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Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn)
J. Mol.
Biol.
144, 467-480
(1980)
Polymerization
of Deoxyhemoglobin Carlem (86 Glu -+ Val, 873 Asp -+ Asn)
The Effect of 873 Asparagine
on the Gelation Hemoglobin
and Crystallization
of
KAZUHIKO ADACHI AND TOSHIO ASAKURA
University
!Phe Children’s Hospital oj Philadelphia Department of Pediatrics Department of Biochemistry and Biophysics of Pennsylvania, Philadelphia, Penn. 19104, IJ.S.A.
(Received
9 June 1980, and in revised form 5 August
1980)
The kinetics of aggregation and the solubility of deoxy Hbt C,,,,, (a& 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of agg re gated with a clear exhibition deoxy Hb S and deoxy Hb A. Deoxy Hb C,,, of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration. The initial hemoglobin concentration required for the aggregation of deoxy HbC uarle. was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb CHsrlemwas approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time Z~~T+SU.S hemoglobin concentration in 1% M-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 40 for deoxy Hb Cm,,-. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb CHarle,,,were unchanged with phosphate concentrations varying from 1.2 M to 2.0 M. The by solubilities of deoxy Hb S and deoxy Hb CHsrlemwere increased exponentially lowering the pH of the medium, with the increase being more conspicuous for Hb Gurw The gels (or aggregates) of Hb C,,,,, were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb CHsrlemcan be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomeric deoxy Hb
4 nuclei G e polymer ti gel *nuclei C *polymer ti crystals
The hemoglobin concentration required for the crystallization of deoxy Hb ‘Amem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb CHsrlemafter aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the 873 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C. t Abbreviations
used: Hb, hemoglobin;
w2-2836/36/366467-14 $02.66/O
Hb Cn, hemoglobin 467
CHarlam.
0 1980 Academic Press Inc. (London)
Ltd
MX
1. Introduction (HI) (‘“) has amino acid mutations at the /IS ((iI 4 I’al) and the p73 Hb (“nartrm (=\sp -+ r\sn) positions. Although the former substitution is identical to that iti Hb S. deoxy Hb PH forms gels less readily than does deoxy Hb S. The minimum gelling concentration (MM”) of deoxy Hb CyHis 36.2 g/d1 in cont,rast to 24.0 g/d1 for, droxy Hh S (Bookchin d al., 1967: Bookchin & 9agel. 1971). indicating that the* second amino acid mutation (873) in Hb PH has an antigelling eff&t. Structural studies of the gels and crystals of deoxy Hb S have shown that thra 873 position is in the contact region. which is complementary t,o the ,96 position in thr. molecule (Wishner d nl.. 1975: Love rt al.. 1978). The difference in the solubility of droxy Hb (‘” from tha,t of deoxy Hb S and the inhibitory effect of th(b deox!. forms of Hb CTHand Hb Korle Bu on t,he gelation of deoxy Hh S (Bookchin c)tnl.. 1967. Bookchin & Bagel, I97 1) also support the idea that the 873 residues are involved in the gelation of deoxy Hb S. Recently. we found that deoxy H b .4 forms gels when a solution of deos~, HI) .\ with a concentration
of ahout
lS(&,
of its solubilit~
is warmed
to 31’(
(Ada&i
k
Xsakura.
1979c). From this we concluded that, the hydrophobic amino wid substit,ut,ion (\-at) at the /36 position is not essent,ial for the gelabion of hemoglobin but, that this substitution accelerates the formation of gels and st)abilizrs thtx conformation of deoxy Hb S gel. We also found that aggregates or gels of tieox> Hb A-Iwere converted to t,hree-dimensional crystals over a period of days (.-\dachi k ,\sakura. 1979c). This period is much shorter than that (3 mont,hs) for crystallization of deoxy Hb S gels (Wishner rt nl.. 1975.1976: Hofrirhter r,f UJ 19766). Kinetic studies of the aggregation and crystallization of deoxy Hb X and deoxy Hb S in concentrated phosphate buffers suggested that t’he size of nut-ltv produced during the delay period is reduced in concentratjrd phosI)hat,e buff&t (.\dachi & &akura. 19796) and that the nuclei for gels and rrystals are differt’nt (,Adachi & &akura. 1980). Although details of the structure of these nuclei are not yet known. their nature may be clarified by studies of abnormal hemoglobins. particularly those that have an amino acid substitution at t,he contact region and affect the gelation of deoxy Hb S. We report here the results of stlldies on this kinetics of the aggregation and crystallization of deoxy Hb (
2. Experimental Procedures Hh C’, was purified from Hb 8(“, blood by the standard chromatographic trchnicloc> OII (‘M-Sephadex and was identified by fingerprmts after tryptic and chymotrypbic digestions and amino acid analysis (Adachi et al., 198Oa). Hb S was purified from .4S blood by column chromatography on DEAE-Sephadex as described elsewhere (Adachi &r Asakura, 1979b). The concentration of hemoglobin was determined spectrophotometrically by the use of millimolar extinction coefficients of mE 555 = A0 for deoxyhemoglobin and mE,,, = ;?3% for carbonmonoxyhemoglobin (on a tetramer basis). Kinetic studies of the aggregation and de-aggregation of hemoglobins were rarrird out turbidimetrically as described elsewhrrt-
POLYMERIZATION
OF DEOXYHEMOGLOBIS
(“Har,em
ais
(Ada&i & Asakura, 1979u,b). The solubihty of hemoglobin was determined by measuring the concentration of dissolved hemoglobin in the supernatant after centrifugation. The pH of concentrated phosphate buffer was determined with a pH electrode after a l@fold dilution of the solution.
3. Results (a) Effect of pH on the kinetics ofaggregation of deoxy Hb Sand deoxy Hb C, in 1.8 Mphosphate
buffeer
Like deoxy Hb S and deoxy Hb A (Adachi & Asakura, 1979b,c), deoxy Hb Cs aggregated with a clear exhibition of a delay time when solutions of an appropriate
concentrat’ion were incubated at 30°C in 1% &f-phosphate buffer. The length of the delay and aggregation times and the degree of aggregation (measured as Al,OO) depended upon the initial hemoglobin concentration; the higher the concentration of Hb (yn>the shorter the delay and aggregation times and the greater the turbidit,y increase. The aggregates could be reliquefied by cooling. The major difference between the aggregation of deoxy Hb C, and that of deoxy Hb S is the relationship between the delay time and the hemoglobin concentration. For aggregation. a higher concentration
of deoxy
Hb C, is required
than of deoxy
Hb S. For instance,
a solution of deoxy Hb S with a concentration of 75 mg/dl exhibits a delay time of about ten minutes in 1.8 M-phosphate (pH 7.36) at 3O”C, whereas a concentration of 290 mg/dl of deoxy Hb Cn is required for the same length of delay time under the same experimental conditions (Fig. l(a)). In other words, if we compare the lengths of delay times for solutions of deoxy Hb C, with those of the same concentrations of deoxy Hb S in 1.8 M-phosphate buffer (pH 7.36), at 30”(‘, the delay time for deoxy Hb Cn is approximately 10’ times longer than that of deoxy Hb S (Fig. I (a)). The logarithmic plotting of the delay time versus hemoglobin concentration in 1% M-phosphate buffer (pH 7.36) shows linear lines with slopes (n-value) of 2.7 for deoxy Hb S and 3.9 for deoxy Hb Cn (Fig. l(a)). To investigate whether this difference can be attributed to the difference in the electrostatic bond involved with COO- of the 1373 asparatic acid, experiments were carried out at four different pH values in l-8 M-phosphate buffer at 30°C. As shown in Figure 1, more concentrated hemoglobin solutions are required for the aggregation of both deoxy Hb S and deoxy Hb Cn at lower pH values. There is a linear relationship between the log of reciprocal delay time and hemoglobin concentration. Under the same pH condition, the lines for Hb Cn lie always to the right side of those for Hb S, indicating that a more concentrated hemoglobin solution is required for the aggregation of deoxy Hb C, to reveal the same length of delay time as compared to Hb S. The slopes (n values) for the delay time of deoxy Hb S and deoxy Hb C, decrease with increase in pH (Fig. l(b)). The aggregation time (tr - td) is also inversely related to hemoglobin concentration and affected by the pR (broken lines in Fig. l(a)). The slopes (n-values) of deoxy Hb Cn are 3.7, 3.9 and 55 at pH 8.0, 7.36 and 7.0, respectively (Fig. l(a)). The difference in n values for delay times between deoxy Hb C, and deoxy Hb S is greater at higher pH values (Fig. l(b)).
