c o e f f i c i e n t w a s accompanied by an i n c r e a s e i n t h e d i f f u s i o n c o e f f i c i e n t , which suggested a d i s s o c i a t i o n of t h e hemoglobin molecule i n t o a new s p e c i e s having h a l f t h e molecular weight of t h e o r i g i n a l hemoglobin molecule, F i e l d and Ogston (2) concluded from c o n s i d e r a t i o n s of t h e r a t e of boundary spread i n t h e u l t r a c e n t r i f u g e t h a t t h e r e a c t i o n w a s revers- i b l e and t h a t t h e d i s s o c i a t i o n equilibrium w a s e s t a b l i s h e d i n l e s s than 200 seconds, It was l a t e r demonstrated ( 3 ) t h a t t h e mass a c t i o n expression obeyed by t h e r e a c t i o n w a s given by:

H I I H I I + H + - - - + H M ~ + H ~ (1)

where Hh i s a hemoglobin h a l f molecule. From s t u d i e s of t h e dependence o f t h e r e a c t i o n on p r o t e i n concentration and hydrogen i o n concentration, apparent equilibrium c o n s t a n t s were calculated. The c o n s t a n t s were

found t o have a s c a t t e r which could not be a t t r i b u t e d t o e r r o r s i n - herent i n t h e method of t h e i r evaluation, Since ferrihemoglobin is r a p i d l y formed i n carbonmonoxyhemoglobin s o l u t i o n s a t a c i d pH (4) i t appeared p o s s i b l e t h a t t h e s c a t t e r i n t h e values found f o r t h e equilibrium c o n s t a n t s w a s due t o contamination o f t h e carbonmonoxyhemo- g l o b i n with ferrihemoglobin, Preliminary work indeed showed t h a t a t equal concentrations carbonmonoxyhemoglobin and ferrihemoglobin d i d d i s s o c i a t e t o d i f f e r e n t e x t e n t s a t pH 5.0.

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It has been demonstrated

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t h a t c e r t a i n a c i d groups i n v a r i - ous hemoglobin d e r i v a t i v e s have a c i d d i s s o c i a t i o n c o n s t a n t s which de- pend upon t h e n a t u r e o f t h e bonding s t a t e o f t h e i r o n i n t h e heme, o r t h e n a t u r e of t h e ligand bonded t o t h e heme group. The d i f f e r e n c e ob- . served i n t h e e x t e n t s of d i s s o c i a t i o n of carbonmonoxyhemoglobin and ferrihemoglobin suggested t h a t t h e molecular d i s s o c i a t i o n r e a c t i o n o f hemoglobin might a l s o be wheme-Linkedll, A study of t h e c o r r e l a t i o n s between t h e r e a c t i o n s o f a molecule with a s t r u c t u r e as complex as t h a t of hemoglobin can be i n s t r u c t i v e i n e l u c i d a t i n g r e l a t i v e p o s i t i o n s o f r e a c t i v e groups i n t h e molecule, It i s with this view i n mind t h a t a study o f t h e d i s s o c i a t i o n p r o p e r t i e s o f s e v e r a l d e r i v a t i v e s of normal a d u l t hemoglobin was undertaken.

Proe edure

The method of study has been t h e determination o f t h e s e d i - mentation c o e f f i c i e n t s of v a r i o u s d e r i v a t i v e s o f normal a d u l t hemoglo- b i n a s a f u n c t i o n o f t h e t o t a l p r o t e i n concentration a t a pH o f

5.0.

The d e r i v a t i v e s which have been s t u d i e d a r e carbonmonoxy-, oxy-, f e r r o - , and ferrihemoglobin. These s t u d i e s were c a r r i e d o u t a t a pH o f f i v e because t h e concentration range i n which d i s s o c i a t i o n occurs c o i n c i d e s with t h e optimum range o f p r o t e i n concentration f o r use o f t h e s o h l i e r e n o p t i c a l of t h e u l t r a c e n t r i f u g e .

It has been shown t h a t t h e measured sedimentation c o e f f i c i e n t , S , i n a system i n d i s s o c i a t i o n equilibrium i s given by

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where S and S. a r e t h e sedimentation c o e f f i c i e n t s of t h e o r i g i n a l

0 1

s p e c i e s and t h e products o f i t s d i s s o c i a t i o n r e s p e c t i v e l y and a is t h e

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degree of d i s s o c i a t i o n . The q u a n t i t y

f

i s t h e r a t i o o f t h e molecular weight o f t h e ith s p e c i e s produced i n t h e d i s s o c i a t i o n t o t h e molecular weight o f t h e o r i g i n a l materials. For t h e c a s e o f d i s s o c i a t i o n i n t o s u b u n i t s having h a l f t h e molecular weight o f t h e o r i g i n a l m a t e r i a l t h e expression f o r t h e sedimentation c o e f f i c i e n t becomes:

where S i s t h e sedimentation c o e f f i c i e n t of t h e h a l f molecule.

H

Preparation o f M a t e r i a l s

Hemoglobin s t o c k s o l u t i o n s were prepared from f r e s h l y drawn r e d blood c e l l s from normal a d u l t donor@. These c e l l s were washed fourtgi-aes with i s o t o n i c s a l i n e s o l u t i o n ; and t h e supernatant from each washing was removed using a s u c t i o n p i p e t t e . The packed r e d blood c e l l s from t h e last wash were mixed with an equal volume o f d i s t i l l e d water and 0.4 volume o f toluene. This mixture, r e d blood c e l l s , water, and toluene was shaken vigorously t o r u p t u r e c e l l w a l l s and t o e f f e c t complete l y s i s , The l y s a t e was c e n t r i f u g e d f o r 15-20 minutes a t

1800

RPM i n a c l i n i c a l c e n t r i f u g e s f t e r which t h e toluene and stroma were removed. The remaining hemoglobin s o l u t i o n was then c e n t r i f u g e d f o r 1-2 hours a t 30,000 BPI4 i n t h e Spinco Model L U l t r a c e n t r i f u g e t o remove t h e last t r a c e s of stroma,

To avoid any d i f f e r e n c e s i n t h e behavior o f t h e d e r i v a t i v e s o f hemoglobin due t o t h e e f f e c t s of t h e technique used t o prepare f e r r o - hemoglobin, a l l o f t h e p r o t e i n was i n i t i a l l y reduced, then t r e a t e d as necessary t o prepare t h e o t h e r d e r i v a t i v e s . The method o f Adams ( 6 ) w a s f i r s t used t o prepare ferrohemoglobin; t h i s method was, however, abandoned because o f t h e d e l e t e r i o u s e f f e c t s of t h e reducing agent

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sodium d i t h i o n i t e a f t e r p a r t i a l oxidation (7).

