(Received for publication, August 19, 1996, and in revised form, November 5, 1996)
From the Departments of Physiology and Biophysics,
¶ Medicine (Division of Hematology), and
Anatomy and
Structural Biology, Albert Einstein College of Medicine,
Bronx, New York 10461
Time-resolved fluorescence methods have been used to show that 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), a fluorescent analog of 2,3-diphosphoglycerate, binds to the central cavity of carboxyhemoglobin A (HbACO) at pH 6.35. A direct quantitative approach, based on the distinctive free and bound HPT fluorescent lifetimes of 5.6 ns and ~27 ps, respectively, was developed to measure the binding affinity of this probe. HPT binds to a single site and is displaced by inositol hexaphosphate at a 1:1 mol ratio, indicating that binding occurs at the 2,3-diphosphoglycerate site in the central cavity. Furthermore, the results imply that low pH HbACO exists as an altered R state and not an equilibrium mixture of R and T states. The probe was also used to monitor competitive effector binding and to compare the affinity of the binding site in several cross-bridged HbA derivatives.
The solvent-accessible central cavity of hemoglobin A
(HbA)1 contains functionally important
binding sites for several classes of allosteric effectors that
facilitate the lowering of oxygen affinity (1, 2). The -subunit end
of the central cavity contains a cluster of eight positive charges that
interact with the negative charges of 2,3-diphosphoglycerate (DPG) (3).
This site also binds a variety of other negatively charged effectors such as inositol hexaphosphate (IHP), inorganic phosphate, chloride, and polyglutamic acid. The other end (
-subunit) of the central cavity contains additional binding sites, particularly for chloride ions. Another class of potent effectors derived from clofibric acid and
bezafibrate (e.g. L35) bind near the middle of the central cavity with their negative charges projecting toward the
-subunit end (4-6). Binding of these effectors is also associated with a
reduction in oxygen affinity. Study of these effectors is of practical
interest since control of oxygen affinity is a necessary component for
the design of acellular Hb-based oxygen carriers (7, 8).
X-ray crystallographic studies are important both in pinpointing effector-binding sites and in characterizing the geometry of the effector-bound site (2). However, other methods must be used to determine the structural and functional interactions that are important in solution and to perform titration studies for obtaining binding constants as a function of solution conditions and/or structural state. Functionally relevant synergistic and antagonistic effects among effectors are also best elucidated through solution studies. Functional characterization of hemoglobin suggests that synergistic and competitive activity can occur when combinations of effectors are bound (4). Of the various allosteric effectors, the interactions of DPG (the natural allosteric effector found in the red blood cell) and its analogs in HbA have been investigated the most extensively. However, the binding of DPG can only be measured indirectly through its effects on ligand reactivity and on spectroscopically accessible chromophores such as the heme groups. In this report, we present an extension of the use of a fluorescent analog of DPG to probe directly the hemoglobin DPG-binding site in a quantitative fashion.
Gibson and MacQuarrie (9, 10) showed that
8-hydroxy-1,3,6-pyrenetrisulfonate (HPT) can be used as a fluorescent
analog of DPG. Utilizing the observation that the fluorescence signal from HPT is highly quenched when bound to HbA, they performed steady-state fluorescence intensity measurements and established that
HPT has a lower affinity for the DPG-binding site than DPG or IHP. A
subsequent study by that group (11), using HPT as a probe of the
DPG-binding site, focused on the detection of ligand-binding intermediates occurring along the R to T
transition pathway. In those initial experiments, the results were
limited in large part by two aspects of the methodology. (i) To monitor
changes in HPT fluorescence in the presence of the highly absorbing
heme, low concentrations of Hb had to be used. As a result,
dissociation of the tetramer produced -dimers in a concentration
high enough to complicate data analysis. (ii) The use of steady-state
fluorescence quenching as a means of obtaining titration data does not
allow for probing of the HPT-bound species or for a direct
determination of the fractions of both bound and unbound effector in a
single measurement. In the present study, these limitations were
overcome using a combination of front-face optical geometry and
fluorescence lifetime measurements. The use of front-face excitation
allows for the observation of HPT fluorescence upon binding to
hemoglobin at high concentrations without inner-filter effects or
substantial dimer formation. Front-face fluorescence techniques have
been used previously to probe the highly quenched fluorescence from tryptophans within both hemoglobins and myoglobins (12).
