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Comparison of the acid denaturation of several hemoglobins which differ in amino acid sequence
ARCHIVES
OF
BIOCHEMISTRY
Comparison
AND
of the Which DANIEL
Department
BIOPHYSICS
472-478
161,
Acid
Denaturation
Differ
in Amino
D. JONES2 oj Chemistry,
of Several Acid
JACINTO
AND
Georgetown Received
(1974)
University,
October
Hemoglobins
Sequence’ STEINHARDT3 Washington,
DC.
20007
11, 1973
There are pronounced differences in kinetic and thermodynamic stability between human and horse hemoglobins. Since the amino acid sequences of the LY, p dimers of horse and human hemoglobins differ in 61 locations, it is difficult to account for them in terms of specific direct or indirect effects of the sequence differences. Rhesus hemoglobin differs from human in only 12 locations and its stability resembles that of human more closely than does horse, although pronounced differences remain. The stabilities of rhesus ferrihemoglobin and deoxyhemoglobin (Hb+ and Hb”) are intermediate between those of the corresponding high-spin forms of horse and human hemoglobin; but there are only small or negligible differences between the low-spin forms (carbonylhemoglobin and oxyhemoglobin) of the two species. The equilibrium isotherm between native and acid unfolded forms of rhesus Hb+ resembles that of horse more than that of human, but it is slightly more stable and slightly less cooperative. The effects of octanol on the rates of unfolding of rhesus ferrihemoglobin are only slightly smaller than with human. There is no effect of octanol on the unfolding rate of any of the CO hemoglobins. Unlike the equilibria of horse and human, octanol is also without effect on the unfolding equilibrium of rhesus ferrihemoglobin, and thus qualifies as a true catalyst of the initial stage of the acid unfolding reaction of the monkey ferriprotein. Differences in stability are tentatively attributed to a limited number of the 12 differences between the two proteins.
Kinetic stability (rate of denaturation) at extremes of pH is an easily measured general property of the homologous proteins of different species which is affected by details of amino acid sequence. Thermodynamic stability (ratio of amounts of denatured to native conformers as a function of pH) is also affected. Such changes may accompany more specific changes in properties which depend on amino acid sequence, such as the sickling potentiality in human sickle-cell hemoglobin caused by a single (nonconservative) substitution of Val 6 for Glu 6 in the
two p chains of the molecule. The differences in stability in that case are small (1) ; very much larger differences in this respect are found between the hemoglobins of human and horse (2) which differ from one another by 61 sequence differences per CY,@ dimer, about one fifth of the total, although many of the substitutions are conservative. Efforts to relate stability differences to sequence differences in these two species have not succeeded, partly because the differences are so numerous. We have, therefore, sought a species which has only a small number of sequence differences relative to human hemoglobin. Such a species is the rhesus monkey (3, 4). The differences in amino acid sequence between rhesus and human hemoglobin are shown in Table I, which also shows the amino acids in horse
1 Supported by NIH Grant HL 12256. 2 Present address : Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973. 3 To whom requests for reprints should be sent at. the Department of Chemistry, Georgetown University, Washington, D.C. 20007. 472 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
COMPARISON
OF
THE
ACID
DENATURATION TABLE
SEQUENCE
DIFFERENCES
OF
I
HUMAN
AND
RHESUS -------71
or-chains Horse6 Rhesus Human
8
19
I----
thr ser thr A6
dY dY ala AB 1
p-chains Horse Rhesus Human
9 ala asn ser A6
jleu dY lieu FdY jasn ala IE 17 E 20 176 87 /his ala jasn dn jala thr 1E20 F3 /Heme-binding / region ___-----
13 ala thr ala A 10
168
33 val leu val B 15 01, B contact _---------
50 asn ser thr Dl
MONKEY”
u Corresponding amino acids for horse hemoglobins are also shown, as well h Rhesus sequence: Ref. (3); horse and human sequences: Ref. (4).
hemoglobin which occur at loci in which human and rhesus differ. The present paper, therefore, reports kinetic and equilibrium data for the acid denaturation of rhesus monkey hemoglobin, Hm.4 The results for this species are compared with previous published results with Hu A (1, 2, 5, 6) and with Hu S (5-7). This study also makes use of the effect of rz-alcohols on the acid denaturation of hemoglobins to broaden the scope of the stability properties studied. Trace amounts of nalcohols (C&C,) effectively enhance ferrihemoglobin (Hbf) denaturation rates (human and horse) when present at concentrations below low3 M, i.e., at molar ratios of alcohol to Hb+ of 100: 1. The effectiveness 4 Abbreviations table:
used
are given
Ferrihemoglobin
oxyhemoglobin
Hu+A Human hemoglobin A Human heHu+S moglobin 8 Hm+ Rhesus hemoglobin Horse hemoHs globin
in the following
Carbonylhemoglobin
Deoxyhemoglobin
OzHuA
COHuA
Hu”A
OzHuS
COHuS
Hu”S
OzHm
COHm
Hm”
OsHs
COHs
Hs”
Hb or a combination of Hb with other symbols refers to any or all species. With human hemoglobin the suffix A is to be understood if no suffix is given.