Ii.
170 O-
pH 8.01 Hb
S
ADAPHI
AND
‘I’. ASABUKA
8.01
7.36 S
CH
7.04 s
8’ I n:2a7 i-
.' rn’d, , 0’
736 Cl+
6.58 S
7.01 CH
d
,I
n=5.5/n:3.9/
1x6.5 /
, n-5.5
.-,I , , , ^-Z.V . a; I, I I I I 1 I I I I I I I 1 I I, I I I -2.5
-1.5
-2.0
-1-O
-0.5
0
log G (g/d11 (a) IO-
\ OX
\ ‘-,d -->
._.-•
a-
-0
-e -----.
Hb CH Hb S
0
I
I 7-O
I
I 0.0
PH (b)
FIG. l(a). Effect of pH on the kinetics of the aggregation of deoxy Hb S and deoxy Hb C, in 1.8 Mphosphate. Hemoglobin solution of various concentrations ((0, n ) Hb 8: (0, 0) Hb C,) at, different pH values were heated from 0°C to 30°C at time zero. The delay times (td) (solid lines) and the aggregation times (tTtd) (broken lines) were measured spectrophotometrically by recording t,he absorbance at 700 nm. - n -- w - , Delay time (td) of deoxy Hb S ; --O---O-P, aggregation time of aggregation time (If-td) of deox? deoxyHbS;-~--~-,delaytime(t,)ofdeoxyHbC~;--O--~----, Hb CH. l/r, reciprocal delay and aggregation times; C,, Hb concn. (b) The relationship between n values and pH. The data for deoxy Hb S and deoxy Hbt’, in 1% M-phosphate buffer are derived from the data in (a).
(b) Effect of pH on the solubility
of deoxy Hb S and deoxy Hb CL,
The solubilities of the oxy and deoxy forms of hemoglobins are known to depend upon the pH of the medium (Singer & Singer, 1953 ; Magdoff-Fairchild et al., 1976 : Goldberg et al., 1977). In contrast to the increase in the MGC and the solubility of deoxy Hb S at higher pH values, the solubility of deoxy Hb S in concentrated phosphate buffer decreases at higher pH values. To investigate the effect of the replacement of Asp by Asn in Hb (1” on its solubility, the solubilities (the supernatant hemoglobin concentrations) of deoxq Hb S and Hb CH at different pH values were determined after the aggregation
POLYMERIZATION
OF DEOXYHEMOGLOBIN
CHarlem
471
P’-’
FIG. 2. Effect of pH on the solubility of deoxy Hb 8 and deoxy Hb CH in 1.8 M-phosphate buffer at 30°C. The solubility was determined by measuring the concentration of soluble hemoglobins in the supernatant after completion of aggregation. Experimental conditions are the same as those shown in Fig. l(a).
.c E
-3-
-5
-
-6-
11 I/T(x103)(K-‘)
FIG. 3. Effect of temperature on the delay time of deoxy Hb C, aggregation in 1% M-phosphate buffer (pH 7.4). Hemoglobin (293k5.7 mg/dl) in 1.8 M-phosphate buffer (pH 7.36) was heated from 0°C to various temperatures. Results are the average of results of 2 to 3 experiments at different temperatures.
reaction had reached a plateau. The solubilities of both deoxy Hb S and deoxy Hb C, increase exponentially as the pH of the medium is decreased, with deoxy Hb C, showing greater increases at lower pH values (Fig. 2). (c) Effect of temperature The kinetics of the aggregation of deoxy Hb S are highly temperature-dependent’ (Hofrichter et al., 197ti,b; Pumphrey & Steinhardt, 1977; Adachi & Asakura, 19793). The lower the temperature, the longer both the delay and aggregation times. A similar relationship is observed for the aggregation of deoxy Hb C, (Fig. 3). The apparent activation energy for the aggregation of deoxy Hb C, in
17%
K. .4DA(‘HI
ASD
‘I’. .ZS.AKC’KA
I.8 hr-potassium phosphate (pH 7.36), calculated from the results showy in Figure 3, ranges from about 25 kcal/mol near 30”(’ t,o as high as 65 kcal/mol near 15°C. These values for the delay time of droxy Hb ( lH are slightly higher than those for pure deoxy Hb S (Adachi K- =\sakura. 19798).
((i)
Effect
of phosph,atp
cotwratratiott
As reported previously, (Ada&i K- Asakura; 1979h). there is a linear relationshil~ between the n value and the phosphate concentration. The I/ values for deoxy Hb S were 2.X. 76 and 155 in 1.X. 1.5 and 1.0 nl-phosphate buffers, while those for deoxy Hb A were 2.3, .56 and 12 in 2.1. 1.8 and 1.2 M-phosphate buffers, respectively (Adachi & Asakura. 1980). From these results we concluded that the sizes of nuclei decrease as the concentration of phosphate buffer is increased. Similar showed experiments with deoxy Hb (‘(a in four different phosphate concentrations in that the n value for Hb Cs was about 3-. and that this value was unchanged phosphate buffers (pH 74) with concentrations bet,ween 1.2 M and 2.1 M (Fig. 4(a) and (b)). These results suggest that the sizes of nuclei of deoxy Hb (‘u are unchanged in these phosphate buffers. The aggregation times (tf- td) for deoxy Hb (‘n also show linear curves in 2.1 41 and 1.8 $f-phosphate buffers (open circles in Fig. 4(a)). but those in 1.5 37and 1.2 Nphosphate buffers do not show linear curves. as the values fluctuate widely in these buffers.
(e)
Ibuggregation
of aggregates
of deozy
Hh
(If,
by cooling. bubbling with carbon Polymers of deoxy Hb (‘u can be reliquefied monoxide gas or oxygen, and dilution of the phosphate buffer, as can aggregates of deoxy Hb S (Adachi $ Asakura, 1979b). The de-aggregation curve for deoxy Hb (‘u is similar to those for deoxy Hb A and deoxy Hb S (Fig. 5), suggesting similar structures for aggregates of these deoxyhemoglobins formed by the nucleation It should be pointed out that the decontrolled aggregation mechanism. out in aggregation curves of aggregates of Hb C, formed by simple salting phosphate buffer above 2.4 M are significantly different from those of deoxy Hb r\ 1979a; Adachi et al.. 19806). Aggregates and deoxy Hb S (Adachi & Asakura: formed according to the nucleation-controlled mechanism appear to differ from those produced by t’he simple salting-out mechanism (Adachi 8i Asakura. 1979a). (f) (m’on,t~ersiott, of the aggregates
(gels)
of deoxy
Hb C, to crystalline
forms
It has been reported that gels of deoxy Hb S are converted to suspensions ot three-dimensional crystals after standing for several months (Wishner et al.. 1975,1976 : Hofrichter et al., 19766). We found that aggregates (or gels) of deoxy Hb $, oxy Hb S and carbonmonoxy Hb S also were converted to crystals, but after only a day or two of standing at 30°C (Adachi & Asakura, 1980). Deoxy Hb Cn formed crystals even more rapidly than the above hemoglobins. In l-5, l-8 and
POLYMERIZATION
OF DEOXYHEMOGLOBIS
CHsrlem
I.8 M
2.0 M phosphate
473
I.5 M
1.2M
’ /
.’
-’
-2.51,
-,
,
,
, -1.5
,
,
,
,
, -1.0
,
/
,
,
,
, -0.5
,
,
,
,
i
, 0
,,, , “0.4
,
,
,
I 0,
log 6 (g/d11
309 \
c
\
\
\
\
\
\ ’ ,Hb S \ ‘0
15-
\
\ ‘\
\
Hb CH -o-oI 0
‘,Hb ‘0,
I I.0
A
\ \
‘\
AL R”LA
1
Phosphate
( M)
(b)
FIG. l(a) Relationship of reciprocal delay time and aggregation time to hemoglobin concentration in different phosphate concentrations. Phosphate concentrations used were 2.1 M, 1.8 M, 1.5 M and 1.2 M (pH 7.4) as shown in the Figure. (0) Delay time: (0) aggregation time. (b) Relationship between 12value and phosphate concentration. The n values of deoxy Hb S (0) and deoxy Hh A (A) are from our previous papers (ildachi & Asakura, 1979b,c).