It w a s found t h a t hemoglobin could be q u a n t i t a t i v e l y reduced by evacuation, The evacuation technique used was as follows:

A s t o c k s o l u t i o n of t h e non-crystallized oxyhemoglobin was placed i n a stoppered serum b o t t l e , After p l a c i n g t h i s serum b o t t l e i n a water bath held a t 37*C,, a hypodermic needle connected t o a water a s p i r a t o r was introduced i n t o t h e b o t t l e through its stopper, The evacuation of oxygen from t h e b o t t l e was c a r r i e d o u t while s w i r l i n g t h e con- t e n t s around t o avoid bubbling of t h e s o l u t i o n , After

15

minutes t h e evacuation was complete, a s i n d i c a t e d by t h e change i n c o l o r of t h e s o l u t i o n from a b r i l l i a n t r e d t o a deep blue red, Before removal of t h e a s p i r a t o r needle, n i t r o g e n was introduced i n t o t h e b o t t l e t o b r i n g t h e pres- s u r e above t h e s o l u t i o n t o a point above atmospheric pressure. The concentration of t h e ferrohemoglobin s o l u t i o n w a s determined using t h e Brice-Pheonix D i f f e r e n t i a l Re- fractometer, The value of t h e s p e c i f i c r e f r a c t i v e index increment w a s taken as 0,00181,

An a l i q u o t of t h e above ferrohemoglobin s o l u t i o n w a s dissolved i n a deoxygenated Acetate-NaC1 b u f f e r (0.1 M NaOAc

,

0,042 M HOAc

,

0.15 M NaC1) of pH 5,0, u = 0.25; this s o l u t i o n was of a concentration a p p r o p r i a t e f o r use i n t h e u l t r a c e n t r i f u g e , For sedimentation s t u d i e s on ferrohemoglobin, a p o r t i o n of t h i s s o l u t i o n was t r a n s f e r r e d , using a hypodermic syringe, t o an u l t r a c e n t r i f u g e c e l l which had been flushed o u t with nitrogen, Other p o r t i o n s of t h i s s o l u t i o n wese used t o prepare t h e oxy-, carb~nmonoxy-, and ferrihemoglobin s o l u t i o n s run i n t h e u l t r a c e n t r i f u g e , Oxyhemoglobin and carbonmonoxyhemoglobin s o l u t i o n s wese prepared by blowing oxygen and carbon monoxide, respectively,,over portions, Ferrihemoglobin s o l u t i o n s were prepared by allowing a por- t i o n of t h e ferrohemoglobin s o l u t i o n of pH 5.0 t o stand f o r 24 hours under oxygen a t room temperature. After 24 hours under t h e s e condi- t i o n s t h e conversion t o ferrihemoglobin i s 95% complete.

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Experimental Method

A l l sedimentation c o e f f i c i e n t determinations were made using t h e Spinco Model E U l t r a c e n t r i f u g e , o p e r a t i n g a t speeds of 52,640 o r 56,100 RPM. A t t h e s e speeds o f r o t a t i o n t h e p r o t e i n i n a l l c a s e s moved through a t l e a s t h a l f t h e l e n g t h of t h e c e l l i n t h r e e hours run- ning time. I n a l l experiments two c e l l s were used, one o f which was f i t t e d with a lo p o s i t i v e wedge shaped window. The s c h l i e r e n diagram corresponding t o t h e s o l u t i o n i n t h i s c e l l was displaced upward on t h e viewing screen o f t h e u l t r a c e n t r i f u g e , t h u s f a c i l i t a t i n g simultaneous sedimentation experiments on i d e n t i c a l o r non-identical s o l u t i o n s . I t was p o s s i b l e i n t h i s way t o make a v i s u a l comparison o f t h e sedimenta- t i o n behavior o f any two d i f f e r e n t hemoglobin d e r i v a t i v e s .

The sedimentation c o e f f i c i e n t , S, is defined as t h e r a t e o f movement o f a dissolved molecule i n u n i t c e n t r i f u g a l f i e l d

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where x i s t h e p o s i t i o n o f t h e molecule a t time t and i s t h e angular v e l o c i t y o f t h e u l t r a c e n t r i f u g e r o t o r . The r a t e of movement o f a s e d i - menting molecule i n t h e bulk of a s o l u t i o n is taken t o be t h e r a t e o f movement o f t h e boundary between s o l u t i o n and solvent. Using t h e second form o f t h e equation f o r S t h e logarithm of t h e boundary p o s i t i o n w a s p l o t t e d as a function o f t h e time. The s l o p e of t h i s l i n e divided by t h e square of t h e angular v e l o c i t y g i v e s t h e sedimentation c o e f f i c i e n t uncorrected f o r t h e v i s c o s i t y and bouyancy o f t h e s o l v e n t , It i s con- venient t o r e f e r t h e v a l u e s o f t h e sedimentation c o e f f i c i e n t t o a standard s o l v e n t , water a t 20°C, The sedimentation c o e f f i c i e n t s were reduced t o t h i s standard by t h e use of t h e equation:

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where :

T = t h e temperature a t which t h e sedimentation experiment i s c a r r i e d out.

T

./Q

⁼t h e r e l a t i v e v i s c o s i t y o f t h e s o l v e n t a t temperature T.

w

72 7

²⁰⁼t h e v i s c o s i t y of water a t temperature T r e l a t i v e t o i t s w

value a t 20°C.

-

V = t h e p a r t i a l s p e c i f i c volume o f t h e sedimenting molecule.

P

⁼t h e d e n s i t y o f t h e solvent a t temperature T.

s

p

20 = t h e d e n s i t y of water a t 20°C.

W

A l l sedimentation experiments were c a r r i e d o u t a t 20°C, The r e s u l t s of t h e sedimentation c o e f f i c i e n t determinations correspond t h e r e f o r e t o t h e d i s s o c i a t i o n e q u i l i b r i a a t t h i s temperature,

The s c h l i e r e n o p t i c a l system of t h e u l t r a c e n t r i f u g e was f i t t e d with a #29 Wratten f i l t e r , and t h e s c h l i e r e n p a t t e r n s were recorded on Eastman 103-F Spectroscopic p l a t e s . The boundary p o s i t i o n s were taken as t h e p o s i t i o n of t h e perpendicular b i s e c t o r of t h e s c h l i e r e n peaks;

i t was n o t found necessary t o apply t h e method of Goldberg ( 9 ) t o t h e determination o f t h e boundary p o s i t i o n . The boundary p o s i t i o n s were measured on enlargements o f t h e photographic p l a t e s such t h a t a measured 1 cm, corresponded t o a d i s t a n c e of 0.05 cm, i n t h e u l t r a c e n t r i f u g e c e l l .

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Results

It i s seen i n equation j t h a t t h e q u a n t i t i e s r e q u i r e d f o r t h e evaluation of t h e degree of d i s s o c i a t i o n a from t h e measured sedimenta- t i o n c o e f f i c i e n t S a r e t h e values of t h e sedimentation c o e f f i c i e n t s o f undissociated hemoglobin and of t h e hemoglobin h a l f molecules. To determine t h e value of t h e sedimentation c o e f f i c i e n t of undissociated hemoglobin, sedimentation measurements of carbonmonoxyhemoglobin were performed a t s e v e r a l concentrations i n a b u f f e r o f pH 7 . 0 , ~ = 0.1

(0,0286 M K ,HPO

,,

^0.0143 ^M^{KH *PO}^,). ^{A t}t h e concentrations chosen f o r t h e s e measurements t h e hemoglobin i s not appreciably d i s s o c i a t e d ; t h e s e measurements do show t h e e f f e c t o f t h e t o t a l p r o t e i n concentra- t i o n on t h e sedimentation c o e f f i c i e n t . I n f i g u r e 1 i s seen a p l o t of t h e sedimentation c o e f f i c i e n t of carbonmonoxyhemoglobin a g a i n s t con- c e n t r a t i o n a t n e u t r a l pH, A t zero concentration t h e value o f t h e sedimentation c o e f f i c i e n t of hemoglobin approaches t h e value 4.50 Svedbergs;

this value is taken t o be t h e sedimentation c o e f f i c i e n t o f undissociated hemoglobin. The concentration dependence of t h e a c i d d i s s o c i a t i o n o f carbonmonoxyhemoglobin has been s t u d i e d a t pH 5.0. I n f i g u r e 2 t h e sedimentation c o e f f i c i e n t o f carbonmonoxyhemoglobin as a f u n c t i o n of t o t a l concentration i s given a t pH 5.0. The value approached by t h e sedimentation c o e f f i c i e n t a t zero concentration a t t h i s pH i s 2.70 Svedbergs. This value i s taken t o be t h e sedimentation c o e f f i c i e n t of t h e hemoglobin h a l f molecule.