473
HEMOGLOBINS
,----__-r
104 arg lys arg G6
1 / I \ I j
as sequence
“--
125 glu gin pro H2 01, @ contact
designations.
of medium length n-alcohols as Hb denaturants is, therefore, nearly as great as that of long chain detergent ions such as dodecylsulfate (Steinhardt and Reynolds, unpublished; Verpoorte, unpublished). On the basis of their results with Hs+, Cassatt and Steinhardt (7) proposed a mechanism of denaturation in which t’he alcohol is postulated to bind at a hydrophobic site in or near the heme pocket. Jones et al. (1) confirmed the general features of the phenomena for Hu+, OnHu, and Hu” but found that octanol has a smaller effect on Hu+ than on Hs+, and that it did not affect COHu at all. Unpublished data obtained by Cassatt and Steinhardt (1972), cited by Jones et al. (l), support the postulated mechanism of action of the alcohols by demonstrating that they quench fluorescence of the dye ANS in the heme pocket of globin. The effects of n-octanol on the acid denaturation rates of Hm+, OzHm, Hm”, and COHm are, therefore, included in this paper. With Hm+, the equilibrium between native and denatured is distinguished from t,hat of Hs+ and Hu+ by its indifference to the presence of octanol. EXPERIMENTAL
PROCEDURES
Materials. A 280.ml aliquot of blood from a. single rhesus monkey (Macaca mulatta) was obtained from Litton Bionetics, Inc., Rockville, MD. After saturation with CO, COHm was crystallized three times as described for COHu (a), and stored frozen in a 4% solution. The stock solutions were diluted to 0.1% before use. 02Hm
474
JONES AND STEINHARDT
was prepared as needed by passing 02 (equilibrated with water vapor) over a solution of COHm which was photodissociated (while cooled under three 303-W lamps). The photodissociation of rhesus COHm was slower than that of COHu. The kinetic and equilibrium measurements were performed as described previously (1). Methods. The stop-flow equipment and prooedures used, and the methods of determining pH were the same as in earlier investigations (1, 2, 5, 6). RESULTS
The pH profiles of the acid denaturation rates of Hu+, Hsf, and Hm+ in the presence and absence of octanol are shown in Fig. 1, in which the data for Hu+ and Hsf from earlier work (1, 5, 6) are represented by continuous lines. In the absence of octanol, the log rate-pH denaturation profile of Hm+ lies between those of Hs+ and Hu+; all three lines have approximately the same slope. The same order holds for the augmentations of the rates in the presence of octano&which are largest for horse, less for monkey, and least for human; the latter is, however, not far below the augmentation for monkey. In round numbers, at pH 3, octanol increases the rate g-fold with horse, 4-fold with rhesus, and only about 3-fold with human ferrihemoglobins. The denaturation equilibria for the same three ferrihemoglobins, as affected by pH
“’
28 3.0 32 34 36 3.8 PH FIG. 1. Dependence of the half-period of acid denaturation of rhesus ferrihemoglobin Hm+ on pH at 25% at 0.02 ionic strength. The light broken line represents the data for human ferrihemoglobin and the light solid line the data for horse ferrihemoglobin. The broken lines represent the data for horse and human ferrihemoglobins in the presence of 1.5 X 10V M octanol.
I
I
I
I
I
3% 4.0 4.2 4.4 4.6 48 PH FIG. 2. The effect of 1.5 X 10-a M octanol on the equilibrium between native and denatured ferrihemoglobin of human, rhesus, and horse as a function of pH at 25°C and 0.02 ionic strength.
and octanol, are given in Fig. 2. As shown earlier (2) the equilibrium for Huf covers a wider pH range than its counterpart for Hs+. Octanol has a much smaller effect (1). The equilibrium for rhesus is intermediate in steepness between those of Hs and Hu and octanol does not affect it. The data for Hm+ are much like those for Hsf when octanol is present except that the curve for Hm+ is displaced to lower pH, i.e., rhesus ferrihemoglobin is thermodynamically more stable than human ferrihemoglobin. Thus the relative magnitudes of the octanol effects on the transition curves fall into a different order than that of the steepnessof the curves with respect to their positions on the pH axis. The octanol effect on the equilibrium is largest for Hsf, and negligible for Hm+. The pH profiles of the acid denaturation rates of human oxyhemoglobin (OzHu) and monkey oxyhemoglobin (OzHm) at 2°C are shown in Fig. 3, and those of normal human deoxyhemoglobin (Hue-two forms) and rhesus deoxyhemoglobin, are shown in Fig. 4. Previously published data for horse oxyhemoglobin (8), which are not shown here, show that the latter is more stable, and that its rate of denaturation has a stronger dependence on pH than either human or rhesus. Human and rhesus oxyhemoglobin are also much alike with respect to their kinetic stabilities except that the rhesus protein has a smaller susceptibility to octanol (Fig. 3). It has been shown elsewhere (1) that human deoxyhemoglobin may be obtained in two forms, one of which, “stable” Hu’, is denatured at acid pH about one-tenth as
CO?VIPARISON
OF
THE
ACID
DENATURATION
rapidly as the other, L’unstable” Hu”. Rhesus deoxyhemoglobin (Fig. 4) is more stable than ‘kmstable” Hu’, but less stable than “stable” Hu” (1). It should be noted that the slope of the pH profile of “stable” Hu” is very close to the corresponding slope for Hm”; although the latter is displaced to higher pH by 0.2 pH units. We may con-
OF
TABLE pH
475
HEMOGLOBINS II
ST WHICH HALF-PERIODS FOR ACID DKNATURATION ARE 1 set AT 25°C OR 2”0 VALUE
Human Rhesus Horse
A*
Hb+
Hb” stable
Hb’ unstable
COHb
OnHb
3.12 3.02 2.956
2.25 2.49 2.75d
2.77
2.55 2.41 2.58”
2.0 2.0 1.6”
-
(1 The data for Hb+ and COHb were taken at 25’C in HCl-KC1 or acetate-KC1 solutions of 0.02 ionic strength. The data for Hb” were taken at 25°C in chloroacetate buffer of 0.025 ionic strength. The OlHb data were taken at 2°C in HCl-KC1 solutions of 0.03 ionic strength. * Ref. (1). May be compared with the Hb+ data of (5) and (6). c Ref. (5). May be compared with the Hb+ data of Ref. (9) or the COHb data of (6). d Ref. (8). Ionic strength 0.05.
