THE JOURNAL OF BIOLOGICAL CIIE~~ISTRY
Vol. 239, No. 2, February 1964
Printed in U.S. A.
Changes in Side Chain Reactivity Accompanying the Binding
of Heme to Sperm Whale Apomyoglobin*
From the Department of Biochemistry, Cornell University Medical Co&e, New York 21, New York
(Received for publication, August 30, 1963)
Considerable attention has been devoted recently to the study present was oxidized by either CuCIZ (9) or K3Fe(CN),r followed
of conformational changes occurring during enzyme-substrate by exhaustive dialysis finally against distilled HzO.
interaction (1, 2). Presumably the nature of the interaction Initially globin was prepared from metMb2 by acetone precipi-
between apoproteins and their prosthetic groups is essentially tation according to the method of Theorell and ikeson (10).
similar to that between enzymes and substrates, with the excep- Subsequently, a modification of Teale’s 2-butanone extraction
tion that no catalytic step occurs subsequent to the initial bind- procedure (11) was employed. An approximately 1 y0 deionized
ing. Studies of the physicochemical changes accompanying the solution of metMb was lowered to pH 1.5 at, 0” and then ex-
binding of heme to sperm whale apomyoglobin would seem to be tracted at 4”, first with an equal volume of 2-butanone (Merck
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of particular interest in t.hat the three-dimensional conformation reagent) and twice more with half-volumes of 2-butanone, al-
of sperm whale metmyoglobin is now known (3), and conforma- though the last extraction was generally found to be superfluous.
tional changes occurring on heme-globin interaction may there- The resulting, slightly straw-colored solution was then immedi-
fore be more amenable to interpretation. ately dialyzed at 4” in 18/32 Visking tubing, usually first against
Kinetic studies of heme-globin interaction suggest that the several changes of dilute NaHC03 (50 mg per liter) and finally
initial product of the reaction must rearrange to form the native against several changes of glass-distilled HzO. The resultant
heme-protein (4). Conceivably this rearrangement involves a solution was concentrated to less than half it,s original volume
change in protein conformation. Optical rotatory dispersion by evaporation through the dialysis membrane, and then cen-
studies of metmyoglobin give values for the a-helix content of trifuged to remove some colored precipitate, t,he protein solution
approximately 75% (5, 6), whereas similar studies of globin itself being completely colorless in good prepa.rations. This
suggest a helix content of 50%.’ In view of the differing Cotton solution was then adjusted to be 0.16 M in KCl, and any addi-
effects in the two proteins, however, it is difficult to assess the tional precipitate was removed by centrifugation. (It was
significance of this disparity. In a preliminary communication found, however, that only when the original NaHC03 dialysis
(7), this laboratory cited discrepancies between the H+ ion was omitted was any additional protein precipitated by KCl, so
titration curves of globin and metmyoglobin which could pos- that this step is unnecessary if NaHC03 dialysis is introduced.)
sibly be interpreted as indications of subtle conformational Many properties of the globin so obtained have already been
differences. The primary aim of the present study is to examine described (7). Two additional observations are also of interest.
in greater detail the significance of the relative reactivities of First, removal of the heme is completely insured only if the pH
globin and myoglobin derivatives to H+ ion. Secondarily the of the initial metMb solution is near 1.5. Second, the stability
observed differences in reactivity of imidazoles to H+ ion in the of the resultant globin is markedly lowered in the presence of
two proteins will be related to changes in reactivity to bromo- heavy metal ions. In the cold, in 0.16 M KCl, the presence of
acetic acid, a reagent for which the reaction with metmyoglobin trace metal ions will result in the gradual precipitation of de-
imidazoles has been recently documented (8). natured globin. In recent preparations, inclusion of a dialysis
step with 1 X lop4 M disodium EDTA before dialysis against
EXPERIMENTAL PROCEDURE redistilled Hz0 resulted in globin preparations which were per-
fectly stable for over 1 month when stored in 0.16 M KC1 at 4”.
Reagents-Sperm whale myoglobin was generously supplied by Apart from stability on standing, however, no differences among
Professor F. R. N. Gurd. All reagents, unless otherwise speci- all the globin preparations in properties such as [ar]:‘, sedimenta-
fied, were analytical grade, and water was glass-distilled. tion constant, or titration were apparent, irrespective of the
Preparation of Globin-Globin was prepared only from met- method of preparation.
myoglobin. To insure that myoglobin was ent,irely in the Fe3+ Globin solutions were standardized either directly by dry
state before removal of heme, all myoglobin preparations were weight determinations or indirectly by the Folin-Lowry (12)
examined spectrophotometrically, and any reduced protein assay, with a standard curve obtained with known weights of
* This investigation was supported by Grant HE-02739 from Combination of Globin with Heme-The heme preparations
the National Heart Institute, United States Public Health Serv- used for recombination studies were either hematin, C grade
ice. A preliminary account of this work was presented at the
145th National Meeting of the American Chemical Society, Sep- (Calbiochem), or analyzed hemin, 8.61% iron (British Drug
tember 9 to 13, 1963, New York. 2 The abbreviations used are: m&Mb, sperm whale metmyo-
1 J. Schellman, personal communication. globin; Mb, myoglobin; metHb, methemoglobin; Hb, hemoglobin.
February 1964 I:‘. Breslow 487
Houses, Ltd., London).3 The combining capacity of globin for central thermostated bath w-as pumped in parallel through the
heme was determined as follows. A small quantity of heme was thermospacers of the Beckman DU spectrophotometer and the
dissolved in 0.05 ml of N NaOH and immediately diluted in water-jacket.ed, covered titration vessel in which the optical
borate buffer, pH 9.2, to a final concentration of 1 mM. Increas- density and pH, respectively, were simultaneously recorded.
ing aliquots of the heme solution were then immediately added The Radiometer TTTla pH meter was used for all studies at 25”
to globin solutions in borate buffer, pH 9.2, ionic strength 0.16. and 41”, and the Radiometer model 4 was used for studies at 9”.
