Kinetics of Hemoglobin-Carbon Monoxide Reactions Measured

Proc. Natl. Acad. Sci. USA Vol. 74, No. 7, pp. 2620-2623, July 1977 Chemistry

Kinetics of hemoglobin-carbon monoxide reactions measured with a superconducting magnetometer: A new method for fast reactions in solution (magnetic susceptibility/flash photolysis/metalloproteins) JOHN S. PHILO Department of Physics, Stanford University, Stanford, California 94305 Communicated by William M. Fairbank, April 25,1977

ABSTRACT A new technique for measuring fast reactions a superconducting quantum interference device (SQUID) in solution has been demonstrated. The changes in magnetic magnetometer that can sense very susceptibility during the recombination reaction of human small magnetic fields. It is hemoglobin with carbon monoxide after flash photolysis have primarily the very low noise and fast response of the SQUID been measured with a new high-sensitivity instrument using magnetometer that make kinetic experiments possible. cryogenic technology. The rate constants determined at 200 (pH The instrument may be operated in two modes. To measure 7.3) are in excellent agreement with those obtained by photo- the total susceptibility of a sample, the change in magnetometer metric techniques [Gray, R. D. (1974) J. Biol. Chem. 249, 2879-2885]. A unique capability of this new method is the de- output is recorded as the sample is inserted into the sensing coil. termination of the magnetic susceptibilities of short-lived re- In this mode, the instrument can resolve a change in volume action intermediates. The magnetic moment of the intermediate susceptibility* of 9 X 1012, which is equivalent to a change of species Hb4(CO)3 was found to be 4.9 ± 0.1 jtB in 0.1 M phos- 0.0001% of the susceptibility of a typical diamagnetic sample phate buffer by partial photolysis experiments. This value agrees (such as a protein in solution). This is an improvement of about with the predictions of two-state allosteric models of coopera- two orders of magnitude over vibrating sample, force, or other tivity in hemoglobin. Possible applications and improvements SQUID systems. The system is calibrated with a sample whose in this technique are discussed. susceptibility is known. In the second operating mode, the Magnetic susceptibility measurements have often been used sample remains fixed within the sensing coil, and changes in by chemists and biochemists to probe the electronic states of susceptibility due to chemical reactions, temperature changes, transition metal ions. Such measurements can yield information etc., are measured. This mode was used for these kinetic ex- on the symmetry and strength of ligand fields and the oxidation periments. The system response time is 300 us. For kinetic ex- state of the ion, as well as on the strength of interactions between periments, the susceptometer is characterized by a white noise clusters of ions. Magnetochemical methods have been partic- spectrum above 5 Hz with a rms level of 1.1 X 10-11 (Hz)-1/2, ularly helpful in studies of metalloproteins and of synthetic or a concentration of a spin 'k species of 0.70,uM(Hz)-1/2. The analogues of their active sites. present sample Dewar configuration permits sample temper- The power of many physical techniques has been greatly atures in the -5 to 100° range; low-temperature experiments extended by their use in kinetic studies to measure reaction rates are possible with slight modifications. Sample temperature may and the properties of intermediate species not present in be regulated to +0.0010. This instrument will be described in equilibrium. However, conventional magnetic susceptibility detail elsewhere. instruments generally lack both the sensitivity and time reso- Flash Photolysis Experiments. For these experiments, the lution necessary for measuring fast reactions in solution. In our HbCO solution is placed in a cylindrical quartz bulb (0.30 cm3) laboratory, we have been applying superconducting technology which is placed within the sensing coil for the duration of the to construct a very-high-sensitivity magnetic susceptibility experiment. A Lucite rod serves as a light pipe to carry the instrument for research in biophysics and chemistry. In addition photolytic flash to the sample. A commercial photographic flash to higher precision and sensitivity for equilibrium measure- unit was used with a flash input energy of 50 J and a pulse du- ments, we hoped through this approach to achieve sufficient ration of 1 ms. Up to 80 mj of visible photons reached the time resolution for kinetic measurements. This paper reports sample. The susceptibility changes after the flash were recorded measurements of the kinetics of the reaction of human adult on a digital signal averager. The interval between flashes was hemoglobin with carbon monoxide and a measurement of the 20 s, and usually 32 or 64 transients were averaged. The system magnetic moment of an intermediate in this reaction. time constant could be varied upward from its lower limit of 300 jss and was normally set at 1 ms. All experiments were done MATERIALS AND METHODS at pH 7.3 and 20' ± 0.1'. Data Analysis. Because the iron in HbCO is diamagnetic and Magnetic Susceptibility Instrument. The essential elements in Hb it is paramagnetic (S=2), the changes in magnetic sus- of the magnetic susceptometer are indicated in Fig. 1. The ceptibility are directly proportional to the change in Hb con- samples are placed within a room temperature Dewar flask and centration.t In all these experiments, at equilibrium the Hb is are magnetized in the homogeneous field (0.43 T) of a superconducting electromagnet. The sample's magnetization pro- * Note that SI units are used here; to convert to cgs units, divide by duces changes in the magnetic field at a superconducting 4ir. sensing coil. These magnetic field changes are transmitted to t Strictly speaking, the proportionality holds only if the magnetic moment of each heme is independent of the ligation state of the Abbreviation: SQUID, superconducting quantum interference de- others. This is not always true (see Results), but this is a small effect vice. and may be ignored for most purposes. 2620 Downloaded by guest on September 25, 2021 Chemistry: Philo Proc. Natl. Acad. Sci. USA 74 (1977) 2621