PH FIG. 3. The rates of denaturation of human and rhesus oxyhemoglobins with and without 1.5 X W3 M octanol as a function of pH at 2°C and 0.02 ionic strength.
‘LIZ: Hu” with octanol * Hm’ with octanol
2.2
2.4
2.6
2.8
3.0
PH
FIG. 4. The effect of pH and 1.5 X lWa M octanol on the half-periods of denaturation of rhesus deoxyhemoglobin at 25°C and 0.025 ionic strength. For comparison, lines representing data for both “unstable” and “stable” human deoxyhemoglobin are also included. The data for “unstable” human deoxyhemoglobin are indicated by the two uppermost lines in the figure.
elude that Hm” as well as the liganded forms is slightly more resistant to low pH than its human analogs. The data for Hs” given in Table II show that horse deoxyhemoglobin is appreciably less stable than either rhesus or human deoxyhemoglobins; and is, in fact, about as unstable as the “unstable” form of Hu” (1). Data for COHu and COHm and also COHs are included in Table II which tabulates the relative stabilities of the various hemoglobin toward acid denaturation by listing the pH values corresponding to a half-period of 1 sec. Comparisons between columns indicate that COHb is usually the most stable for each of the speciesstudied at 25°C (except for ‘ktable” human deoxyhemoglobin). Since the slopes of the pH-log half-period profiles differ from one form to another,5 comparisons of stabilities would differ if pH values were listed for much shorter half-periods, i.e., 100 msec instead of 1 sec. Attention is also invited to the fact 6 An extreme exampIe is provided by the oxyhemoglobins of human and horse, in which the latter has a much steeper pH profile (slope 2.3) than the former. Thus, at low pH OzHu is less stable than OzHs, but at pH above about 3, human oxyhemoglobin is much more stable than horse oxyhemoglobin. The data for 02Hs are given in Ref. 8.
476
JONES AND STEINHARDT TABLE
RATE
ENHANCEMENT
Human Rhesus Horse a Oxyhemoglobin
III
FACTORS FOR DENATURATION OF HEMOGLOBIN AT 25”CP WHEN IS PRESENT. pH IS 3.0 EXCEPT WHERE STATED OTHERUTSE
1.5 X 10m3~ OCTANOL
Hb+
Hb” stable
Hb” unstable
COHb
OzHP
3.2 4.3 7.6
7.0 (pH 2.5) 2.6 -
3.0 -
1.0 1.0 1.1
2.1 1.4 5.9 (pH 2.40)
at 2°C. Sources of data, and conditions,
that the order of stabilities of the various speciesdiffer for each of the liganded forms. Thus, details of the conformations of the protein moiety of the various liganded forms may well differ. The effect of octanol on Hm” is small: a pH displacement of only 0.12 units. This is just half of that produced by octanol on the lesssteep pH profile of Hu’. At a given pH, however, as a result of the difference in slope, the factor by which the rate is changed by octanol is the same with both proteins: about 2.5. Table III showsthe rate enhancement in the denaturation of hemoglobins due to the presence of octanol. The octanol effect for Hm+ is intermediate between those for Hsf and Huf. Octanol appears to have no observable effect on the denaturation rates of the COHb of either human or rhesus, and a smaller effect on Hu”S and Hu”A.
as in Table
II.
In spite of the very much larger number of sequence variances between rhesus and horse hemoglobin, the kinetic stability for Hm+, relative to that of Huf (as measured by the shift in pH for a given denaturation velocity) is increased by more than half the total difference between human and horse. Smaller differences in stability between human and rhesus than those described above are found in one low-spin ferro form COHb, and negligible differences between speciesin the other low-spin form OzHb. 2. A relatively large difference between the high-spin deoxy forms Hm” and Hu’, manifested in part by a higher dependence on pH for Hm” (slope of log rate-pa profile 2.67) as compared with Hu” (slope of log rate-pH profile 0.86). The results with Hm” represent at pH > 2.4 slightly lessresistance to acidity than the “stable” form of Hu’, and considerably greater stability than the DISCUSSION “unstable” form of Hu”. If all forms of Hm The several differences in stability at low are consistently more stable than their Hu pH between rhesus and human hemoglobin analogs, the Hm” data must correspond to may be attributed to effects of a limited the lessstable form of the two forms of Hu” number of amino acid sequence variances (1). 3. The equilibrium between native and between the two proteins: four in the ~1 chains and eight in the ,8 chains. If this at- acid-denatured Hm+ differs sharply from tribution is valid the larger differences that of Huf and Hs+. It is intermediate in between Hs+ and Hu+, rebetween the stabilities of human and horse “cooperativity” hemdglobins niay possibly be ascribed to quiring about 0.45 pH units to go from 90% the larger number of sequence differences native to 20% native, where Hu+ requires between the hemoglobins of the latter two only 0.35 pH units and Hu+ requires 0.90 pH units. The position of the midpoint of species. The differences in stability properties of the Hm+ equilibrium on the pH axis is inhuman and rhesus hemoglobins which re- termediate between those of Hu+ and Hs+, but lies close to that of Hs+. quire explanation are: 4. The effects of octanol on the unfolding 1. The log rate-pH profile for the ferri rate of Hm+ are smaller t’han with OZHU form is intermediate between those of the same form of horse and human hemoglobin. or with Hu’ but only slightly smaller than
COMPARISON
OF
THE
ACID
DENATURATION