Since t h e values of t h e sedimentation c o e f f i c i e n t s of hemoglo- b i n and t h e hemoglobin h a l f molecule have been obtained from d a t a e x t r a p o l a t e d t o zero concentrationi i t is necessary t o c o r r e c t t h e measured sedimentation c o e f f i c i e n t s t o t h e values which would o b t a i n

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F i g u r e 1

0

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at i n f i n i t e d i l u t i o n . It i s seen i n f i g u r e 1 t h a t t h e sedimentation c o e f f i c i e n t o f undissociated hemoglobin i s 4.25 Svedbergs a t a t o t a l concentration of 1%; i t i s assumed t h a t t h e c o n t r i b u t i o n t o t h e t o t a l c o r r e c t i o n o f t h e measured sedimentation c o e f f i c i e n t s due t o t h e whole molecule w i l l amount t o 0.25 ~vedbergs/% to t a l p r o t e i n . The l a c t o - g l o b u l i n molecule has a f r i c t i o n a l c o e f f i c i e n t and a molecular weight c l o s e t o t h a t of t h e hemoglobin h a l f molecule. The e f f e c t o f t h e t o t a l p r o t e i n concentration on t h e sedimentation c o e f f i c i e n t o f t h i s m a t e r i a l amounts t o 0,125 S v e d b e r g s h t o t a l p r o t e i n (10). This same value has been assumed t o hold f o r t h e hemoglobin h a l f molecule.

I n o r d e r t o apply t h e s e c o r r e c t i o n s t o t h e measured sedimenta- t i o n c o e f f i c i e n t s , an approximate value of t h e degree o f d i s s o c i a t i o n w a s c a l c u l a t e d using t h e experimentally determined sedimentation coef- f i c i e n t i n equation

3,

S u b s t i t u t i n g t h e values o f t h e sedimentation c o e f f i c i e n t s o f hemoglobin and t h e hemoglobin h a l f molecule i n t o t h i s equation and rearranging, t h i s equation becomes:

The v a l u e s o f q obtained from t h i s equation were used i n an expression for ^SY,^2O t h e c o r r e c t i o n t o be a p p l i e d Co t h e measured sedimentation coefficient*:

The value o f t h e sedimentation c o e f f i c i e n t obtained by adding t h i s increment t o t h e experimentally determined value i s t h e sedimentation c o e f f i c i e n t c o r r e c t e d f o r f r i c t i o n a l e f f e c t s . I n t a b l e I a r e given t h e r e s u l t s o f t h e sedimentation c o e f f i c i e n t determinations f o r hemoglobin d e r i v a t i v e s and t h e values o f t h e s e q u a n t i t i e s a f t e r applying t h e above

(17)

Table I

Measured Sedimentation C o e f f i c i e n t s (S ) and Their w* 2 0

Corrected Values (S:, 20) of Derivatives o f Normal Human Hemoglobin at pH 5.0

Ekpt. No. Derivative Concentration S S

%

^w*²⁰ ^W,^{2 0}

Carbonmonoxy F e r r i

Ferro

ow

Ferro OXY Ferro 0 XY

Ferro

Carbonmonoxy OXY

F e r r i

~ X Y

FerWro OXY Ferro oxy

Carbonmonoxy

(18)

c o r r e c t i o n . These c o r r e c t e d sedimentation c o e f f i c i e n t s a r e p l o t t e d i n f i g u r e s

3 - 6

f o r t h e s e d e r i v a t i v e s a t s e v e r a l concentrations.

I n f i g u r e

3

a r e shown t h e c o r r e c t e d sedimentation c o e f f i c i e n t s f o r oxyhemoglobin a t d i f f e r e n t concentration a t pH 5.0, The g r e a t s c a t t e r o f t h e s e p o i n t s i s probably due t o t h e extreme l a b i l i t y o f oxyhemoglobin t o o x i d a t i o n t o ferrihemoglobin i n a c i d s o l u t i o n . I n f i g u r e s

4, 5,

and 6 , a r e shown t h e c o r r e c t e d sedimentation c o e f f i c i e n t s o f ferrihemoglobin, ferrohemoglobin, and carbonmonoxyhemoglobin, re- s p e c t i v e l y , a t d i f f e r e n t concentrations,

Of t h e hemoglobin d e r i v a t i v e s shudied, i t appears t h a t a t a given concentration a t pH 5.0 t h e sedimentation c o e f f i c i e n t o f f e r r o - hemoglobin is g r e a t e r than t h a t o f t h e o t h e r m a t e r i a l s , For example, i n experiment 261 which was a simultaneous sedimentation experiment on t h e d e r i v a t i v e s carbonmonoxy- and ferrohemoglobin a t 0,7876 each, t h e c o r r e c t e d sedimentation c o e f f i c i e n t of carbonmonoxyhemoglobin i s

3.78

Svedbergs while t h a t of ferrohemoglobin i s 4.25 S, A d i f f e r e n c e i n t h e values o f t h e sedimentation c o e f f i c i e n t s o f t h e d e r i v a t i v e s may a r i s e from d i f f e r e n c e s i n t h e values o f t h e f r i c t i o n a l c o e f f i c i e n t s o f t h e m a t e r i a l s , I n t a b l e 11 a r e shown t h e r e s u l t s of sedimentation coef- f i c i e n t determinations on t h e d e r i v a t i v e s ferro-, oxy-, and ferrihemoglobin a t a pH o f 7.0, It i s seen t h a t t h e c o r r e c t e d sedimentation c o e f f i c i e n t s o f t h e s e d e r i v a t i v e s a g r e e well with t h e value o f t h e sedimentation c o e f f i c i e n t o f carbonmonoxy hemoglobin a t t h i s pH,

Shape f a c t o r s , t h e r e f o r e , do n o t account f o r t h e d i f f e r e n c e s observed a t pH 5.0, I n t a b l e I I S a r e presented t h e r e s u l t s o f molecular weight determinations by t h e method o f Archibald (11) on ferrohemoglobin and oxyhemoglobin a t pH 5.0, These molecular weights, though n o t i n

(19)

(20)

(21)

-15-

Figure

5

I I

\

- \ .. .. \ z - 61 0

-

_{_I}

( 3 0

0

Lf-l

I I

= I a

I

0

CT

a

W

- LL

I I

0. Ln v m

Ln

-I\

0

-LC)

0

Ln

-a

0

0 m

(22)

-16- Figure

6

(23)

agreement with t h e sedimentation c o e f f i c i e n t d a t a f o r t h e s e m a t e r i a l s a t pH 5.0 and a t t h e concentration 0.5&, do show t h e molecular weight of oxyhemoglobin t o be s i g n i f i c a n t l y l e s s than t h a t of ferrohemoglobin,

Table I1

Measured Sedimentation C o e f f i c i e n t s (Sw,20) and Their Corrected Values (S;, 20 ) of Derivatives of

Normal Human ~ e m o ~ l o b i n a t pH 7.0

Derivative Concentration

%

266 Ferro O,86 4.24 4.46

266 OXY 0.86 4,23 4.45

267 Ferro 0.43 4.37

4.48

267 OXY 0.43 4.34 4.45

282 F e r r i 0.27 4.40 4*47

282 F e r r i 0.54 4.26 4,40

Table I11

Molecular Weights of Oxyhemoglobin and Ferrohernoglobin a t Equal Concentration i n Solutions of pH 5.0

Expt, No. Derivative Concentration % Molecular Weight

269 Ferro 0.56 64,600

A s u i t a b l e parameter f o r t h e demonstration of any i n t r i n s i c d i f f e r e n c e i n t h e molecular d i s s o c i a t i o n behavior of t h e s e d e r i v a t i v e s i s t h e apparent d i s s o c i a t i o n constant K

~ P P ' For t h e purposes of t h e s e c a l c u l a t i o n s , i t has been assumed t h a t t h e d i s s o c i a t i o n of hemoglobin d e r i v a t i v e s i n a c i d s o l u t i o n produces non-identical h a l f molecular spe- e i e s ' m , The apparent d i s s o c i a t i o n constant K i s , i n t h i s case:

aPP

(24)

I n t h i s expression MW

undissoc

.

i s t h e gram molecular weight o f human hemoglobin,

66,800.