AK= 5x101

C,, VACUUM ROOM TEMPERATURE 040 80 100 ~~~~~~~~~160 SPACE z

0 40 100 TIME AFTER FLASH (ims) FIG. 2. Magnetic susceptibility changes after flash photolysis of HbCO. The scale in SI volume susceptibility units is indicated by the vertical segment. The initial negative offscale change is an experimental artifact (see Results). Total [Hb] = 38.5 MM; [CO] = 130 AM; 0.1 M phosphate, pH 7.3; 20°. Average of 64 transients. System time constant = 1 Ms. FIG. 1. Schematic cross section of magnetic susceptometer. from the error matrix and the estimated uncertainties Xm and with and is therefore The [CO]eq. fully saturated CO diamagnetic. the the entire reaction change in susceptibility from its equilibrium value was con- For partial photolysis experiments verted directly to Hb concentration by using the known molar record was used, and it was assumed that the dimers and susceptibility difference between Hb and HbCO in the buffer Hb4(CO)3 intermediates react at the same rate. These experi- used (1). ments were conducted at very low [CO]eq (small A), and K(0) rate of Hb with CO after and the fraction e'/Xn were treated as adjustable parameters. The of recombination photolysis The dimers were assumed to have the same molar susceptibility was analyzed to find an effective value for e', the rate constant for the forward reaction. Such an analysis is complicated by the as do free chains. dissociation of 20-30% of the Hb tetramers into dimers at the Sample Preparation and Reagents. Human Hb A was pu- from whole blood and was concentrations used in these experiments (2). Further, as a rified by the toluene procedure (3) on a G-25 filtration consequence of heme-heme interaction, the apparent value stripped of organic phosphates Sephadex gel column. The fraction of MetHb was initially less than 1%. of e' increases with fractional saturation. However, the primary was obtained from Chemical purpose of these was to demonstrate the validity 2,3-Diphosphoglycerate Sigma experiments was to of the new technique. Therefore, for purposes of comparison Co. as the pentacyclohexammonium salt and converted with the rate constants obtained by spectrophotometric tech- the free acid on a Dowex 50W X 8 (H form) column. 2,2- (Bis-Tris) was niques by Gray (2), a constant value for over the final 70% Bis(hydroxymethyl)-2,2',2"-nitrilotriethanol of the (tetramer) reaction has been assumed. Over this range, obtained from Aldrich Chemical Co., purified sodium di- T. the fast dimer reaction has already gone essentially to com- thionite from J. Baker Chemic4l Co., and CO from Liquid pletion. Because the pseudo-first-order criterion [CO] >> [Hb] Carbonic Corp. were was not always met in these experiments, the more general Solutions containing CO prepared by diluting buffer second-order formulation was used. Differences between the saturated with CO at atmospheric pressure at 200 with de- oxygenated buffers, assuming a CO solubility of 1.0 mM. A few a and f# subunits have been neglected. The rate of HbCO dissociation is very slow and therefore the back reaction may be grains of sodium dithionite were added directly to the syringe neglected. The kinetic equations therefore become: used to fill the sample bulbs to remove residual dissolved 02- Heme concentrations were measured in a Beckman DK2-A [Hbk [1] the extinction of [Hb]o exp ff'[CO]eq t} spectrophotometer by using coefficients [Co]t [Co]o Banerjee et al. (4). in which the subscripts refer to concentrations at times 0 and RESULTS t and at equilibrium. If CO is in great excess, its concentration remains essentially constant, the denominators in Eq. 1 become The recombination reaction after photolysis of the HbCO was equal and the kinetics become pseudo first order. If we define studied in 0.05 M 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotri- K(t) to be the change in volume susceptibility from its equi- ethanol/0.1 M NaCl buffer both with and without added 1 mM in 0.1 M librium value, then K(t) = Xm[Hb]t. With the fact that [COlt diphosphoglycerate, as well as phosphate. The sus- a are in = [Hb]t + [CO°eq and a constant defined as A Xm[CO]eq, Eq. ceptibility changes during typical experiment shown 1 becomes: Fig. 2. The vertical segment represents a fractional change of only 0.0055% of the total susceptibility of the sample and is K(t) K(O) Jt1Atl equivalent to a change in lfb concentration of 3.3 1sM. Fig. 2 K(t) + A K(0) + A exp [-XM [21 clearly illustrates the feasibility of experiments at micromolar concentrations with millisecond time resolution when signal The values of et' were determined from least-squares fits of the averaging is possible, and at somewhat higher concentrations susceptibility data to Eq. 2, et' and K(0) being treated as ad- for nonrepetitive experiments. The initial negative offscale justable parameters. Standard deviations for e' were determined change is due to a spurious (and unexpected) signal from the Downloaded by guest on September 25, 2021 2622 Chemistry: Philo Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Rate constants for combination of Hb with CO 0.6. et (AM-1 s'1) Total Magnetic Photometrict 0.4- In [Hb], 20.00 + 0.10 230 - 20 w 0.3- Buffer* AM pH 7.3 pH 7.0