Fluorescence lifetime measurements of HPT in the presence of HbA reveal two distinct and easily resolved lifetime components that allow unambiguous determination of the fractions of bound and free HPT under various solution conditions (see below). In contrast, conventional methods of saturation equilibrium binding analysis require a full titration data set complete with a plateau in the binding curve to quantify bound and free ligand (13). In addition, quantitative analysis of steady-state measurements comparing binding data under different solution conditions (pH and/or ionic strength) would be hampered due to the variation in the extinction coefficient of HPT at the excitation wavelength and would therefore require a correction factor to account for this dependence. Furthermore, as will be shown below, although the fluorescence intensity of the bound chromophore is significantly diminished compared with that of the free chromophore, the quantum yield is not negligible. This residual intensity complicates steady-state titration measurements that do not normalize to the intensity of fully bound ligand. Aside from obviating the need for these corrections, lifetime measurements of the bound species can also be used to characterize (structurally and dynamically) the binding site as a function of solution conditions or protein modifications. In particular, the fluorescence spectrum of the bound ligand can be obtained, independent of a large background of free ligand, using time-gated spectroscopy. Last, fluorescence lifetime measurements can assist in the determination of multiple binding sites and in the identification of nonspecific binding.
Lifetime fluorescence methods are applied here to the study of effector binding at low pH to the carboxy forms of both native HbA and three intramolecularly cross-bridged derivatives of HbA. Liganded hemoglobin was chosen for several reasons. First, the affinity for DPG-binding site effectors of deoxy derivatives of cooperative HbA is very high. Consequently, as long as the HbA remains in the T state, there is not likely to be substantial variation in the binding properties of the effectors as a function of protein modification. The greatly reduced affinity of liganded Hb for these effectors increases the dynamic range associated with the parameters describing the interaction between the effector and the protein.
A second reason for studying the liganded derivatives is that there has been considerable ambiguity as to the nature of the interaction of effectors with the liganded protein. Most of the work on the role of effectors in regulating ligand binding is predicated on the assumption that the effect arises from preferential binding of the effector to the T state structure. There is, however, considerable evidence that DPG and IHP do indeed bind to the liganded ferrous forms of HbA (14-17). What is not clear and is still being explored is whether the effectors bind exclusively to the small fraction of liganded T state species, thereby shifting the equilibrium from R toward T as binding progresses, or whether the effectors bind with reduced affinity to the R state liganded species, resulting in an altered R state.
The present study directly addresses the issue of whether HPT and IHP bind stoichiometrically to liganded HbA. These data are then used in conjunction with earlier spectroscopic studies to assess the nature of effector-bound liganded HbA. In addition, the HPT-based technique is used to evaluate how three different site-specific cross-linkers influence the DPG-binding site in liganded HbA.
Human adult hemoglobin was purified as described previously (18). A hemolysate was prepared from whole blood obtained from an AC or AA individual using the freeze-thaw technique to remove membranous material. The hemolysate was dialyzed against 10 mM sodium phosphate (pH 6.8), and the hemoglobins were separated on a CM-52 column using a double gradient (10 mM sodium phosphate (pH 6.8) to 50 mM sodium phosphate (pH 8.3)). Purified HbA was collected and verified using cellulose-acetate gel electrophoresis and isoelectric focusing. The hemoglobin was concentrated and dialyzed against 50 mM bis-tris with 100 mM sodium chloride (pH 7.35) and stripped on a Sephadex G-25 column equilibrated with the same buffer. This purified material was then placed on a second Sephadex G-25 column equilibrated with 50 mM Hepes (pH 7.35) and concentrated and equilibrated with the buffer of choice using a Centricon-10 concentrator (Amicon, Inc.). The hemoglobin was stored frozen in liquid nitrogen until used. HbACO was prepared by gently flowing chemically pure CO over the surface of the HbA solution. Deoxy-HbA was obtained by gently passing pure nitrogen or helium into an anaerobic vessel containing the HbA solution, which was then aerobically transferred to the cuvette. Complete conversion to the CO or deoxy forms was verified by the absorption spectrum.