2.1 M-phosphate buffers, aggregates formed and upon further incubation were converted to crystals. In 1.2 M-phosphate buffer, amorphous aggregates and crystals were formed simultaneously (Fig. 6), suggesting that crystallization of deoxy Hb CH is as rapid as gelation in 1.2 M-phosphate buffer. The crystallization of deoxy Hb C, required a longer period of time in higher phosphate concentrations. For instance, a few days of incubation at 30°C (2 to 3 days) are necessary for the conversion of gels to crystals, in 1.8 M-phosphate buffer, whereas incubating for a few hours or overnight results in the crystallization of deoxy Hb C, in 1.5 Mphosphate buffer. A typical example of the aggregation curve of deoxy Hb C, in 1.5 M-phosphate buffer at 30°C is shown in Figure 7. In this reaction, deoxy Hb C, aggregated after a delay time of about 40 minutes. After aggregation, the cuvette
K. ADACHI
474
loo-
l ,\ \
\
AND
T. ASAKI-KA
\\ ‘\\ s
\ o-
, I4
I Phosphate
, ‘0’ I.6
A,
(M)
FIG. 5. De-aggregation of aggregates of deoxy Hb A, deoxy Hb S and deoxy Hb Cu in 1.8 M-phosphate buffer (pH 7.4). Hemoglobin solutions (deoxy Hb S, 0% g/dl: deoxg Hb ii. 240 g/dl: and deoxy Hb tyH 0.190 g/dl) in 1.8 M-phosphate buffer (pH 7.4) were heated from 0°C to 30°C at t,ime zero: after completion of aggregation. the aggregates were melted by dilution of phosphate molarity with the addition of water at 30°C.
was shaken several times and incubated further at 30°C. The second reaction, crystallization, started after 20 minutes further incubation after the first reaction, as indicated by a steep increase in absorption at 700 nm. If the cuvette was not shaken, an overnight incubation was required for crystallization, indicating that shaking accelerated the formation of crystals. The biphasic curves determined turbidimetrically for the aggregation and crystallization reactions can be seen in Figure 7. Unless the experiment was done in phosphate buffer lower than 1.2 M. these two processes were clearly separate. Microscopic observation of the aggregates formed in the first reaction revealed the presence of only amorphous aggregates, while after the second reaction a number of crystals were seen in addition to aggregates. We could see optical birefringence for both amorphous aggregates and crystals of deoxy Hb CH. (g) S’olubility
of deoxy Hb C, before and after crystallization
We reported that the solubilities of deoxy Hb A, deoxy Hb C, oxy Hb A, oxy Hb S, carbonmonoxy Hb A and carbonmonoxy Hb S determined by measuring the hemoglobin concentration in the supernatant at the plateau of the aggregation curve were dependent upon the initial hemoglobin concentration (Adachi & Asakura, 1979b, 1980). On the other hand, the solubilities of deoxy Hb S in the polymerization reaction in I.8 M-phosphate buffer are apparently independent of the initial hemoglobin concentration (Fig. 8). The solubility of deoxy Hb C, in 1.8 M-phosphate and that of deoxy Hb S and deoxy C, in 15 M-phosphate measured after aggregation showed a dependency upon the initial hemoglobin concentration ; this dependency of deoxy Hb Cn was greater than that of deoxy Hb S in I.5 Mphosphate buffer. If the solubility of Hb CH was determined after the
FIG. 6(a) and (b). Photomicrographs of gel and crystals of deoxy Hb C, in 1.2 M-phosphate buffer (pH 74) at 30°C ( x 40). The cuvette containing chilled Hb c, solution (4.5 g/dl) in 1.2 M-phosphate buffer (Na2S,0.,, 5 mg/ml) was heated rapidly to 30°C. A gel that is the mixture of aggregates and crystals formed after about 10 min incubation at 30°C.
Time (min)
FIG. 7. Aggregation and crystallization curve of deoxy Hb C, (071 g/d]) in 1.5 M-phosphate buffer. Aggregation and crystallization of deoxy Hb C, was determined turbidimet,rically at 700 nm 1
Initial
Hb concn (g/dl)
FIG. 8. Relationship between the solubility of deoxy Hb (lH*r,em and the initial hemoglobin concentration after aggregation and crystallization in 1.8 M and 1.5 M-phosphate buffer (pH 7.4). The solubility w&s determined by measuring the concentration of soluble hemoglobin in the supernatant after completion of the aggregation and after crystallization.
crystallization reaction, the value was much lower and the solubility was independent of the initial hemoglobin concentration (Fig. 8). Thus, the solubility of hemoglobin varies widely, depending upon whether or not it is measured after aggregation or crystallization. The solubility after aggregation by the simple salting out method was different from that after aggregation by temperature-jump method (Adachi et al., unpublished data).
4. Discussion (a) Effect of the 873 Am on the aggregation
of Hb C,
Previous studies have shown that the 873 Asp is involved in gelation. since mutation at this position affects the MGC (Bookchin et al., 1967,197O: Bookchin & Nagel, 1971). The 873 Asp + Asn substitution also affected the kinetics of aggregation and the solubility of deoxy Hb Cn in concentrated phosphate buffer:
POLYMERIZATION
OF DEOXYHEMOGLOBIN
CHarlem
411
the aggregation reaction was inhibited and the solubility after aggregation was increased. Studies on the effect of pH on the solubility and the kinetics of aggregation of deoxy Hb S and deoxy Hb Cu showed that the solubilities of both deoxy Hb S and deoxy Hb C, increased exponentially as the pH of the medium was decreased (Fig. 2), and that the inhibitory effect on gelation of the Asp + Asn substitution at the j373 position was greater at lower pH values (Fig. 2). Since the carboxyl group of Asp is protonated at lower pH values, it seems unlikely that an electrostatic bond at the )573 position is involved in this difference (Nagel & Bookchin, 1975). Instead, it is more likely that the Asp + Asn substitution affects hydrogen-bonding of Asp with an adjacent amino acid residue (/?4Thr) on the deoxyhemoglobin molecule, as indicated by the results of X-ray diffraction studies by Wishner et al. (1975). The pH dependency of the aggregation in concentrated phosphate buffer of deoxy Hb S and deoxy Hb C, is the opposite of that in low phosphate buffer (Briehl Kr.Ewert, 1973; Magdoff-Fairchild et al., 1976; Goldberg et al., 1977). Briehl (1976) pointed out that, in studies of solubility in high phosphate buffers, the increasing concentration of HPO:- is responsible for the enhanced condensation rather than the pH itself, since it is HPOi- rather than H,PO, which is primarily responsible for salting out proteins. Our experimental data for deoxy Hb S and deoxy (1, support this hypothesis. (b) Gelation ana’ crystallization
of deoxy Hb C,
Wishner et al. (1975,1976) and Hofrichter et al. (1976b) reported that gels of deoxy Hb S were converted slowly to a suspension of three-dimensional crystals over a period of several months. We found that gels of deoxy Hb A formed in concentrated phosphate buffers were converted to crystals at a rate much faster than those of deoxy Hb S (Adachi & Asakura, 1979c). As shown in this paper, the gel-to-crystal conversion in deoxy Hb C, was even faster than that in deoxy Hb A. These results indicate that crystals of all of these hemoglobins were thermodynamically more stable than gels. We proposed the following scheme for the formation of gels and crystals (Adachi et al., 1980b).