aIx 0.2- A 33.6 0.28 + 0.01 0.29 0.01 o. 45 5 B 41.3 0.211 + 0.006 0.21 0.00 C 38.5 0.232 ± 0.004 0.23 + 0.00 0.10- 0.08- * Buffer: A, 0.05 M 2,2-bis(hydroxymethyl)-2,2,',2'-nitrilotrietha- 0. tX nol/0.1 M NaCl; B, A + 1 mM diphosphoglycerate; C, 0.1 M phosphate. 0.04OslI t Data from Gray (2); = 10.2 - [Hb] ,M. 25 45 65 85 105 TIME (ms ) FIG. 3. Pseudo-first-order kinetic plot of data of Fig. 2, normal- determination of both e'4 and the magnetic moment. However, ized to 15 ms after the flash. The solid line represents a second-order with the value e'4 = 7.0,M-M so1 determined by Antonini and rate constant of 0.23 1AM-1 s1. Gibson (9), a moment of 4.9 I 0.1 MB was found.* This value for the magnetic moment of Hb4(CO)3 is relevant to an understanding of cooperativity in Hb. The magnetic Lucite light pipe. This rapidly decaying signal is independent moment per heme of Hb4 is 5.3 AB under these conditions (1). of the presence of a sample and is believed to be mechanical in The lower moment for Hb4(CO)3 shows clearly the effect of origin. In effect, it increases the dead time of the instrument cooperative interactions on the electronic state of the iron. from 1 to 15 ms. We expect to eliminate this problem in future According to two-state allosteric models of Hb, by the time experiments. Fig. 3 is a pseudo-first-order plot of the normalized three ligands are bound the quaternary structure has shifted kinetics of the experiment in Fig. 2. to the R (high-affinity) conformation with properties essentially Experiments were conducted in each buffer system at various identical to those of isolated a and (3 chains (10). Because the CO concentrations and were analyzed to find values for the rate magnetic moment of a or # chains is 4.9 MB (1), these new constant, e'. The lowest uncertainties for e' were obtained in magnetokinetic data support such models. experiments at low CO concentrations, in which the estimated ±10% uncertainty in equilibrium [CO] had little effect. The root-mean-square deviation of the data from the fits to Eq. 2 CONCLUSIONS AND DISCUSSION was generally less than 1% of the total susceptibility change. The present study has established the capability of this new Comparison of Rates from Magnetic and Spectrophoto- method to determine reaction rates and the unique ability to metric Methods. In Table 1, the best values for the rate constant measure the magnetic susceptibilities of short-lived interme- et' from these experiments are listed. Also listed are values ob- diates. It may prove particularly valuable in metalloenzyme tained by Gray (2) from measurements of optical absorption research when the electronic states of intermediate species are changes. Although the intent of this comparison was primarily unknown. It is important to note that this method can give in- to show that this new magnetic technique yields kinetic data formation on paramagnetic species that are not detectable by of quality comparable to that of optical techniques, the excel- electron paramagnetic resonance. The range of applications lent numerical agreement for the rate constants is very en- will, of course, depend critically on experimental constraints. couraging. In fact, because of the slightly different tempera- The use of this type of system for stopped-flow experiments tures and pH values, the rates would not be expected to agree seems