Three forms of intramolecularly cross-bridged HbA were used in this
study. An -cross-linked HbA was prepared by reacting deoxy-HbA
with bis(3,5-dibromosalicyl)fumarate as described by Walder et
al. (19). The cross-link introduced is between the
-amino
groups of Lys-
99 and is within the central cavity. Two other
derivatives, with cross-links between residues of the
-chains, were
also prepared. Bis(3,5-dibromosalicyl)sebacate reacts with deoxy-HbA at
slightly alkaline pH to introduce intramolecular cross-linking between
the
-amino groups of Lys-
82 as described by Bucci et
al. (20). This selective
cross-linking reaction has been
optimized for the preparation of pure cross-linked
material.2 Deoxy-HbA (1-2 mM)
in 50 mM borate buffer (pH 9.1) was reacted with
bis(3,5-dibromosalicyl)sebacate at 37 °C for 3 h. At the end of
the reaction, glycylglycine was added in 10-fold molar excess over the
cross-linker to scavenge the excess reagent. The reacted Hb was
dialyzed against 10 mM Tris and 20 mM glycine
(pH 8.3). The cross-linked Hb was purified by ion-exchange
chromatography using a Bio-Rad MacroPrep High Q anion exchanger. The
column was equilibrated with the dialysis buffer, and the proteins were
eluted by stepping sodium chloride (30, 75, and 200 mM) in
the same buffer. The ascending side of the 75 mM NaCl
elution peak was used in this study, and >95% of the material was
cross-linked. Intramolecular cross-linking of HbA between the Cys-
93
residues was carried out using bis(maleimide)/polyethylene glycol 2000 (bis-Mal/PEG2000). HbAO2 (0.5 mM) in
phosphate-buffered saline was reacted with 2-fold molar excess of the
cross-linking reagent at room temperature for 1 h. Under these
conditions, nearly quantitative (>99%) intramolecular cross-linking
occurred. The cross-linked material was separated from the excess
reagent by gel filtration and then further purified by CM-cellulose
chromatography. The cross-bridge results in a slight increase in the
oxygen affinity of the protein.3 The
cross-linking reagents bis(3,5-dibromosalicyl)sebacate and bis-Mal/PEG2000 were prepared by Paul Smith of Bioaffinity Systems (Rockford, IL).
HPT was obtained from Eastman Kodak Co. and used without further purification. Stock solutions for titrations were prepared in 50 mM Hepes (pH 6.35). It should be noted that there is a significant change in the absorption spectrum of HPT upon changing the pH from 6 to 8 (9). However, excitation in most cases was at 310 nm, where the effect is small, and no pH dependence of the lowest energy (505 nm) emission spectrum or fluorescence lifetime was observed (see below). The sodium salt of IHP was purchased from Sigma. A 200 mM aqueous IHP solution was passed and recycled for 1 h through a dry Amberlite column as described previously (21). This solution was frozen and stored in aliquots until needed. 20 mM stock solutions used for titration were prepared with the desired buffer. L35 was obtained as a generous gift from Drs. I. Lalezari and P. Lalezari, and a stock solution was made by dissolving L35 in the desired buffer to a final concentration of 20 mM.
Steady-state FluorescenceFront-face fluorescence spectra were measured using an SLM 8000 photon-counting spectrophotometer equipped with a front-face accessory. HbA solutions of 0.6-1 g/100 ml (0.10-0.16 mM in tetramer) were prepared for titration with the corresponding effector. The met (Fe3+) content was <5%. For a review of front-face hemoglobin fluorescence measurements, see Hirsch (12).