At varying time intervals, aliquots were taken and diluted in pH standards used were 0.05 M potassium hydrogen phthalate,
FDTA-acetat’e buffer, pH 5.6 (13), and the optical density at Beckman pH 7 and pH 10 standards, and 0.01 M NaOH in 0.14
409 rnp was compared wit.h a similar series of solutions containing M KC1 with Harned activity coefficients (16). For studies at
heme but no globin. With this method, combination was found 25” and 41”, protein solutions were prepared at room temperature
to be almost complete within 5 minutes and completed within before optical density and pH recording. For studies at 9”,
2$ hours. The combining capacity so determined was 1 mole of solutions were prepared at 0” and then equilibrated at 9” to
hcme per mole of globin, although spurious results were obtained minimize irreversible temperature effects (although this has
if the heme solution was allowed to remain in alkali too long subsequently been found unnecessary). At 41”, a faint turbidity
before addition to globin. was present in globin solutions near neutrality at 0.16 ionic
Isolation of regenerated metMb apparently free of either excess strength, but not at 0.02 ionic strength. Values of percentage
globin or heme was accomplished on a large scale by mixing a of ionization at this temperature were therefore calculated by
1 yh solution of globin with a slight excess of hemin in borate using the molar extinction at pH 7 found for the lower ionic
buffer, pH 8.9, ionic strength 0.16. The resultant solution was strength.
exhaustively dialyzed against HzO, and a slight precipit,ate All optical densities were determined in l-cm covered cuvettes.
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(presumably containing excess heme and some protein) was Temperature regulation was within +O.l” at’ 25” and 41”, and
removed by centrifugation. Dry weight, spectral, and titration within +0.5” at 9”.
studies of the resulting supernatant revealed the product to be Optical Rotation Studies-Optical rotation studies were per-
essentially indistinguishable from native metMb, as will be formed at the sodium D-line with a Schmidt and Haensch polar-
discussed subsequently. Recovery of metMb by this met,hod imeter, model 58, in a room regulated at 19”. Studies of the
was 82 %. effect of pH on rotation of globin at different temperatures were
Ultracentrifugal Xt&ies---Sedimentation rates of globin and performed as follows. An approximately 1% solution of globin
metmyoglobin were determined in a Spinco model E ultracen- in 0.16 M KC1 was adjusted with N NaOH to the desired pH at
trifuge equipped with phase plate schlieren optics and automatic the appropriate temperature. The solution was then trans-
temperature control. With Spectroscopic II-G plates (Kodak) ferred to the covered polarimeter tube which was immersed in a
for metMb studies, usable patterns could be obtained without water bath at the same temperature. The tube was maintained
the use of special filters at concentrations near 1% by increasing in the water bath until immediately before the readings, at which
the exposure time to 7 seconds. time it was quickly dried and readings were taken over a period
Hydrogen Ion Equilibria-The isoionic pH was determined of about 5 minutes and extrapolated to zero t,ime. The temper-
after passage through a Dintzis deionizing column (14). Con- ature of each solution can then probably be regarded as accurate
tinuous potentiometric H+ ion titrations were performed as to only within ~2”.
previously described (13). For globin and metMb, continuous Reaction of Globin. with Brornoacetic Acid-Bromoacetic acid
titration curves were obtained between pH 11 and 3 at 25” and (Fisher, “highest purity”) was recrystallized from toluene and
41” and between pH 12 and 3 at 9”. Several additional poinm petroleum ether.
at more alkaline pH were obt,ained by discontinuous titration. To native globin in 0.16 M KCl, pH 7, enough of a self-buffered
Spectrophotometric studies of phenolic equilibria were made solution (pH 7.9) of K2HP04 and bromoacetic acid was added so
with globin, metMb, CN--metMb, and CO-myoglobin. CO- that the final conditions were 1% globin, 1 M phosphate, and
Mb for this purpose was prepared as described by Hermans (15)) 0.2 M bromoacetic acid, pH 7.4. The mixture was allowed to
stored under CO, and standardized by dry weight determination. stand at room temperature with occasional shaking for 7 days.
C--m&Mb studies were conducted with metMb in the presence Moderate precipitation was generally observed during the course
of 0.01 M NaCN; the cyanide derivative was spect.rophotometri- of the reaction. In one reaction, therefore, precipitate and
tally demonstrated to be stable to pH 12.9 at 25”. supernatant fractions were separated and then individually
In globin, studies of tyrosine ionization were conducted at both exhaustively dialyzed against H20 and lyophilized. Amino acid
245 and 295 rnp with essentially identical titration curves calcula- analyses of the two fractions? indicated no significant differences.
ble at both wave lengths. As previously cited (7), globin tyro- In the subsequent reaction, then, precipitate and supernatant
sine ionization studies at 0.16 ionic strength gave identical results were not separated but dialyzed, lyophilized, and analyzed
whether conducted at 0.05% protein concentration in glycine- together. Amino acid analyses of the two runs gave identical
KC1 buffer or at 0.4% protein concentration in KC1 alone. All results. Tryptic hydrolysis and peptide mapping of the car-
studies reported here, therefore, were conducted at approximately boxymethylated globin preparations were performed by Dr. L. J.
O.OSy, protein concentration in 0.01 M glycine-KC1 buffer, 0.16 Banaszak and Professor F. R. N. Gurd as previously described
ionic strength. The optical densities of all protein solutions (8).
were obtained versus Hz0 ait.h the appropriate buffer blanks One reaction with acid-denatured globin was also investigated.
subsequently subtracted. Here, globin in 0.16 M KC1 was first allowed to stand at pH 2.9
To insure adequate temperature regulation, water from a 4 Amino acid analysis and peptide mapping of carboxymethyl-
ated globin were performed by Professor F. R. N. Gurd and Dr. L.
3 This hemin was a gift of Dr. Nevenka Rumen. Banaszak.
488 Changes Accompanying Heme Binding to Apomyoglobin Vol. 239, No. 2
TABLE I native metMb and in regenerated metMb prepared from 2-
Major absorption bands of nativs and regenerated metmyoglobin butanone-extracted globin are compared. It is readily apparent
that no significant difference exists between the two proteins.
- In Fig. 1, the titration curve of the same regenerated metMb
Sative Regenerated Native Regenerated
is compared with that obtained for native metMb. No signifi-
cant differences between the two proteins are apparent upon
my WJ X104 Xl@ titration from pH 9 to 3 with HCl. The back-titrat.ion curve,
630 630-635 0.35 0.35 obtained by rapid titration from pH 3 with NaOH, is similar to
505 505 0.93 0.91 that obtained with some but not all metMb preparations. The
409 409 16.0 15.9 reason for this behavioral difference among various metMb
280 280 3.1 3.2
I I preparations is not apparent, but as the continuous rapid back-
titration represents a nonequilibrium situation during which
I 1 I I I I some native protein appears to be regenerated, it may be simply
REGENERATED NATIVE a reflection of differences in protein concentration or in the length
30 of time for which t,he protein was allowed to remain at pH 3.
i I pH9+-3 0.0 - 1
In any event, the globin studied appears to have a conformation
pH3+8 000 --- capable of reacting with heme to give metMb. Presumably the
preparations) preparation of globin by the 2-butanone extraction procedure,
which involves exposure to pH 1.5, is not deleterious despite the
fact that globin is denatured at this pH (7), because this de-
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20 naturation, like that of metMb itself, is reversible.