with Hu+. As with COHu there is no effect of octanol at all on the stability of COHm. 5. The effects of octanol on the equilibria of the ferri forms of the three species are highly individual. The effect is small with Hu+, and negligible in Hm+, but there is a very large effect on the cooperativity of the reaction with Hsf (7). Thus, unlike the kinetic diff erenccs, the thermodynamic stability of Hm does not occupy an intermediate position between those of Hu and Hs. To explain the difference in properties of human and monkey hemoglobins on the basis of 12 changes in amino acid sequence between them (roughly 4% of the total possible) we examine those of the I2 positions in which the sequence differences between Hm and Hu are locations in which Hm resembles Hs rather than Hu, since alterations in the positions might result in a conformation of the native protein more like that of the more stable Hs. Thus, in the a! chains, three of the four differences fall in this category (see numbers 19, 68, and 71 in Table I). The last two of these are heme contacts. Two of the three add to the conformational adaptability by substituting gly for ala. The other difference (number S) places ser in place of thr which occurs in both Hs and Hu-this one may, therefore, be trnt~ativrly classified as a neutral substitution. The eight changes in the p chains are not so easily dealt with. Only one of them, number 125 (which is at an a,~ contact), rcprcsents a change toward the sequence of the more stable Hs. This substitut’ion of glutamine for proline, a helix-breaker, might drastically reduce the fragility of the human hemoglobin molecule. Of the other seven, only three (13, 33, 104) are departures in Hm from amino acids found in bot,h Hs and Hu. These three changes are tentat’ively regarded as neutral with respect to stability. The other four (9, 50, 76, 87) may possibly contribute to the stability changes. The last two of these are heme contacts, and one of them (76) is the only location in which a prototropic group, histidine, present, in Hs is absent in both Hm and Hu (there are, however, numerous sequence differences involving protot’ropic groups when only the more widely different Hs and Hu are corn-
OF
HEMOGLOBINS
477
pared). Most of the neutral substitut’ions and three of the others are conservative. Thus, three sequence differences remain which can affect the stability of the native OLchains of Hm and Hu; there are five possiblc such differences in the p chains. These differences involve heme contacts in both chains; and t’he loss of a possible destabilizing pro in the fi chains of Hu. The change at a second (Y,P contact (p 33) may be important, but, for reasons given above, has been tentatively disregarded. It should not be surprising to find that three of the imputed stability-conferring differences between Hm and Hu introduce gly for ala (twice) or for thr (once) in the monkey protein. Every substitution of gly for another residue in a polypeptidc chain enlarges the conformational freedom of the chain without increasing internal strains. Since the human-rhesus differences are larger wit’h the high-spin forms (Hb”, Hb”) than with the lowspin form (OzHb), special significance must be attached to the heme cont’act8s (a 68, o( 71, p 76, p 75) which differ in the two spccirs. oc,p contacts could also be important since such contacts play a part in determining changes in the heme region, when ligands are bound. However, t’he two p chain contacts of Hm are less clearly different from those of Hu, with respect t’o resemblance to the corresponding contacts of the more stable Hs. There is evidence that the kinetic effect of octanol is exerted in the heme pocket (1, 7, also Cassatt and Steinhardt, unpublished). Its effect’ is clearly greater on the high-spin adduct’s in which no covalent bond need be broken for t’he heme to be ejected. The effect of octanol is also somewhat greater on the ferrihemoglobins of t’hc more stab1e species. Thus, t’hr stabilities of the proteins of all three species are more clearly alike in the presence of octanol than in its absencroctanol acts as a partial equalizer of the differences in the ease with which the stabilizing force of the iron-imidazole bond is overcome. However, whatever the mechanism, it affects only a part of the forces included in the activation energy of t,he initial stages of the denaturation reaction. The effect of octanol on the equilibrium between native and denatured hemoglobin
JONES
AND
has been studied mainly in the ferrihemoglobins where the effects are large (1, 7) and so unlike one another (in Hu+ and Hs+) that they remain unexplained. The case of Hm+ contributes yet one other kind of result, in that the alcohol has essentially no effect on the equilibrium, in spite of substantial kinetic effects. This absence of effect on the rhesus equilibrium puts oetanol in the unique class of ligands called catalysts: ligands which affect the velocities of a reversible reaction in both directions to an equal extent. Such a result must depend on having not only a special, and possibly fortuitous, contribution of binding affinities to both native and acid-unfolding forms (4) but also an appropriate ratio of turnover rates for two opposing reactions. Since it has been shown by Polet and Steinhardt (5) that an early stage in the acid denaturation of horse and human ferrihemoglobins involves ej ection of the heme from the heme pocket, it is possible that octanol is equally effective in enhancing both the rate of ejection and the rate of reentry and accommodation in rhesus ferrihemoglobin. It would be reckless to conclude, without further experimental data, that octanol is also without effect on the denaturation equilibria involving other forms of Hu. The
STEINHARDT
energy-barrier to breaking the heme-protein bonds is clearly greater in, for example, carbonylhemoglobin than it is in ferrihemoglobin (6) ; and there is an absence of any effect of octanol on the energy of activation for breaking this bond in carbonylhemoglobin. Clearly there is no possibility of generalizing for all forms of Hu and Hm solely on the basis of the amino-acid sequence differences of the apoproteins. REFERENCES 1. JONES, D. D., MCGRATH, W. P., CARROL, I>., AND STEINHARDT, J. (1973) Biochemistry 12, 3818. 2. STEINHARDT, J., AND HIREMATH, C. B. (1967) J. Biol. Chem. 242, 1294. 3. MATSUDA, G., MAITA, T., OTA, H., AND TAKEI, H. (1970) Znt. J. Protein Res. 2, 99. 4. DAYHOFF, M. 0. (Ed.) (1973) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 5. POLET, H., AND STEINHARDT, J. (1969) Biochemistry 8, 857. 6. ALLIS, J. W., AND STEINHARDT, J. (1970) Biochemistry 9, 2286. 7. CASSATT, J. C., AND STEINHARDT, J. (1971) Biochemistry 10, 3738. 8. CASSATT, J. C., AND STEINHARDT, J. (1971) Biochemistry 10, 264. 9. ALLIS, J. W., AND STEINHARDT, J. (1969) Biochemistry 9, 5075.