Time-resolved FluorescenceThe time-correlated single photon-counting system was described previously (22). Considerable assistance in optimization was obtained using the methods described by Holtom (23). The light source was a mode-locked, frequency-doubled neodymium/YAG laser that synchronously pumped a dye laser with rhodamine 6G as the active medium. The cavity-dumped dye laser output was frequency-doubled from the visible to the near-ultraviolet by focusing the pulse train onto a KDP type I crystal to generate light in the wavelength range 280-315 nm (5-10 ps fwhm (full width at half-maximum)). For front-face fluorometry, the optically thick sample was placed in a triangular quartz cuvette, which was rotated so that the excitation direction was 34° with respect to the surface normal (24). The solution was slowly stirred using a miniature magnetic bar to reduce artifacts from photodamage.
The fluorescence, collected at right angles to the illuminating light, was collimated, passed through a sheet polarizer (at the magic angle) and a cutoff filter, and then focused onto the entrance slit of a 1/8-meter subtractive double monochromator. The monochromator output was directed onto the surface of a microchannel plate photomultiplier tube, and the signal was measured using standard NIM electronics (22). The instrument response function was typically 35 ps fwhm. Deconvolution of the emission decay curves and fitting to a sum of exponentials (Equation 1),
![]() |
(Eq. 1) |
The absorption spectra of HPT at pH
8.0 and 6.35 (Fig. 1) reveal the presence of multiple
transitions with both electronic and vibrational character. At pH 8.0, it is assumed that the band at 455 nm represents the lowest energy
electronic transition, S0 S1, of the deprotonated species (42). The
shoulders at higher energy near 400 and 375 nm are attributable to
transitions of excited vibrational states within this electronic band
or to ground-state absorption of the protonated HPT. Under acidic
conditions, the positions of these features remain unchanged, but their
relative intensities are altered. In both acidic and basic solutions,
there is a near-UV transition around 297 nm, which is assigned to a higher energy electronic transition, S0
S2. All fluorescence measurements in this report were made with excitation in the near-UV region (280-310 nm), which excites primarily the S2 state.
Because we measured spectroscopic parameters of HPT under different solution conditions (e.g. pH), we wished to ensure that there were no anomalous properties of the probe chromophore that would lead to misinterpretation of the data. Consistent with the observations of Gibson and MacQuarrie (9, 10), there is considerable change in the absorption spectrum of HPT as a function of pH.4 Consequently, this could hamper quantitative analysis of equilibrium ligand binding from steady-state spectroscopy when comparing solutions at different pH values, due to the variation in the extinction coefficient at the excitation wavelength. In contrast, lifetime measurements may be used to quantitate the binding of HPT to Hb without the need for correction factors. The shape and peak wavelength of the HPT emission spectrum are independent of protonation state in this pH region (Fig. 1). Note, however, the presence of a shoulder near 440 nm in the spectrum at pH 6.35. As will be argued below, this emission originates from the pH-dependent higher energy transition.
Time-gated fluorescence spectra of bound ( < 200 ps) and free (
> 2 ns) HPT (see below) do not differ significantly. This observation
is crucial to ensuring that the fractional populations of these species
can be extracted from the pre-exponential amplitudes in a
multiexponential decay fit without correcting for spectral changes.
This requirement for invariance as a function of binding is applicable
as well to the extinction coefficient, which is also unchanged due to
binding of the HPT chromophore to the protein.
Experimental constraints of the present study necessitated the use of
near-UV excitation, which populates a higher energy state of HPT, as
opposed to direct excitation in the lowest energy band around 450 nm.
S2 excitation is typically followed by relaxation to the S1 state,
which fluoresces with high quantum yield. Fig. 2
presents the fluorescence decay curves of free HPT in solution at pH
6.35 for excitation at 310 nm and emission at 510 nm. The fitting
parameters for both one- and two-exponential decay models at high and
low pH are given in Table I. It can be seen that the
excited state lifetime (2 = 5.6 ns) is the same at the
two pH values and is in agreement with that previously reported (43). In addition to the fluorescence decay, there is a rise time
(
1), denoted by the negative pre-exponential parameter,
with a time constant of ~100 ps. Although it is difficult to quantify
the amplitude of this component using a simple sum of exponentials decay model, it is clear that the rise time has a larger amplitude, relative to the decay, at pH 6.35. Measurements of decay curves at
emission wavelengths around 450 nm (see Fig. 1) also result in
lifetimes around 100 ps (data not shown). In addition, a time-gated spectrum of the early time fluorescence (
< 200 ps) shows bands at
445 and 505 nm, which are assigned to emission from the absorption transitions at 400 and 450 nm, respectively. A time-gated spectrum of
the long-lived fluorescence shows only the reddest (505 nm) emission.