Ultracentrifugal Studies of Globin and Metmyoglobin-A some-
what crude indication of the relative conformations of globin and
15 c PRECI PlTATlON OF metMb may be obtained from the ratio of their frictional coef-
ficients (.fglobin/fmetMb). F rom t,he known molecular weight of
the two proteins and their sedimentation rates at infinite dilution
under comparable conditions of pH and ionic strength, this
ratio may be obtained from the equation
f- globin = S2o,w(metMb) mol. wt.,l,bi,(=17,197)
fmetMb &Xl,w(globin) mol. wt. metMb(= 17,830)
The sedimentation rates of globin and metMb at several con-
centrations in phosphate-KC1 buffer, ionic strength 0.17, pH
6.74, were determined at 25” and, although unnecessary for
comparative purposes, were corrected to ~20,~ by using values of
0.74 for the partial specific volume and density and viscosity
data interpolated from the International Critical Tables. The
results are shown in Fig. 2. For metMb, .sz~.~ at infinite dilution
is 1.98. For globin, the best value of .sz~,~ at infinite dilution is
6~H7 8 ' 1.87. The measured ratio of frictional coefficients, 1.02, would
1. titration curves of native metMb and seem to suggest that no major conformational difference exists
metMb regenerated from globin and heme. Temperature, 25”; between the two proteins. Theorell and ikeson (10) have
ionic strength, 0.16. For regenerated metMb: l , titration from reported values of szo,W at infinite dilution of 1.84 and 1.96 for
pH 9 to pH 3; 0, back-titration from pH 3. For native metMb: horse apomyoglobin and CO-myoglobin, respectively. On the
--, titration from pH 9 to pH 3; - - -, back-titration of two dif- assumption that the horse and whale myoglobins have the same
ferent preparations from pH 3.
molecular weights, these sedimentation data similarly suggest
that no definitive conformational differences between globin and
for 30 minutes at room temperature and then rapidly adjusted
myoglobin are observable by such gross measurements. Vis-
to pH 7 with NaOH before addition of KtHP04 and bromoacetic
cosity studies by Eylar5 of sperm whale globin and metMb lead
to similar conclusions.
RESULTS AND DISCUSSION Equilibria of Imidazoles with H+ Ions in Globin and in Met-
myoglobilz-Analysis of the titration curve of metMb has shown
Properties of Regenerated Jfetmyoglohin-The ease of regenerat-
that only 6 of the 12 metMb imidazoles are in H+ equilibrium
ing native metMb from globin prepared by acid-acetone pre- in the native protein and that the remainder were masked in the
cipitation has been well documented (10). On the other hand,
unprotonated form and released upon acid denaturation (13).
globin prepared by 2-butanone extraction has not been as thor-
In a preliminary account (7), differences in H+ ion titration
oughly studied. As previously stated (see “Experimental between globin and metMb in the neutral pH range were inter-
Procedure”), we have found no demonstrable differences in the
preted to indicate the release of 2 to 3 additional imidazoles into
globin prepared by these two procedures provided that all ma- H+ ion equilibrium upon removal of heme from metMb. In
terial insoluble in 0.16 M KC1 at pH 7 to 8 is removed. In Table
I, the positions and intensities of the major absorption bands in 5 E. Eylar, personal communication.
February 1964 E. Rreslow 489
some small part, this analysis was based on a tentatively revised
(and now abandoned) assumption of a total of 11 histidine
residues in metMb.6 The following more detailed accounting of
globin imidaaole H+ ion equilibria will rest on the now more
definite total of 12 metMb imidazoles. It should be noted,
however, that there is a discrepancy between the observed
isoionic pH values of metMb and globin and the most recent
amino acid analyses (17), some small part of which may reside
in the imidazole analysis.
In Table II, the current estimate of titratable groups in metMb
is given. The isoionic pH of metMb is 7.86 and is unchanged by
essentially complete guanidination (18). No lysine residues
therefore titrate below pH 7.86, and even if complete depro-
tonation of imidazoles is assumed at this pH (an assumption not
completely warranted by the estimated imidazole log k’),7 it is
apparent that at pH 7.86 the calculated charge should be at
least 1.5. In globin this discrepancy is again seen. The iso- l .80 ’ I I I I I I
I I I I
ionic pH of 8.63 is compatible with that of metMb when the .2 .4 .6 .8 1.0 1.2 I.4
effect of heme removal is allowed for (7). Here, even generously PROTEIN CONCENTRATION (%).
allowing for complete deprotonation of the carboxyls, or-NHz, FIG. 2. Sedimentation of globin and metMb as a function of
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12 imidazoles, and 0 to 1 e-NH2 group at this pH (a number in protein concentration in potassium phosphate-KC1 buffer, ionic
accord with the maximal acid-binding capacity of 34 to 35 (7)), strength 0.17, pH 6.74. l , metMb; 0, globin.
the calculated charge is 1 to 2 at pH 8.63. At present, however,
there is no recourse other than to assume the correctness of the TABLE II
amino acid analysis as given in Table II. Tit&able side chains 0.i sperm - whale metmyo obin
The difference in titration behavior of the native and acid- Log k’ in native
Side chain No. protein at 25’ Reference
denatured forms of met,Mb, i.e. the titration difference curve, is
primarily a reflection of the difference in w, the electrostatic work
factor,7 and the number of imidazoles in H+ ion equilibria in the or-Carboxyl*. ................ 1 (13, 7)
Porphyrin carboxyls*. ....... 2 4.48 (13, 7)
two species (13). As a first approximation, the value of w for
P,y-Carboxyls*. ............. 20 4.48 (13, 7)
a given class of groups in both native metMb and native globin Reactive imidazoles. ........ 6 6.70 (13, 7)
may be considered the same,8 as the sedimentation data indicate Masked imidazoles. ......... 6 (13)
no major conformational differences between the two native a-NH2 ...................... 1 7.80 (13)
proteins. Moreover, w for the two acid-denatured proteins Fe(OH)z+. .................. 1 8.90 (13)
appears to be the same. Analysis of carboxyl ionization in E-NH*. ...................... 19 -10.5 (13)
denatured globin at 0.16 ionic strength and 25” can be shown to Guanidinium ................ 4
give average values of 0.033 and 4.41 for w and log k’, respec-
* Log k’ for carboxyl titration was determined by assuming the
tively, for the 21 globin carboxyls, in good agreement with the
same acidity for all carboxyl groups. Undoubtedly, however,
values of 0.034 and 4.49 obtained for the 23 carboxyls of de-
the a-carboxyl log k’ is lower than t,hat of the ot.her carboxyl
natured metMb (13). A rough approximation of the relative groups.