The q u a n t i t y a i s t h e second approximation of

2

t h e degree o f d i s s o c i a t i o n c a l c u l a t e d using t h e c o r r e c t e d values o f t h e measured sedimentation c o e f f i c i e n t s t a b u l a t e d ( t a b l e I ) i n equa- t i o n

6.

I n t a b l e I V a r e t h e values of t h e apparent d i s s o c i a t i o n con- s t a n t s f o r t h e d i f f e r e n t d e r i v a t i v e s using t h e d a t a o f t a b l e

H,

It appears from t h e apparent d i s s o c i a t i o n c o n s t a n t s i n t a b l e I V t h a t t h e r e i s an e f f e c t of t h e n a t u r e o f a hemoglobin d e r i v a t i v e on t h e e x t e n t of d i s s o c i a t i o n i n a c i d , From t h e v a l u e s o f t h e s e c o n s t a n t s i t may be seen t h a t ferrihemoglobin d i s s o c i a t e s t o t h e g r e a t e s t e x t e n t while a t t h e same concentration ferrohemoglobin i s l e a s t d i s s o c i a t e d , The s c a t t e r of t h e sedimentation c o e f f i c i e n t s o f oxyhemoglobin seen i n f i g u r e

3

i s a g a i n manifested i n t h e d i s s o c i a t i o n c o n s t a n t s c a l c u l a t e d f o r t h i s m a t e r i a l , Since t h e values f o r oxyhemoglobin f a l l between those o f carbonmonoxyhemoglobin and ferrihemoglobin, i t appears t h a t t h e l a t t e r i s t h e contaminant i n t h e oxyhemoglobin experiments,

Discussion of R e s u l t s

The "hemelinked" a c i d groups o f hemoglobin d e r i v a t i v e s have been extensively i n v e s t i g a t e d

(51,

The a c i d d i s s o c i a t i o n c o n s t a n t s f o r t h e s e groups have been determined and found t o vary from one de- r i v a t i v e t o another, Tabulated i n t a b l e V a r e t h e pK values o f t h e s e

a

groups i n t h e d i f f e r e n t d e r i v a t i v e s . Of i n t e r e s t a r e t h e pKa v a l u e s o f t h e f i r s t hemelinked a c i d group, fa^ t h i s group would be most a f - f e c t e d a t pH 5,0 where t h e molecular d i s s o c i a t i o n r e a c t i o n has been

(25)

-19- Table I V

Apparent Dissociation Constants of Derivatives of Normal Adult Hemoglobin i n Ac-NaC1 Buffer pH 5,0, u = 0.25

Ekpt. No. Derivative Concentration % K X 1 0 5

aPP

Ferro Ferro Ferro Ferro Ferro Ferro F e r r i F e r r i F e r r i F e r r i

Carbonmonoxy Carbonmonoxy Carbonmonoxy Carbonmonoxy Carbonmonoxy

(26)

-20-

shown t o be a f f e c t e d by t h e nature of t h e hemoglobin d e r i v a t i v e . It has been shown t h a t t h e molecular d i s s o c i a t i o n r e a c t i o n involves t h e uptake o f a t l e a s t one proton f o r each hemoglobin molecule d i s s o c i a t e d

(3).

I f t h e f i r s t hemelinked a c i d group were t h e group r e c e i v i n g this proton i n t h e d i s s o c i a t i o n r e a c t i o n , i t would be expected t h a t t h e hemoglobin d e r i v a t i v e s would d i s s o c i a t e t o d i f f e r e n t e x t e n t s a t pH 5.0. Further, t h e ferrohemoglobin would be l e a s t d i s s o c i a t e d s i n c e t h e

f i r s t hemelinked a c i d group i n t h i s m a t e r i a l is, a t pH 5.0 only

64%

protonated a s opposed t o

85%

protonation of t h i s group i n t h e o t h e r d e r i v a t i v e s .

Table V

Hemelinked Acid Groups

Hemoglobin Group One Group Two Group Three

Derivative

PKa PKa PKa

F e r r i 5075 6.68 8.01

Carbonmonoxy (5.75)* (6.68)*

Ferro 3-25 7.93

*No values were found f o r t h e pKa values of carbonmonoxyhemoglobin. Since t h e i r o n i s bonded covalently through d2s p 3 bonds i n both oxy and carbonmonoxyhemoglo- b i n i t has been assumed t h a t t h e i r pKa values a r e equal.

I n order t o reasonably compare t h e change of t h e pKa of t h i s f i r s t hemelinked a c i d group with t h e change of t h e apparent d i s s o c i a - t i o n c o n s t a n t s of t h e d e r i v a t i v e s , t h e negative logarithms of t h e s e quantitieshavebeencalculated. ThesepK v a l u e s a r e s h o w n i n

aPP

t a b l e V I . It i s seen t h a t t h e value of t h e pK of ferrohemoglobin, aPP

5.35

0.15, d i f f e r s most from t h e corresponding values f o r t h e o t h e r

(27)

-21-

d e r i v a t i v e s ; and a l s o t h e pK values f o r f e r r i - , oxy-, and carbon- aPP

monoxyhemoglobin a r e equal within t h e l i m i t s o f e r r o r , Table V I

Values of t h e Negative Logarithms o f Apparent D i s s o c i a t i o n Constants o f

Hemoglobin D e r i v a t i v e s

Hemoglobin Average K

Derivative (x 1 0 ~ ) ~PKaPP ~ ~

F e r r i 5.2 2 1.2 4.3 L 0 - 2

OXY

3.1

²0.9 4 - s ~ 0 - 1 5

Carbonmonoxy 2.4 2

0-8

4 - 6 = ~ 0.1'

Ferro 0 - 5

+

0.2

5 . 3 5 ~

0.15

The a c i d c o n s t a n t s o f t h e hemelinked a c i d groups o f hemoglo- b i n were determined by t i t r a t i o n s of t h e d e r i v a t i v e s

(5).

There w a s no c o n s i d e r a t i o n made o f t h e molecular d i s s o c i a t i o n of t h e s e d e r i v a t i v e s i n a c i d s o l u t i o n a t t h e time t h e s e c o n s t a n t s were evaluated. It i s p o s s i b l e t h a t t h e t i t r a t i o n s r e f l e c t n o t only t h e protonation o f t h e hemelinked a c i d groups but a l s o t h e protonation of a c i d groups ex- posed t o r e a c t i o n by t h e d i s s o c i a t i o n . The d i f f e r e n c e between t h e pK values of ferrohemoglobin and t h e o t h e r d e r i v a t i v e s i s t h e r e f o r e

a

not d i r e c t l y comparable t o t h e d i f f e r e n c e s between t h e pK value o f aPP

ferrohemoglobin and t h e o t h e r m a t e r i a l s ,

It i s s i g n i f i c a n t t h a t , i n t h e t e r t i a r y s t r u c t u r e published f o r horse hemoglobin (121, t h e hemelinked h i s t i d i n e i s seen t o form t h e bond between t h e non-identical chains of t h i s m a t e r i a l , Human hemoglobin i s known t o d i s s o c i a t e i n t o non-identical s u b u n i t s i n a c i d s o l u t i o n ( l g ) , and may be expected t o have a s t r u c t u r e similar t o t h a t of horse hemoglobin.

