MASTER QUATENARY STRUCTURE OF METHEMOGLOBIN II. PULSE RADIOLYSIS STUDY OF THE BINDING OF OXYGEN TO THE VALENCE-HYBRID

The pulse-radiolysis·of solutions of adult human methemoglobin was used in order to reduce a singlB.heme-iron within the protein tetramers. T he v a l en c e - h y b r i d s t hu s f o r m ed w e r e r ea c t ed w i t h o xy gen , K i ne t i cs o f t he reactions were btudied. The effects of PH and inositol-hexaphosphate·were examined. The kinetics of the ligatien·of oxygen to.stripped valence-hybrids showed·a single-phase behaviour at the pH range 6.5-9. the pH.was lowered below 6.5 a second, slower.phase became apparent. In the presence of IHP, above pH·& the kinetics of oxygen binding was of a.single-phasd. A# the PH was:lowered a transition to a second, slower phase was noticed. Below PH 7 the slower phase was 'the only detectable one. .The analysti of the relative contribution of. thd!,faster phase to the total reaction as a function of the pH showed a typical trahsition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9.


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Portions of this document may be illegible in electronic image products. Images are produced from the best available original document.  The pulse-radiolysis·of solutions of adult human methemoglobin was used in order to reduce a singlB.heme-iron within the protein tetramers.
The valence-hybrids thus formed were reacted with oxygen, Kinetics of the reactions were btudied. The effects of PH and inositol-hexaphos phate·were examined.
The kinetics of the ligatien·of oxygen to.stripped valence-hybrids showed·a single-phase behaviour at the pH range 6.5-9. As the pH.was lowered below 6.5 a second, slower.phase became apparent. In the presence of IHP, 4' ..
above pH·& the kinetics of oxygen binding was of a.single-phasd. A# the PH was:lowered a transition to a second, slower phase was noticed. Below PH 7 the slower phase was 'the only detectable one. .The analysti of the relative contribution of. thd!,faster phase to the total reaction as a function of the pH showed a typical trahsition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9.
On this basis it is concluded that human adult. stripped methemoglobin resides in an R quartd'rnary structure while the presence of IHP stabilizes the T structure at PH beiow 7.5. This transition between the quaternary · Structures of methemeglebin cannot be accounted for by the switch between the high-spin and the. According to this suggestion the low temperature EPR spectrum of.nitrosyl.
derivatives of various human hemoglobin variants changes markedly when the. tetramer switches its :quaternary structure that is coupled to ' its affinity state.
In a recent.study (13) we suggested that by using the pulse radiolysis technique the. quaternary structure .of ·methemoglobin can be. characterized by following the kinetics of CO bindi g to.its valence-hybrid. The rate constant for CO binding to the valencq-hybrid is dependent upon the pH and the presence of organic phosphates (13,14). The·change in.this rate constant indicates a transition between affinity states (11) that is correlated with a switch between quaternary structures. In this work we further explore the suggestion that the kinetics of Hemeglobin was eluted using a pH gradient of phosphate buffer (6.7 to 8.0). phate were prepared in triply distilled water immediately before irradiation. The pulse radiolysis set-.up has been previously described (17).
Results 0 binding to the IHP-free and IHP-bound valence-hybrids.
The· reaction of the valence-hybrid with oxygen (eq. 2) was spectrophotometrically followed at 435 nm. The·change in transmittance accompanying the process was recorded on a,fast scan oscilloscope and processed by an on-line computer.
At pH 7.5, in the absence of IHP, the reaction of oxygen binding was pseudo first-order with respect to oxygen..The observed rate constant k, was.
found to depend linearly on [02], and the,second-order rate·constant calculated on a tetrameric basis·was, .k2 = 3.54107M-lsec-1. in absorbance between the deoxy and the oxy forms of the valence-hybrid. Figure 1, curve a is a computer-best-fit using a single-phase.first order rate expression (eq..3).
Curve b is a computer best fit of the same data, using a two-phase first order ' rate expression (eq. 4).
where AA is calculated.from eq, 4.and 8AQ is the change.in absorption for the ·oxygenation reaction studied at the same PH in the: absence of IHP. Effect of pH on·the oxygenation reaction of the valence-hybrid, The· oxygen binding reaction to the valence-hybrid (eq. 2), in the absence of IHP, was.studied at the.PH range 6-9, and the dependence·of k on. the PH is shown in fig. 2. Below pH 6..5 in the absence of IHP, the reaction was biphasic and two·rate constants., kf and ks, were evaluated according to eq. 4 ( fig. 2). The pH dependence of ·this reaction was examined in the ·presence of IHP as well.  Table I, The effect of pH on the two rate constants,kf and ks, is illustrated in fig. 2 fig. 3. The data thus treated resulted in a sigmoidal curve of k vs. PH ( fig. 3). This curve resembles those, app similarly obtained, for the binding reaction of CO to the valence-hybrid m previously studied using flash-photolysis ·(19) and Fkllse-radiolysis techniques (13).
Relative contributiens of the slow and fast reacting phases,· Using the data given in Table I  In.the pr6sence of IHP, the pH-dependence of the rate.constant,of oxygen,ligation to·the. valence-hybrid showed a sigmoidal behaviour"that consists of three parts: at low pH .(pH 6 7) at iAtermediate pH (7 < pH < 8), In the absence.of IHP and above pH 6.5 the kinetics of oxygen binding to thB valence-hybrid obeyed a single-phase expression. At PH 6,5 a slower phase.became·apparent. As the.PH was further lowered the biphasic character became more prominent and the relative contribution of the slow phase increased and reached 020% at PH 6.-Both rate constants, those of the fast and of the slow reacting species, are equal to the corresponding ones measured in the presence of.organic phosphates. Thus, it seems that there is an equilibrium between the.two quaternary structures of the methemoglobin even in the absence of IHP. As the PH is lowered, .the T quaternary structure becomes more favourable. This conclusion is similar to the findings with nitrosyl derivatives of various human hemoglobins variants that showed the analogous effect of protons and organic phosphates on the quaternary structure of the hemeglobin tetramer (9)(10)(11)(12).
Comparison between the ligation of oxygen and of carbon-monoxide to the valence-hybrid.
We have shown,that the study of the kinetics of ligation of the valence-, hybrids by the pulse radiolysis technique.can be used to monitor the quaternary structure of the methemoglobin tetramer, The same behaviour is expected when either oxygen or carbon-monoxide is:used. The.analysis and the results shown in this work with·oxygen are more accurate and more.significant as compared to those which were calculated for the reaction with CO (13)..Due to the.fact with 3 different ·rate constants were involved, the analysis for' ilie re#'ct'ign With CO Was less accurate.· Each of the kinetic curves was analyzed 'as if-, it is composed of only two reactions while the third component could' not be taken into account and, therefore, ignored (13). It is impossible to apply the analysis introduced in this study·to the data earlier published, with 00.
"Instedd, if one applies the.former analysis·to .the results in.this preseht study· a different transition ,curve. is evaluated (fig, 3),.This new sigmoidal . curve is characterized by pK = 8 and n = 2,1. These parameters as well as the assymetric shape.of the curve are very similar to those published "e '1'fer'" (13). In conclusion, there is no contradiction ,betweenthe'ttuo·' studies; the difference in the valuesof-the PK and n are due to a.different, more ·meaningful and more accurate analysis introduced in this study, The· R-T transition and the switch in spin states, Methemoglobin can exist in·either of two different.spin states.
Theaquolhet 'form which is .a high spin compound (20), and the.hydroxymet form which is a low spin one (22). The transition between the two spin states takes place at pH above.8 in the absence of organic·phosphates. (22 , 23) and is.shifted to a higher PH in the presence of IHP< . It was suggested· by·Perutz. (3) that the transition.between the two quaternary structures in hemoglobin is coupled to the switch of spin state..This suggestion takes into account the .larger diamet69 of the high-spin compound; the iron in this 0 case, ,cannot fit into the matrix of the porphyrin ring and, thus,. it exerts tension of the molecule that favours the T quaternary structure. It ·is clearly P demonstrated here, that .these two transitions of quaternary structure and spin states do not coincide, but rather·there is a'full pH unit difference between.them.