number of imidazoles released into H+ ion equilibrium in globin
and in metMb can then be obtained from the relative magnitudes near pH 4.6 for metMb. In Table III, the relative heights of
of t,he difference curves alkaline to the pH of denaturation. At
the difference curves (AT*) in the two proteins are shown. If
25” and 0.16 ionic strength, estimat’ion of t’he difference curve is a release of 6 imidazoles upon acid denaturation in metMb is
complicated by precipitation of the denatured protein near pH 6. assumed, the number of imidazoles released in globin upon
The pH of precipitation, however, is increased to above 7 if the
denaturation most closely approximates 3. The number of
t.emperature is lowered to 9”, and more reproducible difference imidazoles estimated by t’his method to be in H+ ion equilibrium
curves are obtained. In Fig. 3, the titration curves of globin in native globin therefore would be 9, as compared to 6 in native
and m&Mb at 9” and 0.16 ionic strength are shown. At this
t,emperature, globin denaturation appears to occur gradually A more accurate measure of the number of imidazoles in Hf
between pH 5.5 and 5.2 as compared to a more abrupt transition ion equilibrium would depend only upon analysis of the titration
6 F. R. N. Gurd, personal communication. curve of the native protein. A preliminary titration curve
7 The terms in Equation 2 are defined as follows: log Ic’ = the analysis based upon the increased number of groups titrating in
intrinsic H+ ion association constant; w = the electrostatic work globin relative to metMb in the neutral pH range suggested that
factor; s!? = the net protein charge; ni = the number of titratable 8 or 9 imidazoles were in H+ ion equilibrium in native globin (7).
groups in a given class; and fiH = the number of protonated groups
This analysis rested on the assumption that the intrinsic pK of
in that class.
8 This assumption is based on the more approximate treatment the t,itratable globin histidines was identical with that found for
of titration curves with a distributed charge model (19). It is the same in metMb. A more precise analysis may be obtained
recognized, however, that with the fixed charge model of Tanford from the equation7
and Kirkwood (21), zu might be expected to vary for different
classes of groups within the same protein. pH = log k’ - wz(0.868) - log[tia/(ni - ;<a)] (2)
490 Changes Accompanying Heme Binding to Apomyoglobin Vol. 239, No. 2
I , I I I I , the alkaline branch of the globin titration curve, but does not
METMb TITRATION. 9%. significantly affect the results. The other log k’ values are those
--- shown in Table II.)
The data obtained suggest that the titration curves are most
GLOBIN TITRATION. 9OC. readily fit with a value of ni = 9 if a single class of histidines is
0 l pii 11-3 assumed. The titration curves shown in Fig. 5 can be shown
0 0 pH 3-8
to be completely reversible under the existent titration conditions
to a value of 2 = +9. However, significant deviations from
25 O--O pH3+7
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-20 ! I I I I
3 4 5 6 7pH 8 9 IO II
FIG. 3. Titration curves of globin and met&lb at 9”, ionic 0
strength 0.16, and partial titration curve of globin at 41”, ionic
strength 0.16. For metMb: --, titration from pH 11.3 to pH 3;
- - -, back-titration from pH 3. For globin at 9”: l , titration
from pH 11.4 to pH 3; 0, back-titration from pH 3. For globin
at 41”: A, titration from pH 8 to pH II.
FIG. 4. Continuous titration of globin at 25” in KCl. At ionic
TABLE III strength 0.16: l , pH 9 to pH 3; 0, back-titration from pH 3. At
Relative magnitudes o.f diference curves in globin and in ionic strength 0.06: n , pH 9 to pH 3; q , back-titration from pH 3.
mctmyoglobin at 9’ and 0.16 ionic strength
7.2 I I I
AvH Globin imidazoles
PH released by
5.8 1.8 4.0 2.7
6.0 1.8 3.6 3.0
6.2 1.7 3.2 3.2
6.5 1.4 2.6 3.2
6.8 1.0 2.0 3.0
7.0 0.5 1.6 1.9
* These values are based on the assumption that the number
of imidazoles released by acid denaturation in metMb is 6.
by the usual plots of log FirI/(ni - pin) against z, assuming vary-
ing values of ni, to determine log k’ and w for the globin histi-
dines. The average of a large number of globin titration curves
at 0.16 and 0.06 ionic strength and 25” are shown in Fig. 4. In
Figs. 5, 6, and 7, the aforementioned plots are calculated from
the data assuming values for ni of 8, 9, and 10, respectively,
at the two ionic strengths. (Values of ni = 7 can readily be I
I I, I I
shown to give unreasonable results.) ijSn was determined 0 2 4 6 8 IO 12
from the number of groups titrating below the isoionic pH, 7
with corrections for e-NH2, a-NHZ, and carboxyl titration
FIG. 5. Plots of pH + log ~&(nd - &H) versus 2 for globin
made by using respective log k’ values of 10.30, 7.80, and 4.48. imidazole groups; ni = 8. l , ionic strength 0.16; 0, ionic
(The e-NH2 log k’ was determined by independent analysis of strength 0.06. Temperature, 25”.
February 1964 E. Breslow 491
linearity in Fig. 5, calculated for ni = 8, begin to occur at z =
+7. Moreover, assuming no major change in molecular dimen-
sions between globin and metMb, the theoretical value of w
calculated from approximate H+ ion titration theory (19) has
been calculated as 0.065 to 0.069 at 0.16 ionic strength and as
0.106 to 0.113 at 0.06 ionic strength (13). For metMb, the
experimentally obtained values of w at these ionic strengths were
0.050 and 0.085, respectively. Tanford and Kirkwood (21) have
pointed out that although significant deviations from the the-
oretically derived value of ZL may be expected if the immediate
charge environment of each ionizing group and the number of
groups in each class are considered, the change of w with ionic
strength should remain relatively constant. The value of 0.050
for the change in w with ionic strength (Au) found for ni = 8
seems somewhat high in view of the theoretical value of 0.042
and the experimental value of 0.035 found for metMb (13). On
the other hand, the data shown in Fig. 6 for n = 9 form a linear
plot exactly to the pH where denaturation begins, and then show
the expected deviations. Moreover, the calculated values of w
at both ionic strengths studied are in essentially perfect agree-
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ment with theory Similar plots for values of n = 10 (Fig. 7) I I I I I
I I I I I
also show the expected deviations from linearity in the known 0 2 4 6 8 IO 12
region of denaturation, and the effect of ionic strength on w is
intermediate between that expected from theory and that found
FIG. 6. Plots of pH + log &(ni - ii;~) versus 2 for globin
for metMb. However, values of w so obtained for n = 10 seem imidazole groups; ni = 9. 0, ionic strength 0.16; 0, ionic
very high, although they might be reconcilable with those of strength 0.06. Temperature, 25”.
metMb with the use of more rigorous titration curve treatment
Reaction of Globin Imidazoles with Bromoacetic Acid-The 7.01
interpretation of the H+ ion titration curves of metMb by ap-
proximate titration theory, indicating the presence of 6 reactive
and 6 unreactive histidine residues, was seemingly substantiated
by the reactivity of the protein to p-nitrophenyl acetate (13).