This assignment explains the apparent difference in rise time amplitude
and the intensity of the 440 nm emission band, depending on pH, since
the pH alters both the ratio of S2 to S1 absorption at the excitation
wavelength of 310 nm and the proportions of protonated and unprotonated
HPT fluorescence (see Fig. 1). All of this evidence indicates that this
rise time is consistent with the rate of relaxation from a higher
excited state (electronic or vibrational) to a vibrationally
thermalized S1. Alternatively, this decay component may represent the
time for photo-initiated proton transfer. The practical implications of
this rise time on the quantitative determination of HPT binding to HbA
are discussed below.
|
An additional potential complication due to the excitation at 310 nm
results from the effect of energy transfer from the intrinsic Trp
residues of Hb to HPT. Because this excitation transfer occurs only to
bound HPT chromophores that lie within the Förster transfer radius, it essentially increases the apparent extinction
coefficient of bound HPT by a factor proportional to the product of the
donor (Trp) extinction coefficient at the excitation wavelength and the
energy transfer efficiency from Trp to HPT under the conditions of the
measurement. This increased apparent extinction coefficient would
overly weight the fraction of bound chromophores in the decay analysis.
In the present case, however, there is reason to assume that this
effect is negligible. First, the extinction of Trp at 310 nm is small,
20 M
1 cm
1 (27),
compared with that of HPT,
4500 M
1
cm
1 (28). Second, in a separate experiment that monitored
the Trp lifetime in the absence and presence of saturating HPT, the
energy transfer efficiency was estimated to be no more than 10%.
Consequently, systematic error due to energy transfer is considered to
be small.
In steady-state fluorescence
measurements, the addition of HPT to deoxyhemoglobin (50 mM
Hepes (pH 7.35)) results in a decrease in the intrinsic tryptophan
fluorescence intensity and the loss of HPT emission (Fig.
3A).5 The
addition of HPT to HbAO2 (above pH 7) results in little or no Trp fluorescence change and the retention of a measurable HPT signal
(Fig. 3B). Furthermore, the Trp fluorescence decrease
observed with deoxyhemoglobin upon addition of HPT is reversible upon
oxygenation of the solution. These results are as expected since
studies by Gibson and MacQuarrie (9, 10) demonstrated that HPT binds with much higher affinity to deoxyhemoglobin than to oxyhemoglobin. HPT
has also been shown by those workers to bind to the T state of hemoglobin with 1:1 stoichiometry (9, 10). However, the measured
dissociation constant is sensitive to pH and ionic strength. HPT is
displaced from deoxy-HbA with the same stoichiometry by DPG and IHP,
with binding affinities in the order IHP > DPG > HPT.
Probe Binding to HbACO
Binding of HPT to HbACO under slightly acidic and alkaline conditions was monitored using the fluorescence at 505 nm. The HPT fluorescence intensity is much more than an order of magnitude lower in the presence of HbACO at pH 6.35 compared with pH 8.0. The decrease is attributed to energy transfer to the heme, and this is confirmed by lifetime measurements showing a picosecond lifetime for bound HPT and a nanosecond lifetime for free HPT (Fig. 4 and Table I). Using the lifetimes, it becomes possible to measure directly the fraction of HPT bound and thereby to establish its dissociation constant. Thus, Equation 1 can be rewritten more explicitly as follows (Equation 2),
![]() |
(Eq. 2) |
As can be seen from Table I, in addition to the lifetime components
representing free and bound HPT, there is an intermediate lifetime
(2 ~ 1 ns, but with large uncertainty due to its small intensity contribution) whose fractional population varies between 4 and 7%. Titration data also reveal that this component is only mildly
dependent on HPT concentration (for a given concentration of HbACO). We
interpret this artifact as arising from nonspecific binding of a
fraction of the HPT to the surface of the Hb. This results in some
fluorescence quenching by the hemes, but less than that for the more
tightly bound, site-specific fluorophores. In all quantitative
analyses, this component is excluded from the fractions of bound and
free HPT. This highlights another advantage of time-resolved
measurements for equilibrium binding studies in that distinct molecular
species can be identified, isolated, and quantitatively measured.