KG
/( nuclei G + polymer $ gel Monomeric deoxy Hb$ nuclei C , ;=t polymer $ crystals
(1) (2)
KC
In this scheme, nuclei G and nuclei C are formed before gelation (or aggregation) and crystallization, respectively. KG and KC are the apparent association constants of the reaction between monomeric hemoglobin and each type of nuclei. This scheme appears to explain the gelation and crystallization of various hemoglobins. Deoxy Hb S has a KG value much higher than that of other hemoglobins. As a result, nuclei G are the more easily formed, and once a sufficient amount of nuclei G have been formed monomeric deoxy Hb S binds to the nuclei to form large aggregates (polymers). The substitution of valine for glutamic acid at the /?S position appears to increase the K, value so strikingly that nuclei G can be produced even where the concentration of deoxy Hb S is only l/40 that of deoxy
178
K. ADACHI
AND
T. ASAKt~rKA
Hb A in 1.8 M-phosphate buffer. The substitution of asparagine for aspartic acid at, the j373 position appears to decrease the KG value, as this additional substitution inhibits gelation: one needs a two or three times more concentrated solution of deoxy Hb C, than of deoxy Hb S to initiate gelation. It is interesting that the substitution of lysine for glutamic acid at the /36 position in deoxy Hb c’ (86 Glu --) Lys) also appears to decrease the KG value, as this substitution markedly accelerates the crystallization of deoxy Hb C (Adachi & Asakura, 1980). Thus, the KG values decrease in the order: Hb S, Hb c‘,, Hb 4 and Hb C. The crystallization reaction is also accompanied by a delay time. The mechanism of this reaction is different from that of gelation because the initial concentration required for crystallization is much smaller than that for gelation, the delay time is usually much longer than that before gelation, and the activation energy for crystallization is greater than that for gelation (Adachi & $sakura, 1979c,1980). The size of a nucleus for crystals estimated from the n value is different from that, for gels in concentrated phosphate buffer (Ada&i & Asakura, 1980). Both KG and K, are specific to each hemoglobin and are affected by various factors including pH, ionic strength. type of salt and temperature. Whether a hemoglobin forms gels or crystals depends upon the relative values of KG and K, and the rate of the formation of nuclei G and nuclei C. It is also related t)o the concentration of monomeric hemoglobin, the so-called critical hemoglobin concentration, which depends upon the surface structure of deoxyhemoglobin and the conditions of the medium. The difference in the critical concentrations for gelation and crystallization is attributed to the great difference in the solubilities of deoxyhemoglobin that are found either after gelation or after crystallization. As reported previously (Adachi & Asakura, 1979c,1980), the solubility of hemoglobin measured after crystallization (S,) is manyfold lower than that measured after gelation (S,j except in deoxy Hb S. The solubility of deoxy Hb 8 after aggregation is exceptionally low compared with that of other hemoglobins and the value does not differ much from the solubility of deoxy Hb S measured after crystallization (Perutz & Liquori, 1951; Pumphrey $ Steinhardt. 1977). This indicates that the concentration of soluble monomeric deoxy Hb S after gelation (or aggregation) is too low for deoxy Hb S to form nuclei C. accounting for the extremely slow conversion of gels of deoxy Hb S to crystals. It should be pointed out that the So of deoxy Hb (‘u depends upon the initial hemoglobin concentration, with a higher )so at, higher initial hemoglobin concentration (Fig. 8), suggesting that the formation of gels does not represent a true equilibrium state (Adachi & Asakura, 1979c). Since the rate of crystallization depends upon So, the rate of crystallization is increased when the initial concentration of deoxy Hb C, is high. The dependence of So upon the initial hemoglobin concentration may be explained by the presence of monomers, oligomers and polymers, particularly during the delay and aggregation times (Adachi & Asakura, 1980). Thus, So corresponds to the total soluble hemoglobin. which is the sum of the concentration in these mixtures. In contrast, after crystallization, the solution contains mainly monomeric hemoglobins, and this value is unaffected by the initial hemoglobin con’cenbration and is specific to each
POLYMERIZATION
OF DEOXYHEMOGLOBIN
GHar,ern
179
hemoglobin. This value may be the true solubility of monomeric hemoglobin. Thus, conversion of gels to crystals of deoxy Hb Cu can be explained as follows : the liquid phase after aggregation is a mixture of monomeric hemoglobin, oligomers and nuclei C. If enough nuclei C are formed, the reaction will shift toward crystallization. As reported previously (Adachi & Asakura, 1980), gels have to be dissociated into monomers before conversion to crystals.
In 1.2 M-phosphate buffer, it appears that nuclei C and nuclei G of deoxy Hb C, are formed at similar rates, and gelation and crystallization take place simultaneously under these conditions. The difference in the solubility of deoxyhemoglobins must be related to the hydrophobic interaction between the amino acid at the /36 (A3) position and Phe 885 (Fl ) and Leu 888 (F4), and to the hydrogen-bonding interaction between the amino acid at the 873 (E17) position and Thr /34 (An4) (Wishner et al.. 1975). (c)
Supermolecular
structure
of gels and aggregates
i\s to the supermolecular structures of deoxy Hb S gels studied by electron microscopy, there appear to be at least two different types of fibers : one composed of six filaments or strands of deoxy Hb S molecules with a diameter of 17 nm. (Finch et al., 1973; Ohtsuki et al., 1977) and another composed of 14 filaments with an average diameter of about 20 nm (Dykes et aE., 1978). Our study, as well as the study reported by Pumphrey & Steinhardt (1977), showed that if a solution of deoxy Hb S was agitated gently in the presence of inositol hexaphosphate, deoxy Hb S formed crystals rather than gels. Dykes et al. (1979) used the method of Pumphrey & Steinhardt (1976,1977) for the preparation of their sample. It appears that under these conditions, crystals of deoxy Hb S rather than gels are formed. Magdoff-Fairchild et al. (1979) reported that fibers of deoxy Hb 8 within SS erythrocytes transform into needle-like crystals when incubated at 37°C for intervals ranging from two days to two weeks and that X-ray diffraction patterns are strikingly similar to those of needles as well as single crystals. The basic structure of fibers of deoxy Hb S has been determined by comparing X-ray diffraction patterns of the fiber with those of monoclinic crystals (form 1) (MagdoffFairchild & Chiu, 1979; Magdoff-Fairchild et al., 1979). More recently Chiu & Magdoff-Fairchild (1980) reported the presence of a new monoclinic crystal, form II, whose cell dimensions are the same as those of form I in deoxygenated sickled erythrocytes. All these results suggest that whether gels or crystals are formed OI whether form I or II crystals are formed depends upon the number of nuclei G formed as well as upon the association constants, KG and Kc, of equations (1) and (2) for the deoxyhemoglobin solution. We are grateful to Janet Fithian for editorial assistance and Billie Corbett for secretarial help in the preparation of the manuscipt. One author (K. A.) was the recipient of Career Development Award 1 K04 HLOO774 from the National Institutes of Health. This work was supported by grants HL-20750 and GM-20138 from the National Institutes of Health.
Ii. AUAC’HI
1x0
ANU T. ASAKUK.4
REFERENCES Adachi, K. d Asakura, T. (1979u). J. Biol. Chem. 254, 4079-4084. Adachi, K. & Asakura, T. (19796). J. Riol. Chem. 254. 7765-7771. Adachi, K. B Asakura, T. (1979c). J. Biol. Chem. 254. 12,273-12,276. Adachi, K. & Asakura, T. (1980). J. Biol. Chem. in the press. Adachi, K.. Kinney. T. R., Schwartz, E. & Asakura, T. (1980a). Hemoglobin, 4; l-26. Adachi. K., Schwartz, E. B Asakura, T. (1980b). Blood, 55, 494-500. Bookchin, R. M. & SageI; R. L. (1971). J. Mol. Biol. 60, 263-270. Bookchin, R. M.. Nagel. R. L. & Ranney. H. M. (1967). J. Biol. C’h,em. 242, 248-255. Bookchin, R. M., Nagel, R. L. & Ranney, H. M. (1970). Biochim. Hiophys. .4&z, 221, 373~ 375. Bookchin. R,. M., Nagel, R. L. & Balazs, T. (1975). ,Vature (/iondonJ, 265, 667+68. Bookchin, R. M., Balazs, T., Nagel, R. L. & Tellez, 1. (1977). :Vatur~ (London), 269,526~527. Briehl, R. W. (1976). In Proc. Symp. Mol. Cell. Aspects of Sickle Cell Disease (Hercules, ,J. 1.. Cottman, G. L., Waterman, M. R. & Schecter, A. N., eds), pp. 145-183, DHEW Pub. no. (NIH) 76-1007, Bethesda. Briehl, R. W. & Ewert. S. (1973). J. Mol. Hiol. 80, 445458. rhiu, C”. (‘. B Magdoff-Fairchild, B. (1980). J. Mol. Biol. 136, 4!&459. Dykes, G.; (‘repeau, R. H. 8r Edelstein, S. J. (1978). LVaturc (London), 272, 5OG510. Dykes, G. G., (‘repeau, R. H. 8: Edelstein. S. J. (1979). J. Mol. Biol. 130, 451472. Finch, J. T.. Perutz, M. F., Bertles, ,J. F. & Dobler, J. (1973). Proc. iVat. Acad. Sri.. I‘.S..-i '70, 718-722.
Goldberg, ,1;1.A.. Husson, M. A. & Bunn, H. F. (1977). J. Kiol. (‘hem. 252, 3414-3421. Hofrichter. ,J.. Ross, P. D. & Eaton, W. ii. (1974). Proc. LVat. Acad. Sci., I’.S.A. 71. 4864 4868. Hofrichter, ,J., Ross, P. D. & Eaton, W. A. (1976a). Proc. ,V’at. Acad. Sci., I:.S.il. 73, 3035 3039.