Inequivalehce between a and B subunits
In a previous study (13) we had.found that when the.valence-hybrid resides in a low-affinity state, ·the kinetics o f CO-binding exhibited a prominent biphasic character. This finding was in accordance with an earlier report by 'Gray and Gibson (24). In the present communication, the reaction of the valence-hybrid with oxygen, rather than with carbon-monoxide, was studied and this biphasicity could not be·noticed.
In a recent article (25) a biphisic behaviour· in the 1.igation of oxyken to the valence-hybrid. was shown even-, in the absence.of IHP. It was claimed (25) that this biphasicity is an expression of a latent chain-inequivalence toward oxygen. .It was suggested ·(25) that the electron does not react indiscriminately with a and B chains during the reduction of methemoglobin ( e<* 1) but rather -that .there is a preference for its .reaction with one specific chain. This suggestion (25)does not agree with two earlier reports (15,17). Also, when the reduction of the·methemoglobin is carried out in the presence of·an excess·of ferricyanide, and the oxidation of the just-formed valence-hybrid is followed spectrgphotometrically -no biphasicity could be detected4. This suggestion (25) by itself cannot provide an explanation for the fact (13,18)

3.
The PH-dependence of the relatiye contribution of the reacting species is a sigmoidal curve that represents a transition between quaternary structures.
The T *R transition is characterized by a P K= 7,5 and a Hill coefficient n= 2.9.

4.
The T * R .transition and the switch in spin state of the ferric iron do not coincide. ·A full pH unit separates between the two phenomena,

5.
No chain inequivalence could be·noticed in the ligation of the valence-hybrid with oxygen even in the.presence of IHP.·In this respect the ligation of CO is different. There, a pronounced biphasicity is clearly seen.
-14 -     The values of ef have been calculated from .the experimental data shown.in Table I