Recent’ carboxymethylation studies of metMb (8) suggest that
8 histidines are available for reaction with bromoacetic acid.
Conceivably this apparent discrepancy is due to the irreversibility
of carboxymethylation, or it might be that more rigorous treat-
ment of the metMb titration curve would disclose the presence 6.2 --
of 1 or more additional reactive histidines with slightly abnormal
log k’ values. Regardless of these difficulties, however, the
difference in relative reartivities of globin and metMb imidazoles
to bromoacetic acid offers another convenient parameter by
LThich subtle changes in the environment of histidine side chains
may be assessed.
Reaction of native globin nith bromoacetic acid was carried FIG. 7. Plots of pH + log Bin/(ni - FCH) versus 2 for globin
out in a manner analogous to that for metMb (8). In Table IV, imidazole groups; n; = 10. l , ionic strength 0.16; 0, ionic
the amino acid analyses of carboxymethylated globin are com- strength 0.06. Temperature, 25”.
pared with the theoretical amino acid composition. Only 1
histidine residue appears to be unreactive in globin as compared residues were unreactive to bromoacetic acid. Upon acid,
with 4 unreactive histidines in metMb. The identity of this alkali, or heat denaturation of metMb (8), and also upon Cu(I1)
unreactive residue was established by peptide mapping of tryp- denaturation,e at least 1 methionine residue becomes reactive to
sin-digested carboxymethylated globin as previously described bromoacetate. Similarly, carboxymethylation of acid-denatured
(8). Only the FG3 histidine could be detected as a nonreactive globin definitively indicates an increased reactivity of methionyl
residue. residues. It would seem, therefore, that the conformation of
One possible cause of the greamr reactivity of globin than of carboxymethylated native globin differs significantly from that
metMb imidazoles to bromoacetic acid could be the increased of the acid- or heat-denatured protein, in which the previously
ease of globin denaturation. The possibility therefore exists internal methionyl residues apparently become exposed.
that the carboxymethylated globin analyzed was no longer On the other hand, urea denaturation of metMb does not seem
“native” in conformation. However, as shown in Table IV, to cause a similar increase in reactivity of methionyl residues (8j,
the methionine content of native globin (as with native metMb) so that absence of methionine reaction may not be a necessary
was unchanged by carboxymethylation; i.e. the methionine indication of native conformation. A preliminary investigation
492 Changes Accompanying Heme Binding to Apomyoglobin Vol. 239, No. 2
IVTABLE directly linked t,o the heme iron and nitrogen atom 1 is hydrogen-
dmino acid analyses oj carbox~methylated native globin* bonded aithin the protein to a peptide bond carbonyl (3). The
change in reactivities of the C1 and EF, or EFs residues, however,
Residues per mole of protein
is not, readily interpretable unless conformational changes in the
Run I vicinity of these residues upon heme removal are postulated
ical which render t,hem more accessible to solvent. In the crystal, at
least, these residues are not directly linked to the heme, nor is it
Lysine ................... 16.7 17.5 19 the heme which appears to block sterically their merger with
Histidine ................ 1.01 0.83 12 solvent .I0
Ammonia .. 7.3 9.35 7 As previously cited (8), the lack of FG3 reactivity in both
Arginine. ................ 4.14 3.40 4 metMb and in globin is not yet interpretable in terms of the
-4spartic acid. ........... 8.5 8.15 8 knorrn protein structure. In metMb it is bonded to a heme
Threonine ................ 5.0 4.70 5 propionate side chain at its N-3 hydrogen position, but is other-
Serine . 5.6 4.57 6 wise exposed to solvent. Although situated in amino acid
Glutamic acid.. . 20.2 20.1 19
sequence between 2 lysyl residues, the t-aminos of these groups
Proline. 4.3 4.06 4
Glycine. . 11.3 11.3 11
do not seem close to the histidine,lO and the expected effect of
Alanine. 16.4 17.6 17 propionate interaction would be to raise log k’. A priori, there-
T:aline 5.08 7.39 8 fore, this histidine would not be expected to fall into the class of
Methionine 2.8 1.83 2 unprot’onated metMb imidazoles masked to Hf ions. More-
Isoleucine. 7.07 8.58 9 over, removal of heme should increase the reactivity of this
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Tyrosine................. 3.14 2.48 3 residue to bromoacetate unless, of course, the postulated con-
Phenylalanine. 6.05 6.01 6 formational changes accompanying this reaction place this
Leucine. 18.2 18 histidine in a more internal position. Alternatively, the lack
3Xarboxymethylhistidine ...... 1.62 2.04 0 of FGs reactivity t,o bromoacet,ate, at least, may be somehow
Dicarboxymethylhistidine ...... 9.1 9.45 0
sterically imposed by amino acid sequence alone, as it, appears
1-Carboxymethylhistidine 0.69 0
e-Dicarboxymethyllysine 0.50 0
similarly unreactive in urea-treated metMb (8).