Hofrichter.
,J., Ross. f’. D. C%Eaton, W. A. (19766). In Proc. Symp. Mol. Cell. Aspects of M. R. & Schechter. A. N.. eds), pp. 185-223, DHEW Pub. no. (NIH) 76-1007, Bethesda. Love, W. E., Fitzgerald, P. &I. D., Hanson, J. C., Roger, W. E. & Ringle, W. E. (1978). (‘hem. (Cauhey, W. S., ed.); pp. 165-177. Biochem. ilspecb of Hemoglobin Abnormalities Academic Press, New York. Magdoff-Fairchild, B. & Chiu, C’. (‘. (1979). Proc. Xat. Acad. Sci., /‘.S.d. 76, 223-226. Magdoff-Fairchild, B., Poillon, W. N., Ting-I, L. & Bertles, ,J. F. (1976). Proc. ,Tat. Acnd. sci., V.S.A 73, 990-994. Magdoff-Fairchild, B., Chiu, (‘. (‘. & Bertles, J. E’. (1979). In Developmercl of !i?herapeutic ilgents for Sickle Cell Disease, Inserm Symp 9 (Rosa, J., Beuzard, Y. & Hercules. ,I., eds), pp. 57-64, Elsevier/North-Holland Biomedical Press, Amsterdam, Nagel, R. L. & Bookchin. R. M. (1975). Sickle Cell ilnemia and oth.er Hemoglobinnputhi~s (Levere, R. D., ed.), pp. 51-66, Academic Press, New York. Ohtsuki, M., White, S. H., Zeitler, E., Wellems, T. E.. Fuller, S. P., Zwick, X, Makinrn. &I. W. & Sigler, P. D. (1977). Proc. IV&. Acad. Sci., V.S.iz 74. 553%5+%2. Peru@ M. F. & Liquori, A. M. (1951). IVature (London), 167, 929-931. Bioyhys. Res. Pommun. 69, 99-105. Pumphrey. ,J. G. & Steinhardt, J. (1976). Biochem. Pumphrey. ,J. G. & Steinhardt, J. (1977). J. Mol. Biol. 112, 359-375. Singer, K. & Singer, L. (1953). Blood, 8, 1008-1023. Wishner. B. (‘.! Ward, K. B., Lattman, E. E. & Love, W. E. (1975). J. Mol. Biol. 98, 179-194. Wishner, B. C’.; Hanson, J. C’.. Ringle, W. M. & Love, W. E. (1976). In Proc. Symp. Mol. Cell. ilspects of Sickle Cell Disease (Hercules, J. I., Cottam, G. L.. Waterman, M. R. rYr Schechter, A. N., eds), pp. 1-31, DHEW Pub. no. (NIH) 76-1007 Bethesda. Sickle rell Disease (Hercules, J. I., Cottam, G. L., Waterman,
Biol.
144, 467-480
(1980)
Polymerization
of Deoxyhemoglobin Carlem (86 Glu -+ Val, 873 Asp -+ Asn)
The Effect of 873 Asparagine
on the Gelation Hemoglobin
and Crystallization
of
KAZUHIKO ADACHI AND TOSHIO ASAKURA
University
!Phe Children’s Hospital oj Philadelphia Department of Pediatrics Department of Biochemistry and Biophysics of Pennsylvania, Philadelphia, Penn. 19104, IJ.S.A.
(Received
9 June 1980, and in revised form 5 August
1980)
The kinetics of aggregation and the solubility of deoxy Hbt C,,,,, (a& 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of agg re gated with a clear exhibition deoxy Hb S and deoxy Hb A. Deoxy Hb C,,, of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration. The initial hemoglobin concentration required for the aggregation of deoxy HbC uarle. was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb CHsrlemwas approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time Z~~T+SU.S hemoglobin concentration in 1% M-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 40 for deoxy Hb Cm,,-. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb CHarle,,,were unchanged with phosphate concentrations varying from 1.2 M to 2.0 M. The by solubilities of deoxy Hb S and deoxy Hb CHsrlemwere increased exponentially lowering the pH of the medium, with the increase being more conspicuous for Hb Gurw The gels (or aggregates) of Hb C,,,,, were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb CHsrlemcan be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomeric deoxy Hb
4 nuclei G e polymer ti gel *nuclei C *polymer ti crystals
The hemoglobin concentration required for the crystallization of deoxy Hb ‘Amem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb CHsrlemafter aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the 873 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C. t Abbreviations
used: Hb, hemoglobin;
w2-2836/36/366467-14 $02.66/O
Hb Cn, hemoglobin 467
CHarlam.
0 1980 Academic Press Inc. (London)
Ltd
MX
1. Introduction (HI) (‘“) has amino acid mutations at the /IS ((iI 4 I’al) and the p73 Hb (“nartrm (=\sp -+ r\sn) positions. Although the former substitution is identical to that iti Hb S. deoxy Hb PH forms gels less readily than does deoxy Hb S. The minimum gelling concentration (MM”) of deoxy Hb CyHis 36.2 g/d1 in cont,rast to 24.0 g/d1 for, droxy Hh S (Bookchin d al., 1967: Bookchin & 9agel. 1971). indicating that the* second amino acid mutation (873) in Hb PH has an antigelling eff&t. Structural studies of the gels and crystals of deoxy Hb S have shown that thra 873 position is in the contact region. which is complementary t,o the ,96 position in thr. molecule (Wishner d nl.. 1975: Love rt al.. 1978). The difference in the solubility of droxy Hb (‘” from tha,t of deoxy Hb S and the inhibitory effect of th(b deox!. forms of Hb CTHand Hb Korle Bu on t,he gelation of deoxy Hh S (Bookchin c)tnl.. 1967. Bookchin & Bagel, I97 1) also support the idea that the 873 residues are involved in the gelation of deoxy Hb S. Recently. we found that deoxy H b .4 forms gels when a solution of deos~, HI) .\ with a concentration
of ahout
lS(&,
of its solubilit~
is warmed
to 31’(
(Ada&i
k
Xsakura.
1979c). From this we concluded that, the hydrophobic amino wid substit,ut,ion (\-at) at the /36 position is not essent,ial for the gelabion of hemoglobin but, that this substitution accelerates the formation of gels and st)abilizrs thtx conformation of deoxy Hb S gel. We also found that aggregates or gels of tieox> Hb A-Iwere converted to t,hree-dimensional crystals over a period of days (.-\dachi k ,\sakura. 1979c). This period is much shorter than that (3 mont,hs) for crystallization of deoxy Hb S gels (Wishner rt nl.. 1975.1976: Hofrirhter r,f UJ 19766). Kinetic studies of the aggregation and crystallization of deoxy Hb X and deoxy Hb S in concentrated phosphate buffers suggested that t’he size of nut-ltv produced during the delay period is reduced in concentratjrd phosI)hat,e buff&t (.\dachi & &akura. 19796) and that the nuclei for gels and rrystals are differt’nt (,Adachi & &akura. 1980). Although details of the structure of these nuclei are not yet known. their nature may be clarified by studies of abnormal hemoglobins. particularly those that have an amino acid substitution at t,he contact region and affect the gelation of deoxy Hb S. We report here the results of stlldies on this kinetics of the aggregation and crystallization of deoxy Hb (
2. Experimental Procedures Hh C’, was purified from Hb 8(“, blood by the standard chromatographic trchnicloc> OII (‘M-Sephadex and was identified by fingerprmts after tryptic and chymotrypbic digestions and amino acid analysis (Adachi et al., 198Oa). Hb S was purified from .4S blood by column chromatography on DEAE-Sephadex as described elsewhere (Adachi &r Asakura, 1979b). The concentration of hemoglobin was determined spectrophotometrically by the use of millimolar extinction coefficients of mE 555 = A0 for deoxyhemoglobin and mE,,, = ;?3% for carbonmonoxyhemoglobin (on a tetramer basis). Kinetic studies of the aggregation and de-aggregation of hemoglobins were rarrird out turbidimetrically as described elsewhrrt-
POLYMERIZATION
OF DEOXYHEMOGLOBIS
(“Har,em
ais
(Ada&i & Asakura, 1979u,b). The solubihty of hemoglobin was determined by measuring the concentration of dissolved hemoglobin in the supernatant after centrifugation. The pH of concentrated phosphate buffer was determined with a pH electrode after a l@fold dilution of the solution.