OF
BIOCHEMISTRY
Comparison
AND
of the Which DANIEL
Department
BIOPHYSICS
472-478
161,
Acid
Denaturation
Differ
in Amino
D. JONES2 oj Chemistry,
of Several Acid
JACINTO
AND
Georgetown Received
(1974)
University,
October
Hemoglobins
Sequence’ STEINHARDT3 Washington,
DC.
20007
11, 1973
There are pronounced differences in kinetic and thermodynamic stability between human and horse hemoglobins. Since the amino acid sequences of the LY, p dimers of horse and human hemoglobins differ in 61 locations, it is difficult to account for them in terms of specific direct or indirect effects of the sequence differences. Rhesus hemoglobin differs from human in only 12 locations and its stability resembles that of human more closely than does horse, although pronounced differences remain. The stabilities of rhesus ferrihemoglobin and deoxyhemoglobin (Hb+ and Hb”) are intermediate between those of the corresponding high-spin forms of horse and human hemoglobin; but there are only small or negligible differences between the low-spin forms (carbonylhemoglobin and oxyhemoglobin) of the two species. The equilibrium isotherm between native and acid unfolded forms of rhesus Hb+ resembles that of horse more than that of human, but it is slightly more stable and slightly less cooperative. The effects of octanol on the rates of unfolding of rhesus ferrihemoglobin are only slightly smaller than with human. There is no effect of octanol on the unfolding rate of any of the CO hemoglobins. Unlike the equilibria of horse and human, octanol is also without effect on the unfolding equilibrium of rhesus ferrihemoglobin, and thus qualifies as a true catalyst of the initial stage of the acid unfolding reaction of the monkey ferriprotein. Differences in stability are tentatively attributed to a limited number of the 12 differences between the two proteins.
Kinetic stability (rate of denaturation) at extremes of pH is an easily measured general property of the homologous proteins of different species which is affected by details of amino acid sequence. Thermodynamic stability (ratio of amounts of denatured to native conformers as a function of pH) is also affected. Such changes may accompany more specific changes in properties which depend on amino acid sequence, such as the sickling potentiality in human sickle-cell hemoglobin caused by a single (nonconservative) substitution of Val 6 for Glu 6 in the
two p chains of the molecule. The differences in stability in that case are small (1) ; very much larger differences in this respect are found between the hemoglobins of human and horse (2) which differ from one another by 61 sequence differences per CY,@ dimer, about one fifth of the total, although many of the substitutions are conservative. Efforts to relate stability differences to sequence differences in these two species have not succeeded, partly because the differences are so numerous. We have, therefore, sought a species which has only a small number of sequence differences relative to human hemoglobin. Such a species is the rhesus monkey (3, 4). The differences in amino acid sequence between rhesus and human hemoglobin are shown in Table I, which also shows the amino acids in horse
1 Supported by NIH Grant HL 12256. 2 Present address : Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973. 3 To whom requests for reprints should be sent at. the Department of Chemistry, Georgetown University, Washington, D.C. 20007. 472 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
COMPARISON
OF
THE
ACID
DENATURATION TABLE
SEQUENCE
DIFFERENCES
OF
I
HUMAN
AND
RHESUS -------71
or-chains Horse6 Rhesus Human
8
19
I----
thr ser thr A6
dY dY ala AB 1
p-chains Horse Rhesus Human
9 ala asn ser A6
jleu dY lieu FdY jasn ala IE 17 E 20 176 87 /his ala jasn dn jala thr 1E20 F3 /Heme-binding / region ___-----
13 ala thr ala A 10
168
33 val leu val B 15 01, B contact _---------
50 asn ser thr Dl
MONKEY”
u Corresponding amino acids for horse hemoglobins are also shown, as well h Rhesus sequence: Ref. (3); horse and human sequences: Ref. (4).
hemoglobin which occur at loci in which human and rhesus differ. The present paper, therefore, reports kinetic and equilibrium data for the acid denaturation of rhesus monkey hemoglobin, Hm.4 The results for this species are compared with previous published results with Hu A (1, 2, 5, 6) and with Hu S (5-7). This study also makes use of the effect of rz-alcohols on the acid denaturation of hemoglobins to broaden the scope of the stability properties studied. Trace amounts of nalcohols (C&C,) effectively enhance ferrihemoglobin (Hbf) denaturation rates (human and horse) when present at concentrations below low3 M, i.e., at molar ratios of alcohol to Hb+ of 100: 1. The effectiveness 4 Abbreviations table:
used
are given
Ferrihemoglobin
oxyhemoglobin
Hu+A Human hemoglobin A Human heHu+S moglobin 8 Hm+ Rhesus hemoglobin Horse hemoHs globin
in the following
Carbonylhemoglobin
Deoxyhemoglobin
OzHuA
COHuA
Hu”A
OzHuS
COHuS
Hu”S
OzHm
COHm
Hm”
OsHs
COHs
Hs”
Hb or a combination of Hb with other symbols refers to any or all species. With human hemoglobin the suffix A is to be understood if no suffix is given.