(28)

PART I1

CARBON-14 LABELLED H B R I D S OF NORMAL ADULT AND S I C K L E CELL EEMOGLOBINS

(29)

A. PREPARATION

AND

ISOLATION OF

CARBON-14

LABELLED HYBRIDS OF NOFZMAL ADULT AND SICKLE CELL HEMOGXQBIN

The d i s e a s e s i c k l e c e l l anemia has received much study s i n c e i t was f i r s t reported i n t h e medical l i t e r a t u r e by Herrick (14) i n 1910, Herrick described t h e c l i n i c a l manifestations of an i n d i v i d u a l a f f l i c t e d with t h e disease, Other workers have described t h e p e c u l i a r change i n shape of t h e red blood c e l l s of such i n d i v i d u a l s under con- d i t i o n s of decreased oxygen pressure and have noted t h e r e v e r s i b i l i t y o f t h i s change on r e t u r n i n g t h e pressure t o normal. The r o l e of oxygen and o t h e r l i g a n d s on t h e heme i n a f f e c t i n g t h i s shape change has been s t u d i e d q u a l i t a t i v e l y and q u a n t i t a t i v e l y f o r many years.

It was n o t u n t i l 1 9 4 9 , however, t h a t t h e p e c u l i a r p r o p e r t i e s of t h e s e r e d blood c e l l s were recognized a s being due t o an abnormality of t h e oxygen c a r r y i n g pigment, hemoglobin (15 )

.

I n t h i s work e l e c t r o p h o r e t i c s t u d i e s were done on t h e hemoglo- b i n from normal i n d i v i d u a l s and on t h e hemoglobin from persons who had t h e d i s e a s e s i c k l e c e l l anemia (15). It w a s found t h a t t h e r e e x i s t e d mobility d i f f e r e n c e s between t h e two hemoglobins i n t h e region of t h e i r i s o e l e c t r i c points. This mobility d i f f e r e n c e corresponded t o a d i f -

ference i n n e t charge of t h e hemoglobins. Since t h i s discovery t h e r e have been i d e n t i f i e d more than IjS d i f f e r e n t human hemoglobins using

t h e e l e c t r o p h o r e t i c technique and o t h e r s (16).

(30)

4 3 -

%he main emphasis i n t h e study of these rqabnomalw'hemoglobins has been i n the elucidation of chemical d i s t i n c t i o n s between t h e s i c k l e c e l l type and normal a d u l t hemoglobin. With the electrophoretic d i f - ference t o account f o r , the f i r s t s t e p taken was a study of the heme groups of t h e two proteins (15). These s t u d i e s indicated no differences i n t h e c r y s t a l s t r u c t u r e s of the prosthetic groups o f t h e proteins, Further electrophoretic s t u d i e s of t h e globin a o i e t i e s from these hemoglobins showed t h a t i t was i n the polypeptide chains t h a t t h e anomalies were t o be found, T j t r a t i o n s t u d i e s were undertaken i n an attempt t o i d e n t i f y t h e m i n o acid residues reajponsible f o r t h e difference i n charge of t h e proteins. Lack of s u i t a b l e s e n s i t i v i t y of t h e t i t r a t i o n s obviated t h e success of t h i s method, Total amino acid analyses (17)

f a i l e d a l s o t o account f o r the differences, These amino a c i d analyses did, however, point out t h e c l o s e s i m i l a r i t y o f amino a c i d composition of t h e two heaoglobins, Ehd group analyses using the method of Sanger demonstrated t h a t the two hemoglobins were each composed of two d i f - ferent polypeptide chains, occurring i n p a i r s and having the same N- terminal sequences (17,18), Using combined paper chromatography and paper electrophoresis on hydrolysates of normal a d u l t and s i c k l e c e l l hemoglobins Ingram (19) was a b l e t o obtain charac t e r i s t i e "fingerprints"

of the two hemoglobins. Elution of the d i f f e r i n g polypeptide s p o t s on t h e "Iingerprintsr8" and a n a l y s i s of these peptides showed the d i f - ference between the hemoglobins t o be due t o the s u b s t i t u t i o n i n s i c k l e c e l l hemoglobin of a v a l i n e residue f o r t h e glutamic a c i d r e s i - due found i n normal a d u l t hemoglobin. This finding accounted quanti- t a t i v e l y f o r the observed difference i n electrophoretic mobility between the two hemoglobins.

(31)

-24-

I n h i s work, Ingram found only 26 peptide fragments f o r both normal a d u l t and s i c k l e c e l l hemoglobin a p a r t from a t r y p t i c h y d r o l y s i s r e s i s t a n t "core". This number of p e p t i d e s was s i g n i f i c a n t i n t h e sense t h a t i t demonstrated again t h e o v e r a l l s i m i l a r i t y o f t h e two hemoglo- b i n s . With such o u t s t a n d i n g s i m i l a r i t i e s o f t h e two hemoglobins, t h e p o s s i b i l i t y e x i s t e d t h a t t h e anomaly w a s confined t o only a small region o f t h e s i c k l e c e l l molecule. It was known t h a t t h e two poly- p e p t i d e c h a i n s , designated as t h e a and t h e

p

c h a i n s , of t h e two hemo- g l o b i n s had t h e same N-terminal sequences, The work o f Ingram, however, l e f t unanswered t h e q u e s t i o n o f t h e chemical i d e n t i t y o f t h e polypeptide c h a i n s having t h e same composition i n t h e two hemoglobins,

I n P a r t I of t h i s Thesis, mention has been made o f t h e a c i d induced d i s s o c i a t i o n o f human hemoglobin. A f t e r passing through a minimum i n d i s s o c i a t i o n i n t h e r e g i o n o f n e u t r a l i t y , hemoglobin under- goes a n o t h e r d i s s o c i a t i o n i n a l k a l i n e s o l u t i o n ; t h i s molecular d i s s o c i - a t i o n i s more s t r o n g l y dependent on t h e hydrogen i o n c o n c e n t r a t i o n (20).

Knowing o f t h e a c i d d i s s o c i a t i o n o f normal a d u l t and s i c k l e c e l l hemoglobins, Singer and I t a n o (21) attempted t o form lthybridstl o f t h e two hemoglobins by d i s s o c i a t i o n o f mixtures o f t h e p r o t e i n s f o l - lowed by d i a l y s i s t o n e u t r a l i t y , Free boundary e l e c t r o p h o r e s i s w a s done on t h e s e recombined mixtures. I n explaining t h e i r l a c k o f suc- c e s s i n forming new e l e c t r o p h o r e t i c s p e c i e s , t h e s e workers suggested t h a t t h e hemoglobins recombined asymmetrically such t h a t , even i n t h e event o f chain exchange, no new e l e c t r o p h o r e t i c s p e c i e s would be formed. It w a s a t t h i s p o i n t t h a t t h e experiments r e p o r t e d i n t h i s Thesis were begun. The f i r s t a t t e m p t s made i n t h i s i n v e s t i g a t i o n a t forming lfhybridsfv c o n s i s t e d i n l a b e l l i n g one o f t h e hemoglobins with

(32)

i o d o a c e t i c a c i d , iodoacetamide, para-chloromercuribenzoic a c i d , o r dimethylnaphthalene sulfonylchloride. Due e i t h e r t o l a c k of s u f f i c i e n t time f o r chain exchange t o occur o r t o unfavorable s t e r i c e f f e c t s o f t h e s e l a b e l s , none of t h e s e e a r l y experiments c l e a r l y demonstrated t h a t chain exchange had taken place. It w a s then decided t o attempt h y b r i d i z a t i o n experiments with r a d i o a c t i v e l y l a b e l l e d hemoglobins.

Indeed, t h e f i r s t evidence o f chain exchange between t h e two hemoglo- b i n s i n t h i s work was obtained using C" l a b e l l e d normal hemoglobin and u n l a b e l l e d s i c k l e c e l l hemoglobin. Because of t h e p o t e n t i a l u s e s o f s u i t a b l y a c t i v e hybrid hemoglobins, a method w a s developed f o r pro- ducing and i s o l a t i n g highly r a d i o a c t i v e hemoglobin hybrids, i n which only one type o f subunit i s r a d i o a c t i v e and t h e o t h e r i s s u b s t a n t i a l l y i n a c t i v e . I n t h e course o f t h i s work chain exchange h a s been observed a f t e r d i s s o c i a t i o n of hemoglobin mixtures i n both a c i d and a l k a l i n e s o l u t i o n .