It is true that the apparent coincidence of t’he increased num-
* These analyses were performed by Professor F. R. N. Gurd ber of imidazoles reacting with Hf ion and bromoacetate upon
and Dr. L. Banaszak. heme removal from metMb is not absolute proof of the identity
of the residues exposed to both reagents. Although the lack
of the relative conformations of native globin and carboxy- of reactivity of the Fs of metMb to both Hf ion and bromo-
methylated native globin by other criteria has been initiated by acetate, and its release in globin, is reasonable, the lack of FG3
ultracentrifugal studies. A 1 7O solution of carboxymethyated reactivity is an enigma. Some additional evidence obtained
native globin at pH 7.5 in 0.16 M KC1 migrated at 25” as a single from hemoglobin mutants, however, would seem to strengthen
symmetrical peak with szo,W = 1.73 compared with an average the case for the identity of the residues reacting with both rea-
value of szo.w for globin under similar conditions of 1.78.g From gent,s. The El residue of metMb is linked in its N-3 position
the molecular weights of the two proteins, the frictional ratio, to the Fes+-bound HzO, and otherwise appears buried inside the
f carborgmethylated globin lfglohin, may be calculated as 1.09. These protein. Diminished reactivity to bromoacetic acid and con-
results suggest some conformational change or reaction of globin ceivably a low enough log k’ to place it in the category of im-
with bromoacetate, but one apparently insufficient to expose idazoles masked to Hf ion might therefore be ant.icipated. Hom-
methionyl residues or to cause the polymerization noted for acid- ever, it appears to react in metMb at least after long exposure t,o
denatured globin (22). Moreover, the similarity in sedimenta- bromoacetic acid (8). The conformational similarity of metMb
tion of metMb and carboxymethyl-metMb has also been noted with the individual chains of methemoglobin is known (3, 231, as
(8) and consideration of the difference in molecular weight of is t’he presence in metHb of a large number of imidazoles buried
these two proteins suggests t,hat a similar minor conformational to H+ ion (20). In hemoglobin, therefore, the histidine cor-
change may occur on reaction of metMb with bromoacetate. responding t,o the ET of metMb would also be expected to be
It would appear, then, that removal of heme from metMb unreactive to H+ ion. A hemoglobin mutant, Hb Zurich, in
results in the exposure of 3 additional histidines to bromoacetic which this hi&dine is replaced by arginine, is spectrally similar
acid and presumably to solvent. The approximate correlation to normal Hb and presumably involves no ma,jor change in
of this conclusion with that obtained from titration data is heme-protein attachment. That CN--metHb Zurich is elec-
gratifying and also adds strength to the thesis that the additional trophoretically inseparable from normal CN--metHb at pH 6.5
histidines released to bromoacetate are those exposed to H+ ion while migrating more slowly towards the anode at pH 8.5 (24)
in the unalkylated protein. The identity of these histidines may would seem only to be explicable in terms of an essentially normal
therefore be tentatively deduced from comparison of the alkyl- log k’ for that histidine in normal hemoglobin. To the extent
ated residues in globin and metnIb. In metMb, the 4 histidyl that metHb and metMb structures are similar, then, the unex-
residues that have been determined as unreactive to bromo- pected reactivity of ET to bromoacetate in metMb may be corre-
acetate are the C1, Fs, FG3, and EFI or EFs, whereas only FGI lated with its reactivit,y to H+ ion in metHb. Moreover, this cor-
is unreactive in globin. The release of Fg upon heme removal relation suggests that on removal of heme from globin, only 1 or
would be anticipated, as nitrogen atom 3 of this histidine is
lo These conclusions are based upon study of a large scale model
g The calculated value of ~20,Wfor globin appears to be slightly of metMb, built from the Kendrew structure, in the laboratory of
lower in KC1 alone than in potassium phosphate buffer. Professor F. R. N. Gurd.
February 1964 IS. Breslow 493
remotely possibly 2 additional histidines, t,he Fs and possibly the
FG3, should be released into H+ ion equilibrium in the absence
of conformational changes. From the titration data alone, then,
the increased reactivity of 3 histidines to H+ ion is indicative of
a conformational difference between globin and metMb.
It is interesting to note here that the shapes of the titration
curves of globin and metMb also suggest another means by
which their relative helix contents might be assessed. The *
greater stability of myoglobin derivatives than of globin to acid b 25 GLOBIN(expt’l)
(and also to alkali) is undoubtedly due in part to stabilization
of the native structure by heme. It appears in Fig. 3, moreover, X
that metMb denaturation occurs over an appreciably narrower :: 20
pH range than does that of globin. If the denaturation may be
viewed rather simply as a polypeptide helix + coil transition, 7
then the treatment of such transitions, as for example by Schell- 15
man (25), may be cited to indicate that the broadening of the
transition in globin could be due to a decreased number of resi-
dues involved in the unfolding process as well as to the absence of
heme stabilization. It would seem, therefore, that a detailed
study of the denaturation of both globin and met&lb could lead
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to an estimate of their relative helical contents.
Phenolic Equilibria-In a preliminary communication (7), the
ionization of the 3 globin tyrosines was compared with the data of 0
Hermans (15) for phenolic equilibria in CO-Mb. The marked B 9 IO II 12 13
difference observed between the two proteins was most simply
attributable to the exposure in globin of the tyrosyl residue found
buried in CO-Mb, although it was noted that a possible conforma- FIG. 8. Spectrophometric titrations of tyrosines in globin, CO-
Mb, and CN--metMb in glycine-KC1 buffer, ionic strength O.lG.
tional change occurred in globin within the pH range of tyrosine At 25”: n , globin; 0, CO-Mb; 0, CN--metMb; - - -, theoretical
titration. The relative tyrosine titration curves of globin and globin titration (see the text). At 9”: A, CN--metMb. Arrows
several myoglobin derivatives have therefore been re-examined indicate pH regions in which increases in optical density could be
under identical conditions. In Fig. 8, the change in molar observed during a 30-minute interval.
extinction at 245 rnp in glycine-KC1 buffer, ionic strength 0.16,
is shown for globin, CO-Mb, and CN--metXb at 25” as well as mately the effect of two negative charges in the Mb derivatives
for CN-metMb at 9”. The not unexpected similarity of the not present in globin, assuming the similarity of log k’ for lysine
CO-Mb and CN--metMb tit,rations is readily apparent, and the in the different proteins.“) It is easily seen that below pH 11 a
discrepancy betlveen globin and Mb phenolic equilibria is similar, large discrepancy exists between the observed and theoretical
although not quite as striking, as that previously deduced from globin tyrosine ionizations; i.e. globin titration cannot be simply
comparison of globin t’itrations from this laboratory with CO-Mb accounted for by the introduction of another tyrosyl residue into
titrations from Hermans. H+ ion equilibrium in the native protein. If indeed the third
As previously cited, the degree of globin ionization is independ- tyrosine is in Hf ion equilibrium in native globin, a shift in log
ent of t,ime within the entire pH range studied, and the titration k’ of the other ionizable tyrosines must also be assumed to occur
curve is essentially reversible from pH 13, although a slight shift upon heme removal.
to approximately 0.2 lower pH unit betn-een pH I1 and 12 on il considerable amount of evidence can be accumulated to she\\
back-titration is discernible (7). In the 1lb derivatives, hom- that the upper third of the globin tyrosine titration curve is
ever, time-dependent increases in optical density can be observed accompanied by a conformational change. The ~~0.~ of a 1%
above pH 12, becoming more rapid as the pH is raised. Above globin solution in 0.16 M KC1 increases sharply from approxi-
this pH, the extent of optical density increase at 245 rnk can be mately 1.8 at pH 11 to 2.1 at pH 11.X at 25” with no subsequent
generally correlated with the extent of denaturation (specifically increase at more alkaline pH. Moreover, increasingly dimin-
observed here by optical density changes at 350 mp), an observa- ished solubility on return to pH 7 occurs as the pH of a globin
tion compatible with Hermans’ thesis that 1 tyrosyl residue in solution is raised above 11, although even after exposure to pH
n-hale CO-Mb is masked to solvent in the native protein. -Us0 13, only a fraction of the total globin is precipitated at pH 7.