3. Results (a) Effect of pH on the kinetics ofaggregation of deoxy Hb Sand deoxy Hb C, in 1.8 Mphosphate
buffeer
Like deoxy Hb S and deoxy Hb A (Adachi & Asakura, 1979b,c), deoxy Hb Cs aggregated with a clear exhibition of a delay time when solutions of an appropriate
concentrat’ion were incubated at 30°C in 1% &f-phosphate buffer. The length of the delay and aggregation times and the degree of aggregation (measured as Al,OO) depended upon the initial hemoglobin concentration; the higher the concentration of Hb (yn>the shorter the delay and aggregation times and the greater the turbidit,y increase. The aggregates could be reliquefied by cooling. The major difference between the aggregation of deoxy Hb C, and that of deoxy Hb S is the relationship between the delay time and the hemoglobin concentration. For aggregation. a higher concentration
of deoxy
Hb C, is required
than of deoxy
Hb S. For instance,
a solution of deoxy Hb S with a concentration of 75 mg/dl exhibits a delay time of about ten minutes in 1.8 M-phosphate (pH 7.36) at 3O”C, whereas a concentration of 290 mg/dl of deoxy Hb Cn is required for the same length of delay time under the same experimental conditions (Fig. l(a)). In other words, if we compare the lengths of delay times for solutions of deoxy Hb C, with those of the same concentrations of deoxy Hb S in 1.8 M-phosphate buffer (pH 7.36), at 30”(‘, the delay time for deoxy Hb Cn is approximately 10’ times longer than that of deoxy Hb S (Fig. I (a)). The logarithmic plotting of the delay time versus hemoglobin concentration in 1% M-phosphate buffer (pH 7.36) shows linear lines with slopes (n-value) of 2.7 for deoxy Hb S and 3.9 for deoxy Hb Cn (Fig. l(a)). To investigate whether this difference can be attributed to the difference in the electrostatic bond involved with COO- of the 1373 asparatic acid, experiments were carried out at four different pH values in l-8 M-phosphate buffer at 30°C. As shown in Figure 1, more concentrated hemoglobin solutions are required for the aggregation of both deoxy Hb S and deoxy Hb Cn at lower pH values. There is a linear relationship between the log of reciprocal delay time and hemoglobin concentration. Under the same pH condition, the lines for Hb Cn lie always to the right side of those for Hb S, indicating that a more concentrated hemoglobin solution is required for the aggregation of deoxy Hb C, to reveal the same length of delay time as compared to Hb S. The slopes (n values) for the delay time of deoxy Hb S and deoxy Hb C, decrease with increase in pH (Fig. l(b)). The aggregation time (tr - td) is also inversely related to hemoglobin concentration and affected by the pR (broken lines in Fig. l(a)). The slopes (n-values) of deoxy Hb Cn are 3.7, 3.9 and 55 at pH 8.0, 7.36 and 7.0, respectively (Fig. l(a)). The difference in n values for delay times between deoxy Hb C, and deoxy Hb S is greater at higher pH values (Fig. l(b)).
Ii.
170 O-
pH 8.01 Hb
S
ADAPHI
AND
‘I’. ASABUKA
8.01
7.36 S
CH
7.04 s
8’ I n:2a7 i-
.' rn’d, , 0’
736 Cl+
6.58 S
7.01 CH
d
,I
n=5.5/n:3.9/
1x6.5 /
, n-5.5
.-,I , , , ^-Z.V . a; I, I I I I 1 I I I I I I I 1 I I, I I I -2.5
-1.5
-2.0
-1-O
-0.5
0
log G (g/d11 (a) IO-
\ OX
\ ‘-,d -->
._.-•
a-
-0
-e -----.
Hb CH Hb S
0
I
I 7-O
I
I 0.0
PH (b)
FIG. l(a). Effect of pH on the kinetics of the aggregation of deoxy Hb S and deoxy Hb C, in 1.8 Mphosphate. Hemoglobin solution of various concentrations ((0, n ) Hb 8: (0, 0) Hb C,) at, different pH values were heated from 0°C to 30°C at time zero. The delay times (td) (solid lines) and the aggregation times (tTtd) (broken lines) were measured spectrophotometrically by recording t,he absorbance at 700 nm. - n -- w - , Delay time (td) of deoxy Hb S ; --O---O-P, aggregation time of aggregation time (If-td) of deox? deoxyHbS;-~--~-,delaytime(t,)ofdeoxyHbC~;--O--~----, Hb CH. l/r, reciprocal delay and aggregation times; C,, Hb concn. (b) The relationship between n values and pH. The data for deoxy Hb S and deoxy Hbt’, in 1% M-phosphate buffer are derived from the data in (a).
(b) Effect of pH on the solubility
of deoxy Hb S and deoxy Hb CL,
The solubilities of the oxy and deoxy forms of hemoglobins are known to depend upon the pH of the medium (Singer & Singer, 1953 ; Magdoff-Fairchild et al., 1976 : Goldberg et al., 1977). In contrast to the increase in the MGC and the solubility of deoxy Hb S at higher pH values, the solubility of deoxy Hb S in concentrated phosphate buffer decreases at higher pH values. To investigate the effect of the replacement of Asp by Asn in Hb (1” on its solubility, the solubilities (the supernatant hemoglobin concentrations) of deoxq Hb S and Hb CH at different pH values were determined after the aggregation
POLYMERIZATION
OF DEOXYHEMOGLOBIN
CHarlem
471
P’-’
FIG. 2. Effect of pH on the solubility of deoxy Hb 8 and deoxy Hb CH in 1.8 M-phosphate buffer at 30°C. The solubility was determined by measuring the concentration of soluble hemoglobins in the supernatant after completion of aggregation. Experimental conditions are the same as those shown in Fig. l(a).
.c E
-3-
-5
-
-6-
11 I/T(x103)(K-‘)
FIG. 3. Effect of temperature on the delay time of deoxy Hb C, aggregation in 1% M-phosphate buffer (pH 7.4). Hemoglobin (293k5.7 mg/dl) in 1.8 M-phosphate buffer (pH 7.36) was heated from 0°C to various temperatures. Results are the average of results of 2 to 3 experiments at different temperatures.
reaction had reached a plateau. The solubilities of both deoxy Hb S and deoxy Hb C, increase exponentially as the pH of the medium is decreased, with deoxy Hb C, showing greater increases at lower pH values (Fig. 2). (c) Effect of temperature The kinetics of the aggregation of deoxy Hb S are highly temperature-dependent’ (Hofrichter et al., 197ti,b; Pumphrey & Steinhardt, 1977; Adachi & Asakura, 19793). The lower the temperature, the longer both the delay and aggregation times. A similar relationship is observed for the aggregation of deoxy Hb C, (Fig. 3). The apparent activation energy for the aggregation of deoxy Hb C, in
17%
K. .4DA(‘HI
ASD
‘I’. .ZS.AKC’KA
I.8 hr-potassium phosphate (pH 7.36), calculated from the results showy in Figure 3, ranges from about 25 kcal/mol near 30”(’ t,o as high as 65 kcal/mol near 15°C. These values for the delay time of droxy Hb ( lH are slightly higher than those for pure deoxy Hb S (Adachi K- =\sakura. 19798).
((i)
Effect
of phosph,atp
cotwratratiott
As reported previously, (Ada&i K- Asakura; 1979h). there is a linear relationshil~ between the n value and the phosphate concentration. The I/ values for deoxy Hb S were 2.X. 76 and 155 in 1.X. 1.5 and 1.0 nl-phosphate buffers, while those for deoxy Hb A were 2.3, .56 and 12 in 2.1. 1.8 and 1.2 M-phosphate buffers, respectively (Adachi & Asakura. 1980). From these results we concluded that the sizes of nuclei decrease as the concentration of phosphate buffer is increased. Similar showed experiments with deoxy Hb (‘(a in four different phosphate concentrations in that the n value for Hb Cs was about 3-. and that this value was unchanged phosphate buffers (pH 74) with concentrations bet,ween 1.2 M and 2.1 M (Fig. 4(a) and (b)). These results suggest that the sizes of nuclei of deoxy Hb (‘u are unchanged in these phosphate buffers. The aggregation times (tf- td) for deoxy Hb (‘n also show linear curves in 2.1 41 and 1.8 $f-phosphate buffers (open circles in Fig. 4(a)). but those in 1.5 37and 1.2 Nphosphate buffers do not show linear curves. as the values fluctuate widely in these buffers.