Procedure

Since hemoglobin d i s s o c i a t e s r e v e r s i b l y i n both a c i d and alka- l i n e s o l u t i o n , hybrid hemoglobins have been formed a f t e r d i s s o c i a t i o n under both conditions. Equimolar mixtures of carbonmonoxy s i c k l e c e l l hemoglobin Hbs(CO), and carbonmonoxy a d u l t hemoglobin HbA(C0) were d i a l y s e d f o r 24 hours a g a i n s t e i t h e r an a c i d o r a l k a l i n e b u f f e r a t 3@C. The a c i d b u f f e r was an a c e t i c acid-sodium a c e t a t e b u f f e r o f pH 5.0 having t h e composition: 0.042 N HAc, 0.10 M NaAc, 0.15 M NaC1.

m e a l k a l i n e b u f f e r was t h e b u f f e r o f Hasserodt and Vinograd (20)

having a pH o f 11.0, and u = 0.35 with t h e composition: 0.033 M N a 2 H P O L , 0,0167 M Ha,POL, 0.15 M NaC1. After d i s s o c i a t i o n of a mixture o f t h e

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-26-

hemoglobins, t h e m a t e r i a l s were d i a l y a e d t o a pH of 7.22, a g a i n s t t h e b u f f e r s p e c i f i e d as developer no, 1 by Allen, Schroeder, and Balog

( 2 2 ) . This developer has t h e composition: 0,024 M NaEL, PO,

,

^0.050

M N a 2 H P 0 , , 0.01 M KCH. The hemoglobin mixtures were s e p a r a t e d chromatographically on IRC-50 columns i n equilibrium with t h i s developer,

I n t h e s e experiments one o f t h e hemoglobins i n t h e mixtures was uniformly l a b e l l e d with carbon-14 c o n t a i n i n g amino a c i d s , Upon forma- t i o n o f hybrids r a d i o a c t i v i t y was t r a n s f e r r e d t o t h e o t h e r i n i t i a l l y non-radioactive hemoglobin, I n experiments u s i n g r a d i o a c t i v e s i c k l e c e l l hemoglobin i t was necessary t o remove t h e f a s t minor components from t h e HbS before h y b r i d i z a t i o n s i n c e t h e s e minor components have t h e same chromatographic p r o p e r t i e s a s FhA (23). This was accomplished by chromatography o f from

50

t o 100 mg, o f t h e r a d i o a c t i v e s i c k l e c e l l hemoglobin on columns i n equilibrium with developer no, 2, Only t h e major s i c k l e c e l l hemoglobin component recovered from t h e s e columns w a s ^Usedi n t h e subsequent h y b r i d i z a L ~ o n experiments.

P r e p a r a t i o n of M a t e r i a l s Unlabelled Normal Adult and S i c k l e C e l l Hemoalobins

The u n l a b e l l e d hemoglobins were prepared from f r e s h l y drawn r e d blood c e l l s from e i t h e r normal o r s i c k l e c e l l anemic donors, This procedure has been described i n P a r t I o f t h i s Thesis.

Uniformly Labelled Adult and S i c k l e C e l l Hemoalobins

It was necessary t h a t t h e r a d i o a c t i v e hemoglobins used i n pre- p a r i n g hemoglobin hybrids be uniformly l a b e l l e d i n t h e a and t h e

p

chains. It was a l s o d e s i r a b l e t h a t t h e amino a c i d chosen f o r t h e l a b e l occur with high frequency i n t h e hemoglobin molecule t o a s s u r e

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-27-

t h e highest p o s s i b l e incorporation of r a d i o a c t i v i t y i n t o t h e hemoglobino A t t h e o u t s e t of t h i s work nothing w a s known of t h e r e l a t i v e composi- t i o n s of t h e a and t h e $ chains; i t w a s f e l t , however, t h a t l e u c i n e would f u l f i l l t h e s e two requirements. This w a s a f o r t u n a t e choice f o r i t has been shown by H i l l and Craig (24) t h a t t h e r e is an equal number of l e u c i n e r e s i d u e s i n t h e two chains, The incorporation of C' -1- l e u c i n e thus a s s u r e s equal s p e c i f i c a c t i v i t i e s i n both chains,

Blood samples containing from

15%

t o 66% r e t i c u l o c y t e s were obtained from donors having acquired hemolytic o r s i c k l e c e l l anemias, After f o u r washings of t h e r e t i c u l o c y t e - r i c h r e d blood c e l l s with cold i s o t o n i c s a l i n e s o l u t i o n t h e incorporation of t h e d b - 1 - l e u c i n e w a s begun using a modification of t h e procedure o f Borsook, e t a1 (25) f o r t h e incorporation of r a d i o a c t i v e amino a c i d s i n t o r a b b i t hemoglobin, This procedure w a s begun within two hours a f t e r t h e red blood c e l l s were drawn, The complete incubation medium was prepared using t h e s t o c k s o l u t i o n s described i n Table V I I , The following amounts of t h e s e s t o c k s o l u t i o n s were added t o each 1 0 m l , of packed r e d blood c e l l s from t h e last wash:

5.85

m l . of amino a c i d mixture without l e u c i n e , 0.60 m l , of glucose s o l u t i o n , 1,2 m l o glutarnine s o l u t i o n ,

1.8

m l , o f NaHCOJ s o l u t i o n , and

l l , 7

m l . of f e r r o u s ammonium s u l f a t e s o l u t i o n . To t h i s mixture w a s added 7 micromoles, 50 uc, of Nuclear-Chicago uniformly l a b e l l e d

d

-1-leucine dissolved i n 0.5 m l , NKM s a l i n e s o l u t i o n .

The complete incubation mixture was separated i n t o 2 m l , por- t i o n s i n 20 m l , beakers which were then placed i n a Dubnoff Shaking Incubator under an atmosphere of 95% C0, -SOB2

.

After incubation f o r four hours, t h e red blood c e l l s were washed f o u r times i n c o l d i s o t o n i c s a l i n e s o l u t i o n , The supernatant s o l u t i o n s r e s u l t i n g from t h e

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Table V I I

S o l u t i o n s Required f o r Incubations

NKM S a l i n e , 0.13

M

N a C 1 , 0,005 M K C 1 , 0,0075 M MgC1,.

This s o l u t i o n was prepared u s i n g r e d i s t i l l e d water, Glucose i n NKM, 0.22 M Glucose i n NKM s a l i n e , This s o l u t i o n was prepared f r e s h l y f o r each incubation.

Ferrous Ammonium S u l f a t e . 0.01 M Fe(NH4 ) k (SO,,

I3

^{i n NKM}

s a l i n e , T h i s s o l u t i o n w a s prepared f r e s h l y f o r each i n - cubation.

Glutamine i n NKM. 0.01 M glutamine i n NKM s a l i n e . This s o l u t i o n may be prepared i n q u a n t i t y , s t o r e d frozen and thawed o u t as needed f o r use,

Sodium Bicarbonate, 0.27 M NaHC05 i n r e d i s t i l l e d water, This s o l u t i o n was prepared f r e s h f o r each incubation.