shown in Fig. 8 is a theoretical curve for globin tyrosine ionization More dramatic evidence of a major globin conformational change
constructed by use of the assumption that all 3 globin tgrosines occurring at 25” above pH 11 is seen from a study of the optical
are titratable in the native protein with log k’ equivalent to that rotation at the sodium D-line with change of pH. At 25”, a
of the 2 titratable tyrosines in the Mb derivatives. Specifically small decrease in [o(h, from -18” at pH 7 to -21” at pH 11
t’he curve was constructed by using the equation occurs, followed by a sharp decrease to -45” at pH 12 and to
-50” at pH 12.5. No further decrease above this pH is demon-
AE, lob in = 3/2 A&O-Y,> (3)
(at pH X - 0.1) (at pH X) I1 Actually, preliminary calculations suggest that log X-’ for
lysine may be slightly higher in metMb than in globin. Consider-
where AE denotes increase in optical density. (The shifting of the ation of this would shift the theoretical globin curve to still more
theoretical curve to 0.1 lower pH unit is to accommodate approxi- alkaline pH.
Changes Accompanying Heme Binding to Apomyoglobin Vol. 239, No. 2
-AH(2’2 - T,)
pHz - PHI = + 0.868 (WI.% - w,.%) (5)
The value of AH for tyrosine ionization, so calculated, should
be insensitive to differences in charge and shape between the two
temperatures. Q’, however, would reflect such differences and,
in the pH region of normal tyrosine ionization, should gradually
decrease with pH because of the higher enthalpy of lysine ioniza-
tion and the consequent larger negative charge on the protein
with increasing temperature at the same degree of tyrosine
ionization. It can immediately be seen from Fig. 9, however,
that near 60% ionization a significant increase of titration curve
pH displacement occurs, indicating an actual increase in Q’ at
higher pH. Between 10 and 50% ionization, an average value
of Q’ between 25” and 9” or between 41” and 25” may be cal-
culated from Fig. 9 as 5.0 kcal or 4.6 kcal, respectively. More-
over, as shown in Fig. 10, no significant change in globin con-
formation at 25” and 9” is demonstrable by optical rotation
below 60% tyrosine ionization. Assuming a value of u = 0.068
at these two temperatures within this pH range, and calculating
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2 at the two temperatures from titration data (see (7) and Fig.
3), AH below 60% tyrosine ionization can be calculated as 6.6
FIG. 9. Tyrosine ionization in glycine-KC1 buffer, ionic strength kcal, in keeping with the usually assigned value of 6 to 7 kcal
0.16 at 41” (O), 25” (0), and 9”(A). All values of percentage for t,he AH of tyrosine ionization. At 41”, approximately half
ionization were calculated at each temperature from the changes the globin is already denatured below pH 10.8 (see the legend for
in molar extinctions at 245 rnp, assuming no tyrosine ionization Fig. 10). By using a value of w = 0.055 (intermediate between
below pH 7 and 100% ionization at the pH values shown, above
which no significant optical density increases seemed to occur. the values for the native and acid-denatured species), an average
The average value of AEw was 11,500 per tyrosine. AH may be calculated from the titration data (see (7) and Fig.
3) between 10 and 50% ionization as 7.1 kcal, suggesting that
the tyrosines ionizing in this pH range are almost equally avail-
strable. The change in rotation appears instantaneously upon
able in native and denatured conformations. Near 83% ioniza-
increasing the pH, and is at least partially and instantaneously
tion, however, the value of Q’ has become 9.8 and 11.3 kcal
reversible upon lowering the pH. In two separate experiments
between 25-9” and 41-25”, respectively. Moreover, it can be
at 25”, the rotation of a globin solution in 0.16 M KCl, exposed
shown (Fig. 10) that at both 25” and 9”, the increase in optical
for 16 hours to pH 12 and 13, fell immediately to -27” and -37”,
levorotation with pH occurs in a pH range coincident approxi-
respectively, upon return to pH 10.8. In one experiment in
mately mith the last third of the total tyrosine titration, and
which globin was exposed to pH 12.5 for only 20 minutes, the
therefore with a midpoint near 83% ionization. For the same
rotation on return to pH 10.8 was also -27”. No further
degree of ionization u-ithin this temperature range, then, no
decrease on prolonged exposure to pH 10.8 was observed.
large difference in w at the two temperatures should be expected,
The approximately parallel correlation of the optical rotation
and the increase in Q’ would seem to be a reflection of a real
change with the last third of the globin tyrosine titration curve
increase in AH of tyrosine ionization (see Equations 4 and 5).
at 25” is a suggestive but not conclusive indication that 1 globin
Presumably the discrepancy in Q’ near 83% ionization, when
tyrosine may also be masked in the native protein and only
calculated within the higher and lower temperature ranges, is a
become ionizable upon denaturation. The possibility must still
reflection of the fact that at 41”, at the lowest pH at which it was
be considered that the coincidence of the conformational change
possible to obtain optical rotations (Fig. lo), the observed rota-
with tyrosine ionization is fortuitous and that all 3 tyrosines
tion indicated considerable denaturation when only 50% of the
are merely equally available in native and denatured st,ates.
tyrosines were ionized. A difference in w for equivalent degrees
Evidence to the contrary, however, may be gleaned from the
of tyrosine ionization may therefore contribute to the increased
comparative globin tyrosine t’itration curves at 9”, 25”, and 41”
value of Q’. In the main, however, the simplest conclusion
(Fig. 9). The pH displacement between temperatures T1 and
invited by the above data is that part of the total tyrosine titra-
Tz of a given degree of tyrosine ionization is related to the appar-
tion is conformationally dependent; or, more specifically, that
ent heat of ionization,r2 Q’, by the equation (13) 1 of the 3 globin tyrosines is ionizable only in the denatured
-&‘Wz - T,) protein.
pHz - pH1 = The apparent reversibility of globin tyrosine titration upon
return from pH 13 (7) remains to be considered. Admittedly, if
and to the intrinsic heat of ionization, AH12, by the equation 1 globin tyrosine is unmasked upon globin denaturation, an
appreciable shift in the observed tyrosine titration to more acid
I2 As defined here, the apparent heat of ionization, Q’, is related pH should be apparent upon back-titration from pH 13. That
to the intrinsic heat of ionization, AH, as the apparent association
constant for H+ ion at a given DH (loa k’ - 0.868 wz) is related to this does not occur is most probably attributable to reversibility
the intrinsic association cons&t (iogk’). Equatiod 5 may there- of denaturation under the titration conditions. As has already
fore be derived directly from Equations 4 and 2. been cited, optical rotation criteria suggest that at least two-
February 1964 E. Breslow
thirds of the alkaline denaturation is instantaneously reversible 100
upon lowering the pH. Quite possibly, under titration condi-
tions, in which the protein concentration is more dilute than in
optical rotation studies, reversibility is essentially complete. It
is also possible that failure to observe complete restoration of the
initial optical rotation upon back-titration is due to imperfect
refolding of the molecule in areas not adjacent to the abnormal
tyrosyl residue. The assumption will therefore tentatively be
made that at 25” and go, the upper third of the globin tyrosine
titration curve is accompanied by a rapid and reversible con-
formational change which releases a previously masked tyrosine
into H+ ion equilibrium.