473
HEMOGLOBINS
,----__-r
104 arg lys arg G6
1 / I \ I j
as sequence
“--
125 glu gin pro H2 01, @ contact
designations.
of medium length n-alcohols as Hb denaturants is, therefore, nearly as great as that of long chain detergent ions such as dodecylsulfate (Steinhardt and Reynolds, unpublished; Verpoorte, unpublished). On the basis of their results with Hs+, Cassatt and Steinhardt (7) proposed a mechanism of denaturation in which t’he alcohol is postulated to bind at a hydrophobic site in or near the heme pocket. Jones et al. (1) confirmed the general features of the phenomena for Hu+, OnHu, and Hu” but found that octanol has a smaller effect on Hu+ than on Hs+, and that it did not affect COHu at all. Unpublished data obtained by Cassatt and Steinhardt (1972), cited by Jones et al. (l), support the postulated mechanism of action of the alcohols by demonstrating that they quench fluorescence of the dye ANS in the heme pocket of globin. The effects of n-octanol on the acid denaturation rates of Hm+, OzHm, Hm”, and COHm are, therefore, included in this paper. With Hm+, the equilibrium between native and denatured is distinguished from t,hat of Hs+ and Hu+ by its indifference to the presence of octanol. EXPERIMENTAL
PROCEDURES
Materials. A 280.ml aliquot of blood from a. single rhesus monkey (Macaca mulatta) was obtained from Litton Bionetics, Inc., Rockville, MD. After saturation with CO, COHm was crystallized three times as described for COHu (a), and stored frozen in a 4% solution. The stock solutions were diluted to 0.1% before use. 02Hm
474
JONES AND STEINHARDT
was prepared as needed by passing 02 (equilibrated with water vapor) over a solution of COHm which was photodissociated (while cooled under three 303-W lamps). The photodissociation of rhesus COHm was slower than that of COHu. The kinetic and equilibrium measurements were performed as described previously (1). Methods. The stop-flow equipment and prooedures used, and the methods of determining pH were the same as in earlier investigations (1, 2, 5, 6). RESULTS
The pH profiles of the acid denaturation rates of Hu+, Hsf, and Hm+ in the presence and absence of octanol are shown in Fig. 1, in which the data for Hu+ and Hsf from earlier work (1, 5, 6) are represented by continuous lines. In the absence of octanol, the log rate-pH denaturation profile of Hm+ lies between those of Hs+ and Hu+; all three lines have approximately the same slope. The same order holds for the augmentations of the rates in the presence of octano&which are largest for horse, less for monkey, and least for human; the latter is, however, not far below the augmentation for monkey. In round numbers, at pH 3, octanol increases the rate g-fold with horse, 4-fold with rhesus, and only about 3-fold with human ferrihemoglobins. The denaturation equilibria for the same three ferrihemoglobins, as affected by pH
“’
28 3.0 32 34 36 3.8 PH FIG. 1. Dependence of the half-period of acid denaturation of rhesus ferrihemoglobin Hm+ on pH at 25% at 0.02 ionic strength. The light broken line represents the data for human ferrihemoglobin and the light solid line the data for horse ferrihemoglobin. The broken lines represent the data for horse and human ferrihemoglobins in the presence of 1.5 X 10V M octanol.
I
I
I
I
I
3% 4.0 4.2 4.4 4.6 48 PH FIG. 2. The effect of 1.5 X 10-a M octanol on the equilibrium between native and denatured ferrihemoglobin of human, rhesus, and horse as a function of pH at 25°C and 0.02 ionic strength.
and octanol, are given in Fig. 2. As shown earlier (2) the equilibrium for Huf covers a wider pH range than its counterpart for Hs+. Octanol has a much smaller effect (1). The equilibrium for rhesus is intermediate in steepness between those of Hs and Hu and octanol does not affect it. The data for Hm+ are much like those for Hsf when octanol is present except that the curve for Hm+ is displaced to lower pH, i.e., rhesus ferrihemoglobin is thermodynamically more stable than human ferrihemoglobin. Thus the relative magnitudes of the octanol effects on the transition curves fall into a different order than that of the steepnessof the curves with respect to their positions on the pH axis. The octanol effect on the equilibrium is largest for Hsf, and negligible for Hm+. The pH profiles of the acid denaturation rates of human oxyhemoglobin (OzHu) and monkey oxyhemoglobin (OzHm) at 2°C are shown in Fig. 3, and those of normal human deoxyhemoglobin (Hue-two forms) and rhesus deoxyhemoglobin, are shown in Fig. 4. Previously published data for horse oxyhemoglobin (8), which are not shown here, show that the latter is more stable, and that its rate of denaturation has a stronger dependence on pH than either human or rhesus. Human and rhesus oxyhemoglobin are also much alike with respect to their kinetic stabilities except that the rhesus protein has a smaller susceptibility to octanol (Fig. 3). It has been shown elsewhere (1) that human deoxyhemoglobin may be obtained in two forms, one of which, “stable” Hu’, is denatured at acid pH about one-tenth as
CO?VIPARISON
OF
THE
ACID
DENATURATION
rapidly as the other, L’unstable” Hu”. Rhesus deoxyhemoglobin (Fig. 4) is more stable than ‘kmstable” Hu’, but less stable than “stable” Hu” (1). It should be noted that the slope of the pH profile of “stable” Hu” is very close to the corresponding slope for Hm”; although the latter is displaced to higher pH by 0.2 pH units. We may con-
OF
TABLE pH
475
HEMOGLOBINS II
ST WHICH HALF-PERIODS FOR ACID DKNATURATION ARE 1 set AT 25°C OR 2”0 VALUE
Human Rhesus Horse
A*
Hb+
Hb” stable
Hb’ unstable
COHb
OnHb
3.12 3.02 2.956
2.25 2.49 2.75d
2.77
2.55 2.41 2.58”
2.0 2.0 1.6”
-
(1 The data for Hb+ and COHb were taken at 25’C in HCl-KC1 or acetate-KC1 solutions of 0.02 ionic strength. The data for Hb” were taken at 25°C in chloroacetate buffer of 0.025 ionic strength. The OlHb data were taken at 2°C in HCl-KC1 solutions of 0.03 ionic strength. * Ref. (1). May be compared with the Hb+ data of (5) and (6). c Ref. (5). May be compared with the Hb+ data of Ref. (9) or the COHb data of (6). d Ref. (8). Ionic strength 0.05.
