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Biochimica et Biophysica Acta, 580 (1979) 405--410 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 31285

F O R M A T I O N OF NUCLEI D U R I N G D E L A Y TIME P R I O R TO A G G R E G A T I O N OF D E O X Y H E M O G L O B I N S IN C O N C E N T R A T E D PHOSPHATE B U F F E R

KAZUHIKO ADACHI a, TOSHIO ASAKURA a and MICHAEL L. McCONNELL b

aThe Children's Hospital of Philadelphia, Department of Pediatrics and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104 and bChromatix, Inc., 560 Oakmead Parkway, Sunnyvale, CA 94086 (U.S.A.) (Received April 23rd, 1979)

Key words: Hemoglobin S; Polymerization; Nucleus; Preaggregation complex; Laser light scattering

Summary The delay time prior to aggregation of deoxyhemoglobin S was found to be shortened significantly if monomeric deoxyhemoglobin S was added near the end of the expected delay time. This suggests the formation of a preaggregation complex of deoxyhemoglobin S, probably so-called 'nuclei', near the end of the delay time. In fact, the molecular weight of d e o x y h e m o globin S as measured by a low angle laser light scattering p h o t o m e t e r increased exponentially during the delay time prior to the visible aggregation of deoxyhemoglobin S. These results support the nucleation-controlled aggregation mechanism for the aggregation of deoxyhemoglobin S in concentrated phosphate buffer.

The sickling of erythrocytes that is associated with sickle cell anemia is caused by the polymerization of d e o x y Hb S [1 ]. Kinetic studies of the gelation o f d e o x y Hb S or the sickling of red blood cells have shown the existence of a delay period prior to the onset of the polymerization process [2--9]. Hofrichter et al. [ 5] proposed from their kinetic studies that gelation or red cell sickling occurs in two different equilibrium stages, nucleation and fiber formation, and that a delay time is required for the production of nuclei. Although this hypothesis explains the unique kinetic behavior of Hb S gelation, little is known a b o u t the formation of nuclei or a b o u t the size and the nature of nuclei. Recently we found that diluted solutions of deoxygenated Hb S and Hb

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CHarlem in concentrated phosphate buffer aggregate with a clear demonstration of a delay period [12]. The length of this delay period depends on the hemoglobin concentration, phosphate concentration, and temperature [12, 13]. These phenomena are similar to those of the gelation of concentrated d e o x y Hb S solution. Since this procedure requires only one hundredth the amount of Hb S compared to the classic gelation technique, it is possible to use instruments that are suitable only for dilute protein solutions. In this communication we describe experimental results that suggest the formation of nuclei during the delay time. These results were obtained by studying the effect of the addition of monomeric Hb S at different points in the delay period and by measuring the molecular weight of Hb S molecules with a low angle laser light scattering p h o t o m e t e r [ 14]. Sickle hemoglobin was isolated by column chromatography on DEAESephadex [12] from blood drawn from patients who received exchange transfusion. Concentration of deoxyhemoglobin was determined spectrophotometrically using the millimolar coefficient, mEsss = 50 (tetramer basis). The kinetics of the aggregation of Hb S were measured turbidimetrically at 700 nm in an anaerobic photometric cuvette with a Perkin-Elmer 124D double beam s p e c t r o p h o t o m e t e r as described elsewhere [ 12]. Changes in the molecular weight of deoxy-Hb S due to the formation of nuclei during the delay time were measured with a KMX-6 low angle laser light scattering p h o t o m e t e r (Chromatix, 560 Oakmead Parkway, Sunnyvale, CA 94086). By utilizing a 633 nm helium-neon laser (2 mW), the fluorescence problem as well as problems due to sample absorption are minimized. Since the light scattering is measured at a low angle and at low hemoglobin concentrations, the molecular weight of hemoglobin can be calculated from the equation [14] 1 Mr =