(e)
Ibuggregation
of aggregates
of deozy
Hh
(If,
by cooling. bubbling with carbon Polymers of deoxy Hb (‘u can be reliquefied monoxide gas or oxygen, and dilution of the phosphate buffer, as can aggregates of deoxy Hb S (Adachi $ Asakura, 1979b). The de-aggregation curve for deoxy Hb (‘u is similar to those for deoxy Hb A and deoxy Hb S (Fig. 5), suggesting similar structures for aggregates of these deoxyhemoglobins formed by the nucleation It should be pointed out that the decontrolled aggregation mechanism. out in aggregation curves of aggregates of Hb C, formed by simple salting phosphate buffer above 2.4 M are significantly different from those of deoxy Hb r\ 1979a; Adachi et al.. 19806). Aggregates and deoxy Hb S (Adachi & Asakura: formed according to the nucleation-controlled mechanism appear to differ from those produced by t’he simple salting-out mechanism (Adachi 8i Asakura. 1979a). (f) (m’on,t~ersiott, of the aggregates
(gels)
of deoxy
Hb C, to crystalline
forms
It has been reported that gels of deoxy Hb S are converted to suspensions ot three-dimensional crystals after standing for several months (Wishner et al.. 1975,1976 : Hofrichter et al., 19766). We found that aggregates (or gels) of deoxy Hb $, oxy Hb S and carbonmonoxy Hb S also were converted to crystals, but after only a day or two of standing at 30°C (Adachi & Asakura, 1980). Deoxy Hb Cn formed crystals even more rapidly than the above hemoglobins. In l-5, l-8 and
POLYMERIZATION
OF DEOXYHEMOGLOBIS
CHsrlem
I.8 M
2.0 M phosphate
473
I.5 M
1.2M
’ /
.’
-’
-2.51,
-,
,
,
, -1.5
,
,
,
,
, -1.0
,
/
,
,
,
, -0.5
,
,
,
,
i
, 0
,,, , “0.4
,
,
,
I 0,
log 6 (g/d11
309 \
c
\
\
\
\
\
\ ’ ,Hb S \ ‘0
15-
\
\ ‘\
\
Hb CH -o-oI 0
‘,Hb ‘0,
I I.0
A
\ \
‘\
AL R”LA
1
Phosphate
( M)
(b)
FIG. l(a) Relationship of reciprocal delay time and aggregation time to hemoglobin concentration in different phosphate concentrations. Phosphate concentrations used were 2.1 M, 1.8 M, 1.5 M and 1.2 M (pH 7.4) as shown in the Figure. (0) Delay time: (0) aggregation time. (b) Relationship between 12value and phosphate concentration. The n values of deoxy Hb S (0) and deoxy Hh A (A) are from our previous papers (ildachi & Asakura, 1979b,c).
2.1 M-phosphate buffers, aggregates formed and upon further incubation were converted to crystals. In 1.2 M-phosphate buffer, amorphous aggregates and crystals were formed simultaneously (Fig. 6), suggesting that crystallization of deoxy Hb CH is as rapid as gelation in 1.2 M-phosphate buffer. The crystallization of deoxy Hb C, required a longer period of time in higher phosphate concentrations. For instance, a few days of incubation at 30°C (2 to 3 days) are necessary for the conversion of gels to crystals, in 1.8 M-phosphate buffer, whereas incubating for a few hours or overnight results in the crystallization of deoxy Hb C, in 1.5 Mphosphate buffer. A typical example of the aggregation curve of deoxy Hb C, in 1.5 M-phosphate buffer at 30°C is shown in Figure 7. In this reaction, deoxy Hb C, aggregated after a delay time of about 40 minutes. After aggregation, the cuvette
K. ADACHI
474
loo-
l ,\ \
\
AND
T. ASAKI-KA
\\ ‘\\ s
\ o-
, I4
I Phosphate
, ‘0’ I.6
A,
(M)
FIG. 5. De-aggregation of aggregates of deoxy Hb A, deoxy Hb S and deoxy Hb Cu in 1.8 M-phosphate buffer (pH 7.4). Hemoglobin solutions (deoxy Hb S, 0% g/dl: deoxg Hb ii. 240 g/dl: and deoxy Hb tyH 0.190 g/dl) in 1.8 M-phosphate buffer (pH 7.4) were heated from 0°C to 30°C at t,ime zero: after completion of aggregation. the aggregates were melted by dilution of phosphate molarity with the addition of water at 30°C.
was shaken several times and incubated further at 30°C. The second reaction, crystallization, started after 20 minutes further incubation after the first reaction, as indicated by a steep increase in absorption at 700 nm. If the cuvette was not shaken, an overnight incubation was required for crystallization, indicating that shaking accelerated the formation of crystals. The biphasic curves determined turbidimetrically for the aggregation and crystallization reactions can be seen in Figure 7. Unless the experiment was done in phosphate buffer lower than 1.2 M. these two processes were clearly separate. Microscopic observation of the aggregates formed in the first reaction revealed the presence of only amorphous aggregates, while after the second reaction a number of crystals were seen in addition to aggregates. We could see optical birefringence for both amorphous aggregates and crystals of deoxy Hb CH. (g) S’olubility
of deoxy Hb C, before and after crystallization
We reported that the solubilities of deoxy Hb A, deoxy Hb C, oxy Hb A, oxy Hb S, carbonmonoxy Hb A and carbonmonoxy Hb S determined by measuring the hemoglobin concentration in the supernatant at the plateau of the aggregation curve were dependent upon the initial hemoglobin concentration (Adachi & Asakura, 1979b, 1980). On the other hand, the solubilities of deoxy Hb S in the polymerization reaction in I.8 M-phosphate buffer are apparently independent of the initial hemoglobin concentration (Fig. 8). The solubility of deoxy Hb C, in 1.8 M-phosphate and that of deoxy Hb S and deoxy C, in 15 M-phosphate measured after aggregation showed a dependency upon the initial hemoglobin concentration ; this dependency of deoxy Hb Cn was greater than that of deoxy Hb S in I.5 Mphosphate buffer. If the solubility of Hb CH was determined after the
FIG. 6(a) and (b). Photomicrographs of gel and crystals of deoxy Hb C, in 1.2 M-phosphate buffer (pH 74) at 30°C ( x 40). The cuvette containing chilled Hb c, solution (4.5 g/dl) in 1.2 M-phosphate buffer (Na2S,0.,, 5 mg/ml) was heated rapidly to 30°C. A gel that is the mixture of aggregates and crystals formed after about 10 min incubation at 30°C.
Time (min)
FIG. 7. Aggregation and crystallization curve of deoxy Hb C, (071 g/d]) in 1.5 M-phosphate buffer. Aggregation and crystallization of deoxy Hb C, was determined turbidimet,rically at 700 nm 1
Initial
Hb concn (g/dl)
FIG. 8. Relationship between the solubility of deoxy Hb (lH*r,em and the initial hemoglobin concentration after aggregation and crystallization in 1.8 M and 1.5 M-phosphate buffer (pH 7.4). The solubility w&s determined by measuring the concentration of soluble hemoglobin in the supernatant after completion of the aggregation and after crystallization.
crystallization reaction, the value was much lower and the solubility was independent of the initial hemoglobin concentration (Fig. 8). Thus, the solubility of hemoglobin varies widely, depending upon whether or not it is measured after aggregation or crystallization. The solubility after aggregation by the simple salting out method was different from that after aggregation by temperature-jump method (Adachi et al., unpublished data).