PH FIG. 3. The rates of denaturation of human and rhesus oxyhemoglobins with and without 1.5 X W3 M octanol as a function of pH at 2°C and 0.02 ionic strength.
‘LIZ: Hu” with octanol * Hm’ with octanol
2.2
2.4
2.6
2.8
3.0
PH
FIG. 4. The effect of pH and 1.5 X lWa M octanol on the half-periods of denaturation of rhesus deoxyhemoglobin at 25°C and 0.025 ionic strength. For comparison, lines representing data for both “unstable” and “stable” human deoxyhemoglobin are also included. The data for “unstable” human deoxyhemoglobin are indicated by the two uppermost lines in the figure.
elude that Hm” as well as the liganded forms is slightly more resistant to low pH than its human analogs. The data for Hs” given in Table II show that horse deoxyhemoglobin is appreciably less stable than either rhesus or human deoxyhemoglobins; and is, in fact, about as unstable as the “unstable” form of Hu” (1). Data for COHu and COHm and also COHs are included in Table II which tabulates the relative stabilities of the various hemoglobin toward acid denaturation by listing the pH values corresponding to a half-period of 1 sec. Comparisons between columns indicate that COHb is usually the most stable for each of the speciesstudied at 25°C (except for ‘ktable” human deoxyhemoglobin). Since the slopes of the pH-log half-period profiles differ from one form to another,5 comparisons of stabilities would differ if pH values were listed for much shorter half-periods, i.e., 100 msec instead of 1 sec. Attention is also invited to the fact 6 An extreme exampIe is provided by the oxyhemoglobins of human and horse, in which the latter has a much steeper pH profile (slope 2.3) than the former. Thus, at low pH OzHu is less stable than OzHs, but at pH above about 3, human oxyhemoglobin is much more stable than horse oxyhemoglobin. The data for 02Hs are given in Ref. 8.
476
JONES AND STEINHARDT TABLE
RATE
ENHANCEMENT
Human Rhesus Horse a Oxyhemoglobin
III
FACTORS FOR DENATURATION OF HEMOGLOBIN AT 25”CP WHEN IS PRESENT. pH IS 3.0 EXCEPT WHERE STATED OTHERUTSE
1.5 X 10m3~ OCTANOL
Hb+
Hb” stable
Hb” unstable
COHb
OzHP
3.2 4.3 7.6
7.0 (pH 2.5) 2.6 -
3.0 -
1.0 1.0 1.1
2.1 1.4 5.9 (pH 2.40)
at 2°C. Sources of data, and conditions,
that the order of stabilities of the various speciesdiffer for each of the liganded forms. Thus, details of the conformations of the protein moiety of the various liganded forms may well differ. The effect of octanol on Hm” is small: a pH displacement of only 0.12 units. This is just half of that produced by octanol on the lesssteep pH profile of Hu’. At a given pH, however, as a result of the difference in slope, the factor by which the rate is changed by octanol is the same with both proteins: about 2.5. Table III showsthe rate enhancement in the denaturation of hemoglobins due to the presence of octanol. The octanol effect for Hm+ is intermediate between those for Hsf and Huf. Octanol appears to have no observable effect on the denaturation rates of the COHb of either human or rhesus, and a smaller effect on Hu”S and Hu”A.
as in Table
II.
In spite of the very much larger number of sequence variances between rhesus and horse hemoglobin, the kinetic stability for Hm+, relative to that of Huf (as measured by the shift in pH for a given denaturation velocity) is increased by more than half the total difference between human and horse. Smaller differences in stability between human and rhesus than those described above are found in one low-spin ferro form COHb, and negligible differences between speciesin the other low-spin form OzHb. 2. A relatively large difference between the high-spin deoxy forms Hm” and Hu’, manifested in part by a higher dependence on pH for Hm” (slope of log rate-pa profile 2.67) as compared with Hu” (slope of log rate-pH profile 0.86). The results with Hm” represent at pH > 2.4 slightly lessresistance to acidity than the “stable” form of Hu’, and considerably greater stability than the DISCUSSION “unstable” form of Hu”. If all forms of Hm The several differences in stability at low are consistently more stable than their Hu pH between rhesus and human hemoglobin analogs, the Hm” data must correspond to may be attributed to effects of a limited the lessstable form of the two forms of Hu” number of amino acid sequence variances (1). 3. The equilibrium between native and between the two proteins: four in the ~1 chains and eight in the ,8 chains. If this at- acid-denatured Hm+ differs sharply from tribution is valid the larger differences that of Huf and Hs+. It is intermediate in between Hs+ and Hu+, rebetween the stabilities of human and horse “cooperativity” hemdglobins niay possibly be ascribed to quiring about 0.45 pH units to go from 90% the larger number of sequence differences native to 20% native, where Hu+ requires between the hemoglobins of the latter two only 0.35 pH units and Hu+ requires 0.90 pH units. The position of the midpoint of species. The differences in stability properties of the Hm+ equilibrium on the pH axis is inhuman and rhesus hemoglobins which re- termediate between those of Hu+ and Hs+, but lies close to that of Hs+. quire explanation are: 4. The effects of octanol on the unfolding 1. The log rate-pH profile for the ferri rate of Hm+ are smaller t’han with OZHU form is intermediate between those of the same form of horse and human hemoglobin. or with Hu’ but only slightly smaller than
COMPARISON
OF
THE
ACID
DENATURATION