Kc/Ro 2A: c

where c is hemoglobin concentration, -~0 is the difference between the Rayleigh factor of the solution and the Raleigh factor due to the solvent alone and A 2 is a viriat coefficient. The K value is an optical constant, dependent on the protein and angle of observation. We used K = 2 . 3 4 6 . 1 0 -7 ( m o l . c m 2.g-~) at 0 = 4.87 ° . Diluted solutions of d e o x y Hb S in concentrated phosphate buffer aggregate with a clear demonstration of a delay period [12, 13]. The length of the delay period and the amount of aggregates were determined by the change of turbidity at 700 nm. The higher the hemoglobin concentration, the shorter the delay time and the stronger the turbidity (amplitude). The delay time was also affected by phosphate concentration and temperature [12]. From these results, it was concluded that d e o x y Hb S in concentrated phosphate buffer aggregates according to the nucleation mechanism similar to that proposed for the gelation of concentrated d e o x y Hb S solution [4, 5, 1 0 ] . In order to determine if the soluble form of aggregates, the so-called nuclei, are actually formed during the delay time, the molecular properties of d e o x y Hb S during the delay time were investigated by t w o methods: (1) b y

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looking at the effect of additions of monomeric Hb S on the kinetics of the aggre~ ion of d e o x y Hb S at different points during the delay time and (2) b y measuring the change in the molecular weight of d e o x y Hb S with a low angle laser light scattering photometer. The kinetics of aggregation of two concentrations of d e o x y Hb S in 1.8 M potassium phosphate buffer, pH 7.34, are shown by d o t t e d lines in Fig. 1. In these experiments, samples of d e o x y Hb S were prepared at 0 ° C and were heated rapidly to 30 ° C [12]. The sample containing 90 mg/dl d e o x y Hb S aggregated after a delay time of a b o u t 5 min (dotted line A), while the sample containing 50 mg/dl d e o x y Hb S began to aggregate after a lengthy delay time of a b o u t 40 min ( d o t t e d line F). If nuclei are not formed during the delay time and if the kinetics of the aggregation of d e o x y Hb S depend only on the concentration of total hemoglobin, the additions of monomeric Hb S (40 mg/dl) into the diluted sample (50 mg/dl) at different points of the delay time should provide a kinetic curve similar to that of deoxy Hb S with a starting hemoglobin concentration of 90 mg/dl (dotted line A in Fig. 1 ). The kinetic curves, however, are strongly affected b y whether the additional Hb S is introduced at the beginning or the end of the delay time (Fig. 1). In these experiments, 10 pl of concentrated o x y Hb S (8 g/dl in 40 mM TrisHC1, pH 7.8) were introduced at 2, 11, 27 and 35 min after the start of the reaction. The oxy Hb S was monomeric and was deoxygenated instantly after introduction because the solution contained excess sodium dithionite (10 mg/2 ml). The results show that the delay time was affected strongly by the time at which the Hb S was added (Fig. 1, solid lines). If oxy Hb S was added 2 min after the reaction was started, the d e o x y Hb S, which then had a concentration of 90 mg/dl, aggregated after 2 min delay time. In contrast, if the same a m o u n t of oxy Hb S was added at the 35 min point, the d e o x y Hb S aggregated w i t h o u t showing any further delay time. These results suggest that the states of d e o x y Hb S are different at the beginning and the end of the delay period. It is more likely that nuclei are formed at the end of the delay period; therefore, the addition of 40 mg/dl of monomeric Hb S caused an instant aggregation. Of course, the delay time and the degree of aggrega-

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Fig. 2. E f f e c t o f t h e a d d i t i o n o f d i f f e r e n t a m o u n t s o f H b S o n the aggregation of d e o x y H b S d u r i n g t h e d e l a y t i m e . S o l u t i o n s (2 m l ) c o n t a i n i n g 50 m g / d l d e o x y H b S a n d 1 0 m g s o d i u m d i t i o n i t e in 1.8 M potassium p h o s p h a t e b u f f e r , p H 7 . 3 4 , w e r e h e a t e d r a p i d l y f r o m O°C t o 3 0 ° C . D i f f e r e n t a m o u n t s o f c o n c e n t r a t e d o x y H b S (8 g ] d l in Tris-HC1, 4 0 m M, p H 7 . 8 ) w e r e i n t r o d u c e d a t t h e 2 5 m i n p o i n t o f d e l a y t i m e t o t h e s o l u t i o n . T h e f i n a l h e m o g l o b i n c o n c e n t r a t i o n s o f e a c h s a m p l e w e r e A, 1 2 0 m g ] d l . ; B, 9 0 m g ] d l ; C, 8 0 m g / d l ; D, 7 0 m g / d l ; a n d E, 6 0 m g ] d l .