4. Discussion (a) Effect of the 873 Am on the aggregation
of Hb C,
Previous studies have shown that the 873 Asp is involved in gelation. since mutation at this position affects the MGC (Bookchin et al., 1967,197O: Bookchin & Nagel, 1971). The 873 Asp + Asn substitution also affected the kinetics of aggregation and the solubility of deoxy Hb Cn in concentrated phosphate buffer:
POLYMERIZATION
OF DEOXYHEMOGLOBIN
CHarlem
411
the aggregation reaction was inhibited and the solubility after aggregation was increased. Studies on the effect of pH on the solubility and the kinetics of aggregation of deoxy Hb S and deoxy Hb Cu showed that the solubilities of both deoxy Hb S and deoxy Hb C, increased exponentially as the pH of the medium was decreased (Fig. 2), and that the inhibitory effect on gelation of the Asp + Asn substitution at the j373 position was greater at lower pH values (Fig. 2). Since the carboxyl group of Asp is protonated at lower pH values, it seems unlikely that an electrostatic bond at the )573 position is involved in this difference (Nagel & Bookchin, 1975). Instead, it is more likely that the Asp + Asn substitution affects hydrogen-bonding of Asp with an adjacent amino acid residue (/?4Thr) on the deoxyhemoglobin molecule, as indicated by the results of X-ray diffraction studies by Wishner et al. (1975). The pH dependency of the aggregation in concentrated phosphate buffer of deoxy Hb S and deoxy Hb C, is the opposite of that in low phosphate buffer (Briehl Kr.Ewert, 1973; Magdoff-Fairchild et al., 1976; Goldberg et al., 1977). Briehl (1976) pointed out that, in studies of solubility in high phosphate buffers, the increasing concentration of HPO:- is responsible for the enhanced condensation rather than the pH itself, since it is HPOi- rather than H,PO, which is primarily responsible for salting out proteins. Our experimental data for deoxy Hb S and deoxy (1, support this hypothesis. (b) Gelation ana’ crystallization
of deoxy Hb C,
Wishner et al. (1975,1976) and Hofrichter et al. (1976b) reported that gels of deoxy Hb S were converted slowly to a suspension of three-dimensional crystals over a period of several months. We found that gels of deoxy Hb A formed in concentrated phosphate buffers were converted to crystals at a rate much faster than those of deoxy Hb S (Adachi & Asakura, 1979c). As shown in this paper, the gel-to-crystal conversion in deoxy Hb C, was even faster than that in deoxy Hb A. These results indicate that crystals of all of these hemoglobins were thermodynamically more stable than gels. We proposed the following scheme for the formation of gels and crystals (Adachi et al., 1980b).
KG
/( nuclei G + polymer $ gel Monomeric deoxy Hb$ nuclei C , ;=t polymer $ crystals
(1) (2)
KC
In this scheme, nuclei G and nuclei C are formed before gelation (or aggregation) and crystallization, respectively. KG and KC are the apparent association constants of the reaction between monomeric hemoglobin and each type of nuclei. This scheme appears to explain the gelation and crystallization of various hemoglobins. Deoxy Hb S has a KG value much higher than that of other hemoglobins. As a result, nuclei G are the more easily formed, and once a sufficient amount of nuclei G have been formed monomeric deoxy Hb S binds to the nuclei to form large aggregates (polymers). The substitution of valine for glutamic acid at the /?S position appears to increase the K, value so strikingly that nuclei G can be produced even where the concentration of deoxy Hb S is only l/40 that of deoxy
178
K. ADACHI
AND
T. ASAKt~rKA
Hb A in 1.8 M-phosphate buffer. The substitution of asparagine for aspartic acid at, the j373 position appears to decrease the KG value, as this additional substitution inhibits gelation: one needs a two or three times more concentrated solution of deoxy Hb C, than of deoxy Hb S to initiate gelation. It is interesting that the substitution of lysine for glutamic acid at the /36 position in deoxy Hb c’ (86 Glu --) Lys) also appears to decrease the KG value, as this substitution markedly accelerates the crystallization of deoxy Hb C (Adachi & Asakura, 1980). Thus, the KG values decrease in the order: Hb S, Hb c‘,, Hb 4 and Hb C. The crystallization reaction is also accompanied by a delay time. The mechanism of this reaction is different from that of gelation because the initial concentration required for crystallization is much smaller than that for gelation, the delay time is usually much longer than that before gelation, and the activation energy for crystallization is greater than that for gelation (Adachi & $sakura, 1979c,1980). The size of a nucleus for crystals estimated from the n value is different from that, for gels in concentrated phosphate buffer (Ada&i & Asakura, 1980). Both KG and K, are specific to each hemoglobin and are affected by various factors including pH, ionic strength. type of salt and temperature. Whether a hemoglobin forms gels or crystals depends upon the relative values of KG and K, and the rate of the formation of nuclei G and nuclei C. It is also related t)o the concentration of monomeric hemoglobin, the so-called critical hemoglobin concentration, which depends upon the surface structure of deoxyhemoglobin and the conditions of the medium. The difference in the critical concentrations for gelation and crystallization is attributed to the great difference in the solubilities of deoxyhemoglobin that are found either after gelation or after crystallization. As reported previously (Adachi & Asakura, 1979c,1980), the solubility of hemoglobin measured after crystallization (S,) is manyfold lower than that measured after gelation (S,j except in deoxy Hb S. The solubility of deoxy Hb 8 after aggregation is exceptionally low compared with that of other hemoglobins and the value does not differ much from the solubility of deoxy Hb S measured after crystallization (Perutz & Liquori, 1951; Pumphrey $ Steinhardt. 1977). This indicates that the concentration of soluble monomeric deoxy Hb S after gelation (or aggregation) is too low for deoxy Hb S to form nuclei C. accounting for the extremely slow conversion of gels of deoxy Hb S to crystals. It should be pointed out that the So of deoxy Hb (‘u depends upon the initial hemoglobin concentration, with a higher )so at, higher initial hemoglobin concentration (Fig. 8), suggesting that the formation of gels does not represent a true equilibrium state (Adachi & Asakura, 1979c). Since the rate of crystallization depends upon So, the rate of crystallization is increased when the initial concentration of deoxy Hb C, is high. The dependence of So upon the initial hemoglobin concentration may be explained by the presence of monomers, oligomers and polymers, particularly during the delay and aggregation times (Adachi & Asakura, 1980). Thus, So corresponds to the total soluble hemoglobin. which is the sum of the concentration in these mixtures. In contrast, after crystallization, the solution contains mainly monomeric hemoglobins, and this value is unaffected by the initial hemoglobin con’cenbration and is specific to each
POLYMERIZATION
OF DEOXYHEMOGLOBIN
GHar,ern
179
hemoglobin. This value may be the true solubility of monomeric hemoglobin. Thus, conversion of gels to crystals of deoxy Hb Cu can be explained as follows : the liquid phase after aggregation is a mixture of monomeric hemoglobin, oligomers and nuclei C. If enough nuclei C are formed, the reaction will shift toward crystallization. As reported previously (Adachi & Asakura, 1980), gels have to be dissociated into monomers before conversion to crystals.
In 1.2 M-phosphate buffer, it appears that nuclei C and nuclei G of deoxy Hb C, are formed at similar rates, and gelation and crystallization take place simultaneously under these conditions. The difference in the solubility of deoxyhemoglobins must be related to the hydrophobic interaction between the amino acid at the /36 (A3) position and Phe 885 (Fl ) and Leu 888 (F4), and to the hydrogen-bonding interaction between the amino acid at the 873 (E17) position and Thr /34 (An4) (Wishner et al.. 1975). (c)
Supermolecular
structure
of gels and aggregates
i\s to the supermolecular structures of deoxy Hb S gels studied by electron microscopy, there appear to be at least two different types of fibers : one composed of six filaments or strands of deoxy Hb S molecules with a diameter of 17 nm. (Finch et al., 1973; Ohtsuki et al., 1977) and another composed of 14 filaments with an average diameter of about 20 nm (Dykes et aE., 1978). Our study, as well as the study reported by Pumphrey & Steinhardt (1977), showed that if a solution of deoxy Hb S was agitated gently in the presence of inositol hexaphosphate, deoxy Hb S formed crystals rather than gels. Dykes et al. (1979) used the method of Pumphrey & Steinhardt (1976,1977) for the preparation of their sample. It appears that under these conditions, crystals of deoxy Hb S rather than gels are formed. Magdoff-Fairchild et al. (1979) reported that fibers of deoxy Hb 8 within SS erythrocytes transform into needle-like crystals when incubated at 37°C for intervals ranging from two days to two weeks and that X-ray diffraction patterns are strikingly similar to those of needles as well as single crystals. The basic structure of fibers of deoxy Hb S has been determined by comparing X-ray diffraction patterns of the fiber with those of monoclinic crystals (form 1) (MagdoffFairchild & Chiu, 1979; Magdoff-Fairchild et al., 1979). More recently Chiu & Magdoff-Fairchild (1980) reported the presence of a new monoclinic crystal, form II, whose cell dimensions are the same as those of form I in deoxygenated sickled erythrocytes. All these results suggest that whether gels or crystals are formed OI whether form I or II crystals are formed depends upon the number of nuclei G formed as well as upon the association constants, KG and Kc, of equations (1) and (2) for the deoxyhemoglobin solution. We are grateful to Janet Fithian for editorial assistance and Billie Corbett for secretarial help in the preparation of the manuscipt. One author (K. A.) was the recipient of Career Development Award 1 K04 HLOO774 from the National Institutes of Health. This work was supported by grants HL-20750 and GM-20138 from the National Institutes of Health.
Ii. AUAC’HI
1x0
ANU T. ASAKUK.4
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