T o t a l Amino Acid Solution. This s o l u t i o n contained t h e following amounts of amino a c i d s expressed as mg, per 1000 m l , of NKM s a l i n e s o l u t i o n : 1-alanine

180,

l - a r g i - n i n e 100, 1 - a s p a r t i c a c i d

380,

1-cysteine 5 0 , g l y c i n e 400, I - h i s t i d i n e HC1.40 500, 1-hydroxyproline 150, l- i s o l e u c i n e 52, (1-leucine 5Z), 1 - l y s i n e H C 1 330, 1- methionine 50, 1-phenylalanine 260, 1-proline 160, 1- s e r i n e 175, 1-threonine 200, 1-tryptophan 60, 1 - t y r o s i n e 150, and 1-valine 370. This s o l u t i o n was heated t o a b o i l t o d i s s o l v e a l l amino a c i d s a f t e r which t h e pH w a s a d j u s t e d t o 7.0 with

5

M NaOH. This s t o c k s o l u t i o n w a s s t o r e d frozen i n small l o t s which were thawed o u t as needed f o r i n c u b a t i o n s , For t h e i n c u b a t i o n s i n which d 4 - 1 - l e u c i n e was i n c o r p o r a t e d i n t o hemoglobin t h i s s t o c k s o l u t i o n was made up omitting 1-leucine,

c e n t r i f u g a t i o n o f each o f t h e s e washings was consolidated and d i s p o s e d o f with r a d i o a c t i v e waste, The f u r t h e r p r e p a r a t i o n of t h e r a d i o a c t i v e hemoglobins w a s t h e same as previously described i n t h i s Thesis. The r e s u l t i n g hemoglobin s o l u t i o n s were d i a l y z e d a g a i n s t f o u r 2 l i t e r changes o f d i s t i l l e d water t o remove t h e last t r a c e s o f unincorporated d 4 - 1 - l e u c i n e . Depending roughly on t h e percentage o f r e t i c u l o c y t e s i n t h e r e d blood c e l l s incubated, t h e s p e c i f i c a c t i v i t i e s obtained i n t h e l e u c i n e l a b e l l e d hemoglobins ranged from l 9 O O t o 10,000 cpm/mg,

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-29-

The s p e c i f i c a c t i v i t i e s of hemoglobin samples from s e v e r a l of t h e s e p r e p a r a t i o n s a r e given i n t a b l e V I I I ,

Table V I I I

S p e c i f i c A c t i v i t i e s o f Hemoglobins i n S e v e r a l P r e p a r a t i o n s

M a t e r i a l

9fo

R e t i c u l o c y t e s Label S p e c i f i c A c t i v i t y c pm/mg

~ b

s 18.9

l e u c i n e 1660

Hb A 40.0 l e u c i n e 7000

Hb S 34,O l e u c i n e 2700

Hb A 24,4 l e u c i n e 2800

Hb A 66.0 Leucine 11000

Hb S 18.9 Alga1,Hyd.

5

100

Hb S 18.9 Methionine 112

Hb S

18 ,

9 Tryptophan

135

Hb S 18.9 Fe

5 9

₂₆₆₀

Hemoglobin was prepared using amino a c i d s o t h e r than

d

-1- l e u c i n e . Also one p r e p a r a t i o n was made using an a l g a l h y d r o l y s a t e and one u s i n g pe5' as t h e l a b e l . The s p e c i f i c a c t i v i t i e s obtained i n t h e s e p r e p a r a t i o n s a r e a l s o given i n t a b l e V I I I , The d e t a i l e d method o f p r e p a r a t i o n o f t h e s e l a b e l l e d hemoglobins i s given i n Appendix 111.

The hemoglobins l a b e l l e d r e s p e c t i v e l y with methionine and tryptophan were t o be used i n forming hybrids; t r y p t i c d i g e s t i o n o f t h e s e hybrids and f i n g e r p r i n t i n g by t h e method o f Ingram (19) were planned, Radioautograms o f t h e f i n g e r p r i n t s would show t h e d i s t r i b u - t i o n o f t h e r e l a t i v e l y r a r e amino a c i d s , methionine and tryptophan,

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-30-

between t h e a and t h e $ chains. During t h e course of t h i s work, a r e - p o r t of t h e s u c c e s s f u l s e p a r a t i o n of t h e a and t h e f3 chains and t h e amino a c i d analyses of t h e i n d i v i d u a l chains w a s published (24), The program f o r s o l u t i o n of t h e problem using t h e methionine and tryptophan l a b e l l e d hybrids w a s t h e r e f o r e abandoned.

Chroma t o graphy Preparation o f Resin f o r Chromatoaraphy

Two pounds o f IRC-50 Amberlite r e s i n were t r a n s f e r r e d i n a hood t o a j a r containing -16 l i t e r s of d i s t i l l e d water. The suspension of r e s i n i n water w a s s t i r r e d vigorously f o r f i v e minutes using an e l e c t r i c s t i r r e r a f t e r which t h e r e s i n w a s allowed t o s e t t l e and t h e supernatant decanted o f f , This process was repeated a t o t a l of f o u r times. The p r e c i p i t a t e from t h e last wash with d i s t i l l e d water was placed on a l a r g e r Buchner funnel and washed with

6

l i t e r s of

4

N HC1. With t h e r e s i n now i n i t s a c i d form i t w a s again washed with d i s t i l l e d water t o remove excess HCl.

The r e s i n of t h e c o r r e c t s i z e f o r use i n t h e chromatographic columns was separated and i s o l a t e d by t h e hydraulic method of Hamilton (26), a s described by Moore and S t e i n (27). This method c o n s i s t e d i n t h e d i f f e r e n t i a l f l o t a t i o n of t h e r e s i n p a r t i c l e s i n a Corning 6400 c o n i c a l separatory funnel. P a r t i c l e s of a s i z e smaller than 250 mesh were removed by f l o t a t i o n a t a flow r a t e of

315

ml./min.; those o f a s i z e from 250 t o 200 mesh were removed by f l o t a t i o n a t a r a t e of 525 ml,/min, The apparatus used i n t h e s e s e p a r a t i o n s w a s previously c a l i - b r a t e d using r e s i n p a r t i c l e s of 200-250 mesh which had been separated by screening,

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-31-

The r e s i n p a r t i c l e s of t h e c o r r e c t s i z e were changed i n t o t h e sodium form by suspension i n 2 M NaOH, Excess base was removed by suspending t h e r e s i n again i n 4 x 10 l i t e r s of d i s t i l l e d water. After t h e last of t h e s e four suspensions, t h e supernatant gave only a s l i g h t l y a l k a l i n e r e a c t i o n with u n i v e r s a l litmus paper. The r e s i n w a s then sus- pen(ied5nth.e chromatographic developer with which i t w a s t o be used,

-

Preparation of Columns f o r Chromatoaraphy

The chromatographic columns were prepared by t h e method of Allen, Schroeder, and Balog (221, and were t h e same dimensions as those o f t h e s e authors, The columns were e q u i l i b r a t e d a t 6OC, by allowing approximately one l i t e r of developer no, 1 t o flow through t h e r e s i n bed,

Separation of Mixtures of Normal Adult and S i c k l e C e l l Hemo~lobins Mixtures of t h e two hemoglobins c o n t a i n i n g from 20 t o 5 0 mg, t o t a l p r o t e i n i n 2 t o

3

m l , of developer no, 1 were layered on t o p of t h e r e s i n beds of t h e e q u i l i b r a t e d columns. The normal a d u l t hemoglo- b i n w a s e l u t e d at 6OC, reaching a maximum of concentration a t 22 m l , e l u a t e , and t a i l i n g o f f t o inappreciable c o n c e n t r a t i o n s a t

40

through 50 m l , The flow of developer w a s then stopped, a f t e r which t h e tempera-

t u r e o f t h e jacketed columns w a s r a i s e d t o 2 8 0 ~ . Allowing from

15

t o 20 minutes f o r t h e r e s i n t o reach t h i s temperature, t h e flow of developer w a s again s t a r t e d . The s i c k l e c e l l hemoglobin w a s desorbed a t t h i s temperature, reaching a maximum of concentration a t about 5 2 m l , e l u a t e ,

The concentrations of t h e chromatographic f r a c t i o n s were measured s p e c t r o m e t r i c a l l y with a Beckman Model DU spectrophotometer, The o p t i c a l d e n s i t i e s of t h e s o l u t i o n s were measured a t t h e wavelengths of