tion depend on the a m o u n t of d e o x y Hb S added. As shown in Fig. 2, the delay time was shortened and the intensity of the turbidity increased if higher concentrations of Hb S were added. To determine if the 'nuclei' were formed during the delay time, the molecular weight of d e o x y Hb S was measured directly with a low angle laser light scattering photometer. The low angle laser light scattering p h o t o m e t e r is known to be a sensitive means to measure changes in the molecular weight of dissolved proteins. If nuclei are formed during the delay time, the molecular weight of d e o x y Hb S should increase prior to the aggregation which can be detected by a spectrophotometer. Before the low angle laser light scattering p h o t o m e t e r was used for investigating the nuclei formation of d e o x y Hb S, the instrument was used to determine the molecular weight of Hb A dissolved in 0.18 M potassium phosphate buffer, pH 7.34. Since the dust particles in the 'viewed' volume produce spikes, or noises, the hemoglobin was flowed (0.25 ml/min) through a millipore filter (pore size 0.22 pro). The molecular weight of d e o x y Hb A under these conditions was calculated as 66 000 in 0.18 M phosphate buffer by extrapolating the hemoglobin concentration to zero. For experiments with deoxy Hb S the solution containing d e o x y Hb S was passed through a 0.44 gm millipore filter immediately before the measurement at 24 ° C. As shown in Fig. 3A, the laser light scattering intensity, G(O ), increases exponentially with time, indicating that the molecular weight of d e o x y Hb S increases rapidly during delay time at room temperature (24 ° C). Similar experiments with Hb A showed no such change, and even for d e o x y Hb S, no scattering change occurred if the experiment was done at 3.1°C (Fig. 3B and C). These results clearly indicate that the changes detected by t h e low angle laser light scattering m e t h o d during the delay time are related to the aggregation of d e o x y Hb S, a demonstration of nuclei formation. Although the nucleation controlled mechanism for the aggregation of d e o x y Hb S has been well accepted, little work has been done to demon-

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Fig. 3. Light scattering of d e o x y h e m o g l o b i n s in 1.9 M pot a s s i um phosphate buffer, pH 7.34. A, de oxy Hb S 41 rng/dl at ro om t e m p e r a t u r e (24~'C); B, d e o x y Hb A 57 mg/ dl at room t e m p e r a t u r e (24°C); C, d e o x y Hb S 57 mg/dl at 3.1°C.

strate the presence of 'preaggregated deoxy Hb S' or 'nuclei' during the delay time. Waterman and Cottman [ 15 ] suggested by measuring transverse water proton nucleation time that no polymer formation occurs during the delay time. Elbaum et al. [16] reported the self-association of diluted deoxy Hb S by Rayleigh light scattering in low phosphate buffer. Wilson et al. [17] observed the linear increase in the intensity of the quasielastic light scattering during pregelation of deoxy Hb S and suggested the linear condensation mechanism in which any size polymer can combine with any other size polymer. The kinetic experiment shown in this paper suggests that the equilibrium states of the preaggregation of deoxy Hb S are different with time during the delay period. The shortening or lack of further delay time when deoxy Hb S is added at the end of expected delay time suggests that nuclei are formed during the delay time. More direct evidence for the existence of nuclei is provided by the low angle laser light scattering experiment. The intensity of the G(O ) of deoxy Hb S increases exponentially with time (Fig. 3A), suggesting that the association of deoxy Hb S occurs during the delay time. Subsequent aggregation will occur by the addition of monomeric hemoglobin to the nuclei [18]. The dependence of the delay and aggregation times upon the concentration of additional hemoglobin monomers suggests that the aggregation reaction after nucleation may be controlled by the equilibrium between monomers and nuclei of deoxy Hb S. These results support the nucleation-controlled mechanism for the aggregation of deoxy Hb S in concentrated phosphate buffer [12, 13] which was proposed for the mechanism of gelation of deoxy Hb S by Hofrichter et al. [4, 5]. This work was supported by Grants HL-20750 and H1-18226 from the National Institutes of Health. We are grateful to Dr. Elias Schwartz for help-