The rise time component of the fluorescence decay for near-UV
excitation of the unbound HPT described above ( ~ 100 ps) is similar to the picosecond decay of the bound species. However, these
cannot be distinguished in the fitting of the decay curve to a simple
sum of exponentials. It is our observation that the negative amplitude
merely cancels a portion of the positive amplitude for the picosecond
(bound) lifetime. This implies that the fraction of bound HPT measured
is a lower limit to the true value. This complication may become
significant only when the amount of unbound HPT is much larger than
that of the bound species, and data presented here are not corrected
for this effect.6
A titration of HbACO at pH 6.35 with HPT by monitoring the amplitudes of the picosecond and nanosecond lifetime components as described above is shown in the binding curves of Fig. 5. Nonlinear fitting to the single-site binding expression (Equation 3)
![]() |
(Eq. 3) |
Competitive Effector Binding by IHP
By monitoring the
fraction of HPT bound to HbA, the result of adding other allosteric
effectors can be measured. The steady-state emission spectrum of
HPT/deoxy-HbA (pH 7.35) in the absence and presence of IHP is shown in
Fig. 6. The increase in HPT fluorescence is indicative
of release of HPT upon addition of IHP. This observation is consistent
with the earlier results of Gibson and MacQuarrie (9). Fluorescence
lifetime measurements allow the fraction of bound HPT, for a given
initial ratio of [HPT] to [HbACO], to be monitored as a function of
a second added effector. The results for IHP are shown in Fig.
7A. The stoichiometric displacement of HPT
from HbACO by IHP, with a sharp end point at 1 eq of IHP/tetramer, is
consistent both with HPT binding at the DPG-binding site and with IHP
having a much higher affinity for that site relative to HPT. MacQuarrie
and Gibson (10), using a steady-state measurement, obtained a similar
result with both IHP and DPG. Although it is possible that HPT binds at
a different site and is released from HbACO due to an IHP-induced
conformational change, this explanation is very unlikely. IHP binding
to HbACO at low pH induces changes that show up in the heme resonance
Raman spectrum of the CO photoproduct (16, 17) and the steady-state
ultraviolet resonance Raman spectrum,7
which indicate a shift toward a more T-like tertiary
structure. Thus, if the HPT-binding site were different from the
IHP-binding site, the IHP-bound HbA would be expected to bind HPT more
tightly, based on the difference in binding affinities between the
T state deoxy and R state CO derivatives of HbA.
It follows with a rather high degree of certainty, therefore, that HPT
and IHP are both binding to the same DPG-binding site.
Nature of IHP-bound HbACO
Earlier studies have demonstrated
that the addition of IHP to solutions of liganded HbA (CO and
O2) has a clear influence on the structure (16, 17) and
reactivity (15) of the liganded species. Significantly, IHP decreases
the yield of geminate recombination on the picosecond and nanosecond
time scales (29, 30), but not to the extent seen in the T
state species (31). Upon addition of IHP, the visible resonance Raman
spectra of the transient forms of HbA generated within 30 ps (32) and
10 ns (16, 17) of photodissociating the parent HbACO show a decrease in
the frequency of the iron-proximal histidine stretching mode from 230 cm1 to ~226 cm
1. This shift was shown to
originate from the
-chains (17). The corresponding frequency for
T state photoproducts is ~220-222 cm
1.