feasible, although the amount of noise introduced by the exactly. On the basis of known values for the heat of activation flow system will have to be determined experimentally. Tem- (5), Bohr effect (6), and pH dependence of the dissociation rate perature jump experiments should also be possible. The present (7), the rates for our experimental conditions should be about instrument in no way represents the best performance attain- 5% smaller than those from Gray's data. However, this differ- able. Both microsecond response times and higher sensitivities ence is much smaller than the uncertainty arising from the +20 are possible with existing technology. temperature uncertainty in Gray's experiments. Magnetic Moment of Hb4(CO)3. An important feature of This work would not have been possible without the continued kinetic studies is the possibility of measuring properties of in- support of W. M. Fairbank and the earlier work of E. P. Day. The termediate author also wishes to thank C. Yen for her help in purifying the he- species not present in equilibrium. To demonstrate moglobin and W. Little for the use of his spectrophotometer. This work this capability, the magnetic moment of Hb4(CO)3 was mea- was funded in part by the National Science Foundation, Air Force sured. As a result of the cooperative interactions in the Hb tet- Office of Scientific Research, Office of Naval Research, and the Center ramer, the affinity for the fourth ligand is very high, and very for Materials Research, Stanford University. little Hb4(CO)3 exists at equilibrium in partially saturated so- The costs of publication of this article were defrayed in part by the lutions. Partial photolysis experiments in 0.1 M phosphate were payment of page charges from funds made available to support the conducted in which only 7.0% of the CO was removed. The research which is the subject of the article. This article must therefore predominant species produced is Hb4(CO)3, and the recom- be hereby marked "advertisement" in accordance with 18 U. S. C. bination occurs very rapidly with a rate constant corresponding §1734 solely to indicate this fact. to e'4 in the Adair formulation (8). The reaction records were analyzed to determine the molar susceptibility (and therefore 1. Alpert, Y. & Banerjee, R. (1975) Biochim. Biophys. Acta 405, the magnetic moment) of Hb4(CO)3. Technical difficulties 144-154. during these first experiments prevented measurements over a sufficient range of CO concentration to permit independent t This value is proportional to (e'4)"2. Downloaded by guest on September 25, 2021 Chemistry: Philo Proc. Natl. Acad. Sci. USA 74 (1977) 2623

2. Gray, R. D. (1974) J. Biol. Chem. 249, 2879-2885. & Rossi-Fanelli, A. (1963) J. Biol. Chem. 238,2950-2957. 3. Drabkin, D. L. (1946) J. Biol. Chem. 164, 703-723. 7. Sharma, V. S., Schmidt, M. R. & Ranney, H. M. (1976) J. Biol. 4. Banerjee, R., Alpert, Y., Leterrier, F. & Williams, R. J. P. (1969) Chem. 251, 4267-4272. Biochemistry 8,2862-2867. 8. Adair, G. S. (1925) J. Biol. Chem. 63,529-541. 5. Gibson, Q. H. & Roughton, F. J. W. (1957) Proc. R. Soc. London 9. Antonini, E. & Gibson, Q. H. (1960) Biochem. J. 76,534-538. Ser. B 146,206-224. 10. Monod, J., Wyman, J. & Changeux, J. P. (1965) Biochem. J. 76, 6. Antonini, E., Wyman, J., Brunori, M., Bucci, E., Fronticelli, C. 534-538. Downloaded by guest on September 25, 2021