with Hu+. As with COHu there is no effect of octanol at all on the stability of COHm. 5. The effects of octanol on the equilibria of the ferri forms of the three species are highly individual. The effect is small with Hu+, and negligible in Hm+, but there is a very large effect on the cooperativity of the reaction with Hsf (7). Thus, unlike the kinetic diff erenccs, the thermodynamic stability of Hm does not occupy an intermediate position between those of Hu and Hs. To explain the difference in properties of human and monkey hemoglobins on the basis of 12 changes in amino acid sequence between them (roughly 4% of the total possible) we examine those of the I2 positions in which the sequence differences between Hm and Hu are locations in which Hm resembles Hs rather than Hu, since alterations in the positions might result in a conformation of the native protein more like that of the more stable Hs. Thus, in the a! chains, three of the four differences fall in this category (see numbers 19, 68, and 71 in Table I). The last two of these are heme contacts. Two of the three add to the conformational adaptability by substituting gly for ala. The other difference (number S) places ser in place of thr which occurs in both Hs and Hu-this one may, therefore, be trnt~ativrly classified as a neutral substitution. The eight changes in the p chains are not so easily dealt with. Only one of them, number 125 (which is at an a,~ contact), rcprcsents a change toward the sequence of the more stable Hs. This substitut’ion of glutamine for proline, a helix-breaker, might drastically reduce the fragility of the human hemoglobin molecule. Of the other seven, only three (13, 33, 104) are departures in Hm from amino acids found in bot,h Hs and Hu. These three changes are tentat’ively regarded as neutral with respect to stability. The other four (9, 50, 76, 87) may possibly contribute to the stability changes. The last two of these are heme contacts, and one of them (76) is the only location in which a prototropic group, histidine, present, in Hs is absent in both Hm and Hu (there are, however, numerous sequence differences involving protot’ropic groups when only the more widely different Hs and Hu are corn-
OF
HEMOGLOBINS
477
pared). Most of the neutral substitut’ions and three of the others are conservative. Thus, three sequence differences remain which can affect the stability of the native OLchains of Hm and Hu; there are five possiblc such differences in the p chains. These differences involve heme contacts in both chains; and t’he loss of a possible destabilizing pro in the fi chains of Hu. The change at a second (Y,P contact (p 33) may be important, but, for reasons given above, has been tentatively disregarded. It should not be surprising to find that three of the imputed stability-conferring differences between Hm and Hu introduce gly for ala (twice) or for thr (once) in the monkey protein. Every substitution of gly for another residue in a polypeptidc chain enlarges the conformational freedom of the chain without increasing internal strains. Since the human-rhesus differences are larger wit’h the high-spin forms (Hb”, Hb”) than with the lowspin form (OzHb), special significance must be attached to the heme cont’act8s (a 68, o( 71, p 76, p 75) which differ in the two spccirs. oc,p contacts could also be important since such contacts play a part in determining changes in the heme region, when ligands are bound. However, t’he two p chain contacts of Hm are less clearly different from those of Hu, with respect t’o resemblance to the corresponding contacts of the more stable Hs. There is evidence that the kinetic effect of octanol is exerted in the heme pocket (1, 7, also Cassatt and Steinhardt, unpublished). Its effect’ is clearly greater on the high-spin adduct’s in which no covalent bond need be broken for t’he heme to be ejected. The effect of octanol is also somewhat greater on the ferrihemoglobins of t’hc more stab1e species. Thus, t’hr stabilities of the proteins of all three species are more clearly alike in the presence of octanol than in its absencroctanol acts as a partial equalizer of the differences in the ease with which the stabilizing force of the iron-imidazole bond is overcome. However, whatever the mechanism, it affects only a part of the forces included in the activation energy of t,he initial stages of the denaturation reaction. The effect of octanol on the equilibrium between native and denatured hemoglobin
JONES
AND
has been studied mainly in the ferrihemoglobins where the effects are large (1, 7) and so unlike one another (in Hu+ and Hs+) that they remain unexplained. The case of Hm+ contributes yet one other kind of result, in that the alcohol has essentially no effect on the equilibrium, in spite of substantial kinetic effects. This absence of effect on the rhesus equilibrium puts oetanol in the unique class of ligands called catalysts: ligands which affect the velocities of a reversible reaction in both directions to an equal extent. Such a result must depend on having not only a special, and possibly fortuitous, contribution of binding affinities to both native and acid-unfolding forms (4) but also an appropriate ratio of turnover rates for two opposing reactions. Since it has been shown by Polet and Steinhardt (5) that an early stage in the acid denaturation of horse and human ferrihemoglobins involves ej ection of the heme from the heme pocket, it is possible that octanol is equally effective in enhancing both the rate of ejection and the rate of reentry and accommodation in rhesus ferrihemoglobin. It would be reckless to conclude, without further experimental data, that octanol is also without effect on the denaturation equilibria involving other forms of Hu. The
STEINHARDT
energy-barrier to breaking the heme-protein bonds is clearly greater in, for example, carbonylhemoglobin than it is in ferrihemoglobin (6) ; and there is an absence of any effect of octanol on the energy of activation for breaking this bond in carbonylhemoglobin. Clearly there is no possibility of generalizing for all forms of Hu and Hm solely on the basis of the amino-acid sequence differences of the apoproteins. REFERENCES 1. JONES, D. D., MCGRATH, W. P., CARROL, I>., AND STEINHARDT, J. (1973) Biochemistry 12, 3818. 2. STEINHARDT, J., AND HIREMATH, C. B. (1967) J. Biol. Chem. 242, 1294. 3. MATSUDA, G., MAITA, T., OTA, H., AND TAKEI, H. (1970) Znt. J. Protein Res. 2, 99. 4. DAYHOFF, M. 0. (Ed.) (1973) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 5. POLET, H., AND STEINHARDT, J. (1969) Biochemistry 8, 857. 6. ALLIS, J. W., AND STEINHARDT, J. (1970) Biochemistry 9, 2286. 7. CASSATT, J. C., AND STEINHARDT, J. (1971) Biochemistry 10, 3738. 8. CASSATT, J. C., AND STEINHARDT, J. (1971) Biochemistry 10, 264. 9. ALLIS, J. W., AND STEINHARDT, J. (1969) Biochemistry 9, 5075.