Both the geminate rebinding and Raman studies are interpretable in
terms of two models. One possibility is that IHP binds to a
T state fraction of the liganded Hb molecules. The Raman and
geminate rebinding results then suggest a mixture of IHP-bound T state liganded Hb and IHP-free R state liganded
Hb. A second possibility is that there is a single population of
IHP-bound liganded Hb having a strained R state structure,
resulting from IHP-induced conformational changes in the direction of
the R to T transition. The results presented here
show that, at low pH, the addition of a slight excess of IHP results in
a single homogeneous population of IHP-bound HbACO. In conjunction with
the Raman and geminate rebinding results, this indicates that the
second model is much more likely. IHP-bound HbACO remains in a strained
or altered R state, in contrast to the HbANO derivative,
which is readily and unambiguously switched to the T state
upon addition of IHP at low pH (33, 34) as reflected in the 222 cm1 frequency for the iron-histidine Raman band of the
10-ns photoproduct (16, 35).
X-ray crystallographic
studies reveal that the clofibric acid derivative L35 binds well away
from the DPG-binding site at a position within the central cavity in
the region of Lys-99 and extending to the C terminus of the
-subunits (4). If effector binding at either site does not globally
alter the Hb structure, it is expected that simultaneous occupation of
the sites by the two effectors can occur. If central cavity
communication is important or multiple binding sites for L35 exist,
then synergistic or antagonistic effects may result. Fig. 7B
demonstrates that the addition of L35 to a solution of HbACO partially
saturated with HPT does not cause additional binding or release of the
HPT below [L35]/[HbA] = 2.5. At higher concentrations, however,
displacement of the HPT begins to be observed.
L35 is a potent clofibrate-derived allosteric effector that exerts a considerable pull toward the T state when bound to HbACO. This effect has been observed directly by monitoring the iron-His stretching frequency of the photoproduct occurring within 10 ns of photodissociation of HbACO in the presence of L35. The spectrum of the photoproduct, which reflects the influence of the initial tertiary/quaternary structure of the ligand-bound globin upon the five-coordinate, high-spin heme in the photoproduct, shows an iron-proximal histidine stretching frequency shifted toward the T state value.8
The results of the present study indicate that, in excess, L35 can
cause HPT to be displaced from HbACO. Since L35 binding to its site in
the interior of the central cavity results in a more T-like
structure, one would anticipate an increase in HPT binding upon
addition of L35 (synergistic effect). The observed decrease in HPT
binding coincident with the addition of excess L35 is most easily
explained if one assumes that L35 can also weakly bind in the vicinity
of the DPG-binding site. A secondary binding site for L35 has been
observed along the length of the G helix of the -subunits in x-ray
crystallographic studies (4). It is plausible that this site is
sufficiently close to the DPG site to alter HPT binding either through
electrostatic effects or through a local conformational change. In
either case, occupancy of this second site reduces HPT binding.
In
the quest for stable tetrameric, low affinity hemoglobins,
cross-bridged hemoglobin derivatives have been synthesized by a number
of methods (7). Cross-bridges have been constructed between residues
lying at different locations or depths within the central cavity. The
accessibility or affinity of the DPG-binding site toward effectors in
the presence of such modifications may be determined by HPT binding
studies as shown by a comparison of the fraction of bound HPT for
wild-type HbACO and three cross-bridged derivatives (Fig.
8). In these measurements, the ratio of HPT to Hb
tetramer was fixed at 1:1. In all cases, the amount of nonspecifically bound HPT was constant at 3-5%.
The 99-cross-linked sample (Fig. 8, XL
99) shows the
smallest change in HPT binding compared with HbA. This cross-bridge is
located well into the interior of the central cavity, and the spacer
arm is short and rigid, which should preclude any direct steric or
electrostatic effect on the binding of HPT to the DPG-binding site.
Earlier functional (36, 37) and spectroscopic (38) studies of this
cross-bridged Hb were interpreted as showing that the R
state is destabilized. If so, enhanced HPT binding is anticipated. The
present results, which reveal no increased effector binding, are more
consistent with the interpretation that the cross-bridge influences
local, but not global, tertiary structure as suggested by Ferrone and
co-workers (39).