If the abnormal globin tyrosine is identical with that found
buried in myoglobin derivatives, the question remains as to the
underlying cause of the striking differences in globin and myo-
globin tyrosine ionizations. To a certain extent, of course, this
difference is a reflection of the diminished stability of globin to
alkaline pH. But it is apparent from Fig. 8 that if only 2 tyro-
sines are in Hf ion equilibrium in native globin (below pH 11)
and in native myoglobin (below pH IL’), a significant decrease in
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log k’ of these 2 tyrosines occurs when the heme is removed.
Hermans (15) has concluded that in CO-Mb 1 of these 2 tyrosines
has a higher log k’ than the other, but he did not take into account
the effect of protein charge on pK, in calculating these data.
Assuming, therefore, the identity of log k’ of both ionizable
tyrosines and a value of AE245 per tyrosine of 11,500 for both 7/ 0 9 IO II 12 13 14 -
proteins, the apparent ionization midpoint of the tyrosines at PH
25” can be estimated as 10.2 in globin and as 11.1 in myoglobin FIG. 10. Correlation of optical rotation changes in globin at
derivatives. Moreover, the potentiometric titration curves of alkaline pH with tyrosine titration curves. The ionic strength
CN--metMb and CO-Mb can probably be assumed to be identi- for all studies was 0.16. The upper 40% of the spectrophotome&ic
cal with those for metMb at 25” between pH 10 and 12 (13), and tyrosine ionization curves are shown for 9”, 25”, and 41” (--).
At 9” (0) and 25” (o), the optical rotations are plotted as the
2 for these proteins can therefore be calculated within this pH percentage of the total rotational change at the sodium D-line in
range. With a value of w = 0.06 for both globin and myoglobin alkali, assuming no change near pH 7. At 41” (A), rotations are
derivat.ives, Equation 2 can be used to calrulate log k’ for these plotted as percentage of the total change from pH 10.85, the lowest
2 tyrosines as approximately 9.9 in globin and 10.5 in myoglobin pH at which it was possible to obtain readings, although [(Y], at
this pH at 41” was -35” as compared with -15 to -18” observed
derivatives. This latter value is in good agreement with that
for globin samples near pH 7 at 9” and 25”.
found by Hermans for the 2 reactive tyrosines of guanidinated
metMb.i3 Although both values of log k’ fall within the range
often described as “normal” for protein tyrosines, the difference are directly linked to the heme (3). Conceivably, then, this
between them is significant. difference is due to an increase in dielectric in the vicinity of the
Further evidence supporting a difference in log k’ between the ionizable tyrosines when the heme is removed. Whether hhis
2 freely titratable tyrosines of globin and CN--metMb may be alteration in dielectric is due to conformational changes ac-
found in the relative values of Q’ for the two proteins between companying heme withdrawal or whether 1 of the ionizable
25’ and 9”. From the data in Fig. 8, Q’ for CN--metMb can be tyrosines is close enough to the heme to be directly influenced by
shown to rise gradually from 5.2 kcal at values of AE2d5 x 1OP an expected local dielectric change when the heme is removed
near 6, to 6.4 kcal at values near 18 (a pH region in which CN-- cannot be stated at the present time.
metMb is completely stable and in which the third tyrosine may For emphasis, several pertinent comments should be added,
be presumed buried). By using a value of 11,500 for AEea5 per some of which are of particular relevance to the interpretation
tyrosine in globin, the data in Fig. 9 may be recalculated in the of tyrosine titration curves. It would seem from the data that
same AEt45 interval to show a decrease from 5.2 to 4.2 kcal. neither seeming reversibility, lack of time-dependent changes,
Although the differences in pH displacement represented by nor marked titration curve inflections are a necessary indication
bhese differences in Q’ are only of the order of 0.05 pH unit, the of the accessibility of all tyrosines to solvent in the native protein.
reproducibility of the results, coupled with the expected decrease Rapid and reversible equilibria between two conformations must
in tyrosine Q’ (in globin) as the pH is increased, lends additional be considered. Second, if the difference in tyrosine titration
support to a difference in log k’ between at least 1 of the 2 titrat- between globin and myoglobin is indeed due to a decrease in
able tyrosines in the two proteins.14 In metMb, no tyrosines log k’ of the freely titratable tyrosines, it is interesting to note
how a difference of 0.6 in log k’ can be appreciably magnified by
u J. Hermans, Jr., personal communication.
14 Spectrophotometric titrations of metMb itself were also con- of Fe(OH)2+ ionization (log k’ = 8.9 at 25”), further investigation
ducted at 245 rnp and indicated a large shift in the apparent titra- is required to ascertain whether any of the apparent shift is due
tion curve to more acid pH relative to CN--metMb. Although to an unexpected difference in tyrosine acidity between metMb
some of this difference may be attributed to the ultraviolet effects and CN--metMb.
496 Changes Accompanying Heme Binding to Apomyoglobin Vol. 239, Ko. 2
the effect of protein charge to greater differences in apparent Acknou&dgments-The author is particularly grateful to
acidity. Conceivably,, large differences in apparent react.ivity Professor Frank R. N. Gurd for his gifts of sperm whale myo-
of tyrosines in relat.ed proteins to reagents other than Hf ion globin, and to both Professor Gurd and Dr. L. Banaszak for the
may also arise from smaller differences in intrinsic reactivities. amino acid analyses and peptide mapping of carboxymethylated
Finally, it should be emphasized t,hat although 1 tyrosine has globin. The able technical assistance of Miss Priscilla Anderson
been concluded to remain masked when the heme is removed is also gratefully acknowledged.
from myoglobin, the reversibility of the globin tyrosine titration
curve cannot make this conclusion unequivocal without demon- REFERENCES
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