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-32-

415 mu o r

539

mu, corresponding t o absorption maxima of carbonmonoxyhemoglobin; o r , i n c a s e s where i t w a s believed t h a t t h e hemoglobin f r a c t i o n s were contaminated with ferrihemoglobin cyanide, t h e measurements were made a t t h e carbonmonoxy-ferrihemoglobin cyanide i s o b e s t i c p o i n t , 522 mu,

Measurement of Radioactivity i n Hemoglobin Samples

The s p e c i f i c a c t i v i t i e s of hemoglobin samples and chromatographic f r a c t i o n s were measured using a Nuclear-Chicago gas flow Geiger c o u n t e r , Model D-47, equipped with a ffmicromilfl window. For counting, t h e hemoglobin samples were placed on p l a n c h e t t e s i n volumes o f 0.5 o r 1 , O m l , o f developer. The s o l u t i o n s were evaporated t o dryness with a h e a t lamp b e f o r e counting. Generally, no more than 0.5 mg, o f p r o t e i n w a s placed on t h e s e planchettes. A s may be seen from f i g u r e

7,

no correc- t i o n s were r e q u i r e d f o r s e l f a b s o r p t i o n by t h e hemoglobin. No correc- t i o n s were made f o r absorption by t h e b u f f e r salts s i n c e t h e s e were always present i n constant amounts.

R e s u l t s o f Hybridization Experiments

Early experiments i n d i c a t e d t h a t hybrids were formed only a f t e r d i s s o c i a t i o n a t a c i d pH, These experiments c o n s i s t e d i n allowing mix- t u r e s o f C' -1-leucine l a b e l l e d normal a d u l t hemoglobin and i n a c t i v e s i c k l e c e l l hemoglobin t o s t a n d a t pH 4,5 o r 1 1 , O f o r a time o f one hour. A f t e r d i s s o c i a t i o n of t h e m a t e r i a l s a t t h e r e s p e c t i v e pH values, t h e s o l u t i o n s were dialyzed a g a i n s t a b a r b i t a l b u f f e r o f pH

8.6,

u = .01, The hemoglobins were s e p a r a t e d by f r e e boundary electropho- r e s i s i n t h e Spinco Model H, c u r r e n t

8

^{m a}f o r

5

hours. Good resolu- t i o n o f t h e hemoglobins was obtained i n both t h e ascending and descend- i n g limbs, The hemoglobin samples were removed from t h e r e g i o n s

(40)

(41)

-34-

corresponding t o t h e f a s t and slow components using t h e sampling mecha- nism o f t h e Spinco instrument. Due t o t h e small amount of pure normal a d u l t o r s i c k l e c e l l helnoglobin which could be recovered by t h i s method, i t w a s not p o s s i b l e t o determine a c c u r a t e l y t h e amounts o f hemoglobins u l t i m a t e l y counted. Attempts were made t o count comparable amounts of t h e two hemoglobins however. The r e s u l t s of experiments o f t h i s type a r e s e e n i n t a b l e

IX.

It may be seen t h a t t h e r e i s an a p p r e c i a b l e t r a n s f e r o f a c t i v i t y t o t h e i n i t i a l l y u n l a b e l l e d s i c k l e c e l l hemoglo- b i n only a f t e r d i s s o c i a t i o n a t pH

4.5.

S e p a r a t i o n s of t h e hybridized mixtures o f t h e hemoglobins by f r e e boundary e l e c t r o p h o r e s i s d i d n o t allow t h e i s o l a t i o n o f u s e f u l amounts o f hybrids and were abandoned i n favor o f chromatographic

s e p a r a t i o n s . I n t h e course o f t h i s work i t was found t h a t by allowing hemoglobin mixtures t o remain a t pH 11.0 f o r 24 hours a t 3 O C . a s i g n i -

f i c a n t t r a n s f e r of a c t i v i t y t o t h e previously u n l a b e l l e d hemoglobin would occur. I n t h e s e experiments t h e hemoglobins were l a b e l l e d as d e s c r i b e d i n t h i s Thesis and were separated chromatographically as described. The r e s u l t s of a t y p i c a l h y b r i d i z a t i o n experiment a f t e r d i s s o c i a t i o n a t pH 11.0 and t h e chromatographic s e p a r a t i o n of t h e hemo- g l o b i n s i n t h e mixture i s seen i n f i g u r e

8.

Also seen i n t h i s f i g u r e a r e t h e r e s u l t s of a chromatographic s e p a r a t i o n of an u n d i s s o c i a t e d mixture o f t h e same m a t e r i a l s . The s i c k l e c e l l hemoglobin, uniformly l a b e l l e d with @ 4 - ~ - ~ e u c i n e , and t h e u n l a b e l l e d normal a d u l t hemoglo- b i n used i n t h e s e experimentswere s u b s t a n t i a l l y f r e e d chromatographically of minor components p r i o r t o t h e s e h y b r i d i z a t i o n experiments. I n

f i g u r e 9 a r e shown t h e r e s u l t s o f chromatographic s e p a r a t i o n s o f d4-1- l e u c i n e l a b e l l e d normal a d u l t hemoglobin and u n l a b e l l e d s i c k l e c e l l

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-35 -

Table I X

Hybridization o f C' Labelled Normal Adult Hemoglobin with Unlabelled S i c k l e C e l l Hemoglobin

Net A c t i v i t y Net A c t i v i t y T o t a l A c t i v i t y a p t o No, Normal Adult S i c k l e C e l l Expected

(CPN (CPM) (CPM)

hemoglobin i n t h e absence o f h y b r i d i z a t i o n and a f t e r d i s s o c i a t i o n a t pH 5,0, I n experiments using l a b e l l e d normal hemoglobin i t w a s not necessary t o remove minor components p r i o r t o h y b r i d i z a t i o n s i n c e t h e s e minors do n o t contaminate t h e chromatograph of t h e s i c k l e c e l l hemo- g l o b i n f r a c t i o n , I n s p e c t i o n o f t h e chromatograms of t h e hybridized mixtures and comparison with t h e chromatograms of t h e unhybridized mixtures show t h a t t h e r e a r e no new chromatographic s p e c i e s formed as t h e r e s u l t o f chain exchange,* Observed, however, i s a t r a n s f e r o f r a d i o a c t i v i t y i n t o t h e i n i t i a l l y unlabelled hemoglobins as a r e s u l t o f t h e d i s s o c i a t i o n s ,

I n t a b l e X a r e summarized t h e r e s u l t s o f s e v e r a l h y b r i d i z a t i o n experiments, Experiments 1

-

⁷show t h e r e s u l t s of chain exchange a f t e r a l k a l i n e d i s s o c i a t i o n , while experiments 1 0

- 13

show t h e re- s u l t s o f subunit exchange a f t e r a c i d d i s s o c i a t i o n . Experiments

8, 9,

*The chromatography protocol s h e e t s from which t h e d a t a o f t h e s e f i g u r e s were taken a r e presented i n Appendix IV.

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(44)

(45)

Table X

R e s u l t s o f Hybridizakion Experiments

Expt

.

S p e c i f i c A c t i v i t y S p e c i f i c A c t i v i t y Percent

No

.

pH of Reactants of Products Hybridization

HbA HbS Hb A HbS

*Only samples of t h e most c o n c e n t r a t e d chromatographic f r a c - t i o n s o f t h e products were counted.

**Only t h e r e s u l t i n g HbS hybrid was assayed f o r r a d i o a c t i v i t y ,

***This hemoglobin mixture was allowed t o remain a t pH = 5.0 f o r 5 days.

and 1 4 demonstrate t h e r e s u l t s of chromatographicseparations of undis- s o c i a t e d hemoglobin mixtures, The s p e c i f i c a c t i v i t i e s given i n t h i s t a b l e a r e t h e averages o f s p e c i f i c a c t i v i t i e s of hemoglobin samples from chromatographic f r a