The 82-cross-linked Hb (Fig. 8, XL
82) shows a marked
decrease in the binding of HPT relative to native HbA and the
-cross-linked species. The location of the cross-bridge is
directly across the DPG-binding site, and Lys-
82 directly
contributes to the binding of effectors at this site (40). Thus, both
the location of the cross-bridge and the site of attachment of the
cross-bridging reagent favor a decrease in effector binding at the
DPG-binding site. The presence of bound HPT, however, indicates that
binding is not completely blocked in this derivative. This may be
related to the flexibility of the spacer arm of the sebacate
cross-bridge between the
-chains. The results are also consistent
with a reduced binding affinity due to the loss of the contribution of
the positive charges of the Lys-
82 side chains to the stability of
the bound complex.
The bis-Mal/PEG2000 derivative of HbACO (Fig. 8, XL93) is
a newly developed cross-bridged Hb (41) and shows a substantial increase in the binding affinity for HPT relative to native HbACO. Functional studies of this Hb have shown that it retains both cooperativity and near-normal oxygen affinity despite the fact that the
site of attachment is Cys-
93. Most simple modifications of this site
produce high affinity hemoglobins with drastically reduced or
eliminated cooperativity. Visible and ultraviolet resonance Raman
spectroscopy studies9 on bis-Mal/PEG2000
HbA reveal that, upon switching from the deoxy to the carboxy
derivative, the
1
2-interface undergoes
transitions that are very similar to those that occur when HbA
undergoes the T to R quaternary switch. There are
indications from the UV resonance Raman spectrum that the presence of
the PEG enhances the hydrogen bonding between the penultimate tyrosine
(residue
145) and its hydrogen-bonding partner on the FG corner in
the CO-ligated derivative. An increase in this interaction is expected
to impart T-like properties to the R state of the
liganded form of the protein. Visible Raman spectroscopy supports this
interpretation in that the spectrum of the 10-ns photoproduct of the CO
derivative shows a drastically lowered iron-proximal histidine
stretching frequency, which is indicative of increased
T-like strain in the R quaternary state. Also
consistent with these findings is the observation that geminate rebinding is reduced in this derivative compared with HbA and the two
other cross-bridged hemoglobins. The geminate yield is expected to
decrease for the more T-like CO-bound derivatives of HbA.
The emerging picture for the bis-Mal/PEG2000 HbACO derivative is that
the cross-bridge-enhanced interaction between Tyr-
145 and the FG
corner of the
-chain of the same subunit favors a shift of the F
helix toward the EF corner (the T state disposition of this
helix) as is reflected in the iron-His stretching frequency. The shift
of the F helix toward the EF corner enhances effector binding at the
DPG-binding site by creating a more T-like site, and this is
reflected in the increased affinity for HPT.
The present study demonstrates how lifetime measurements in a front-face fluorescence scattering geometry can be used to optically probe the DPG-binding site even under conditions of low effector binding affinity. This technique opens the door to direct optical study of long-range communication pathways in normal, modified, and mutant hemoglobins. It is now possible to monitor changes at the DPG-binding site upon ligation, deligation, effector binding at other loci, and changes in quaternary and tertiary structure. The extent of HPT binding can be correlated with other spectroscopic measurements of tertiary and quaternary structure as well as with the equilibrium distribution of R and T states. These results on HPT binding to HbACO at pH 6.35 have also set the stage for nanosecond pulse-probe time-resolved fluorescence studies to follow the time evolution of structural changes at the DPG-binding site subsequent to photodissociation of either an R or a T state CO derivative with bound HPT.
A combination of front-face detection and lifetime measurements has been used to show that (i) HPT binds to a single site in HbACO at pH 6.35, but not at much higher pH; (ii) HPT is easily displaced by IHP binding, generating a strained R state of HbACO; (iii) the clofibrate-derived effector L35 displaces HPT when present in excess; and (iv) HPT binding can be enhanced or blocked by different chemical cross-bridging modifications. The overall picture that emerges from this study is that HPT binds to the DPG-binding site and that this binding can be used to probe the central cavity as a function of solvent- or ligand-induced changes within the protein molecule.