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ful discussions, to Phillip. J. Christ for skillful technical assistance, to Janet Fithian for editorial assistance and to Billie Corbett for secretarial help in the preparation of the manuscript.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Perutz, M. a n d Mitchison, J.M. ( 1 9 5 0 ) N a t u r e 166, 6 7 7 - - 6 7 9 Malfa, R. a n d S t e i n h a r d t , J. ( 1 9 7 4 ) B i o c b e m . Biophys. Res. C o m m u n . 59, 8 8 7 - - 8 9 3 Harris, J.W. a n d Bensusan, H.B. ( 1 9 7 5 ) J. Lab. Clin. Med. 86, 5 6 4 - - 5 7 5 H o f r i c h t e r , J., Ross, P.D. a n d E a t o n , W.A. ( 1 9 7 4 ) P r o c e e d i n g s of the First N a t i o n a l S y m p o s i u m on Sickle Ceil Disease, B e t h e s d a , DHEW P u b l i c a t i o n No. (NIH) 7 5 - - 7 2 3 , p. 43 H o f r i c h t e r , J.~ Ross, P.D. a n d E a t o n , W.A. ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 71, 4 8 6 4 - - 4 8 6 8 Ross, P.D., H o f r i c h t e r , J. a n d E a t o n , W.A. ( 1 9 7 7 ) J. Mol. Biol. 115, 1 1 1 - - 1 3 4 Moffat, K. a n d Gibson, Q.H. ( 1 9 7 4 ) B i o c h e m . Biophys. Res. C o m m u n . 61, 2 3 7 - - 2 4 2 E a t o n , W.A., H o f r i c h t e r , J., Ross. P.D., T s e h u d i n , R.G. a n d Becker, E.D. ( 1 9 7 6 ) Biochem. Biophys. Res. C o m m u n . 69, 5 3 8 - - 5 4 7 R a m p l i n g , M.W. a n d Sirs, J . A . ( 1 9 7 3 ) Clin. Sci. Mol. Med. 45, 655---664 Minton, A.P. ( 1 9 7 4 ) J. Mol. Biol. 82, 4 8 3 - - 4 9 8 M i n t o n , A.P. ( 1 9 7 5 ) J. Mol. Biol. 95, 2 8 9 - - 3 0 7 A d a c h i , K. a n d A s a k u r a , T. ( 1 9 7 8 ) J. Biol. Chem. 253~ 6 6 4 1 - - 6 6 4 3 Adachi, K. a n d A s a k u r a , T. ( 1 9 7 9 ) J. Biol. Chem., in the press K a y , W. ( 1 9 7 3 ) Anal. Chem. 45, 2 2 1 A - - 2 2 5 A W a t e r m a n , M.R. a n d C o t t m a n , G.L. ( 1 9 7 6 ) Biochem. Biophys. Res. C o m m u n . 73, 639---645 E l b a u m , D., Nagel, R.L. a n d Herskovits, T.T. ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 7 6 5 7 - - 7 6 6 0 Wilson, W.W.~ L u z z a n a , M.R., P e n n i s t o n , J.T. a n d Charles, S.J., Jr. ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 71, 1 2 6 0 - - 1 2 6 3 Oster, G. ( 1 9 4 7 ) J. Colloid. Sci. 2, 2 9 1 - - 2 9 9

Formation of nuclei during delay time prior to aggregation of deoxyhemoglobin S in concentrated phosphate buffer.

405 Biochimica et Biophysica Acta, 580 (1979) 405--410 © Elsevier/North-Holland Biomedical Press BBA Report BBA 31285 F O R M A T I O N OF NUCLEI D...
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