Ethane diol was added to aqueous solutions of bovine and
human met(FeUI)- and CO-haemoglobin in order to extend the
temperature range for nuclear magnetict relaxation measurements
below freezing point. No significant difference in the relaxation
rates was found on adding ethane diol except in the case of human
met- and CO-haemog1obin, which may be . ascribed to chang·es
in the hydration sheath. The energies of activation derived :firom
Arrhenius plots of the relaxation rates for bovine Hb are also
independent (within ± 50/o) of ethane diol. It is concluded that the
gross conformation of the haem pocket is not altered by addition
of ethane diol, so that measurements can be done down to - 30 °c

J. Brnjas-Kraljević

S . Vuk-Pavlović

S. Maričić

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CROATICA CHEMICA ACTA CCACAA 45 (4) 619-625 (1973) CCA-809 YU ISSN 0011-1643 547.979 Original Scientific Paper Proton Magnetic Relaxation in Ethane Diol-Water Solutions of Haemoglobin J. Brnjas-Kraljevic, S. Maricic, amd . S . Vuk-Pavlovic Institute of Biology and Institute of Physics, University of Zagreb, 41000 Zagreb, Croatia, Yugoslavia Received May 28, 1973 Ethane diol was added to aqueous solutions of bovine and human met(FeUI)- and CO-haemoglobin in order to extend the temperature range for nuclear magnetict relaxation measurements below freezing point. No significant difference in the relaxation rates was found on adding ethane diol except in the case of human met- and CO-haemog1obin, which may be . ascribed to chang·es in the hydration sheath. The energies of activation derived :firom Arrhenius plots of the relaxation rates for bovine Hb are also independent (within ± 50/o) of ethane diol. It is concluded that the gross conformation of the haem pocket is not altered by addition of ethane diol, so that measurements can be done down to - 30 °c. INTRODUCTION The temperature range within which proteins 'in solution can be studied in their native state is limited by denaturation and fireezing. There are several reasons in favour of extending the lower temperature region for studies of haemoprotein solutilons by proton magnetic relaxation: (a) The interpretation of the results in terms of stereochemical parameters requires extrapolation frrom low to higher temperature data: The accuracy of this procedure1 would increase if more data were available at low temperatures. (b) An extension of measurements to temperatures below zero centigrades is sometime neces-sary for an unequivocal determiination of the type of the temperature depen-dence of the relaxation rates, (c) The rate of exchange of protons between the haem pocket and bulk solvent may be easier to study when diminished by lowering the temperature. (d) A marker molecule with nonexchangeable protons which would not enter the haem pooket and be inert towards hae-moproteins is needed also for magnetic susceptibility measurements by NMR2 and for the elucidation of the molecular model of the p11oton relaxation in haemoprotei:n so1utions3• It ha:s already been found by spectroscopic4 and other studies5 that ethane diol has hardly any influence upon (haemo)protein molecules. The purpose of this paper is to discuss evidence in favour of using ethane diol for freezing point depression of haemoglobin solutions without perturbing significantly the haem environment. EXPERIMENTAL Materials Bovine haemoglobin was prepared from freshly collected cattle blood, and human haemoglobin from the blood supplied by the focal blood bank. The procedure 620 J. BRNJAS-KRALJEVIC ET AL. of Cameron and George6 was followed as closely as possible. The oxyhaemoglobin dialysed against 0.1 M NaCl at pH = 6 was oxidized by addition of KaFe(CN)6• Three aliquots of the ferrihaemoglobin solution were subject to the final dialysis against 0.1 M NaCl+ 5 X 10-5 M EDTA: (a) without addition of ethane diol; (b) with 10 vol. -O/o ethane diol, and (c) with 25 vol. 0/o ethane diol (which in case of human Hb was 35 vol. 0/o) . Ethane diol was analytical grade (»Merck«) and was used as supplied. Carboxyhaemoglobin was obtained by bubbling CO through predialysed oxy-haemoglobin. The solutions for the final dialyses were also saturated with CO. The absorption spectra in the visible range showed changes neither in respect to the ethane diol presence nor in comparisons before and after the relaxation measurements. The haem concentrations were calculated from the extinction coef-ficients given in the literature7 • Methods The proton magnetic longitudinal relaxation times, Ti, were measured with three pulsed spectrometers (made in the »Jozef Stefan« Institute, Ljubljana). The first one operating at 15 MHz was not phase-coherent and the :Bree inducation decays after a n/2 - n/2 pulse sequence were read off. the oscilloscope screen. Those operating at 32 and 24 MHz were phase-coherent, with a digital readout. The sample temperature, achieved by thermostatted precooled nitrogen stream, was recorded with a Y. S. I. thermistor telethermometer directly immersed into the sample. The T1 measurements were taken at 10-20 temperatures between - 30 and + 40 °c in two ways: (a) thermal equilibrium within 1 °c at any given temperature; (b) slow continuous heating from the lowest temperature. There was less than 50/o difference between the (a) and (b) recording. RESULTS All the measurements were :performed with solutions close to pH= 6. The concentrations (per haem) of six independent bovine ferrihaemoglobin preparations were between 2.8 and 6.6 mmol/l. The paramagnetic contribution to the relaxation rate (1/T 1 ) normalized to unit haem-concentration was derived TABLE I The molar (per haem) proton magnetic relaxation rates (T1-1/s-1 mo1-1 l) due to dissolved bovine ferrihaemoglobin (i n 0.1 M NaCl, pH =6) with addition of ethane dial I I vol. O/o ethane diol vdMHz temp./°C ----0·--------1-0-------2-5---0 275, 394, 363 366, 251 , 459 247, 174, 479 329 ± 20 359 ± 60 299 ± 92 15 40 657, 606, 815 896, 658, 935 563, 714, 880 693 ± 60 829 ± 86 719 ± 94 ti 364 ± 60 470 ± 86 420 ± 94 0 I 290, 318, 334 252, 368, 379 444, 454, 418 314 ± 12 333 ± 40 439 ± 10 32 40 755, 762, 1000 629, 835, 849 901, 800, 1025 839 ± 80 771 ± 70 908 ± 65 I I ti 525 ± 80 438 ± 70 469 ± 65 MAGNETIC RELAXATION IN HAEMOGLOBIN 621 as already described1• The data from the low (0 °q and high temperature range (40 °q are collected in Table I together with the difference between the two which is related to the stereochemistry of the haem pocket. The activation energies in Table II are obtained by assuming the Arrhenius relation for the thermally activated relaxation rates in the middle temperatures region. TABLE II The energies of activation (Ea/kcal moi-1) for the thermally activated proton magnetic relaxation rates due to dissolved bovine ferrihaemoglobin (0.1 M NaCl, pH=6) with addition of ethane diol. I ethane diol I vol. 01~ 0 10 25 15 5.78; 4.99; 3.80 4.85 ± 0.57 3.84; 3.80; 4.23 3.96 ± 0.13 4.11; 3.46; 4.88 4.15 ± 0.20 32 4.99; 3.46 ; 4.99 4.48 ± 0.44 3.80; 3.46; 3.65 3.64 ± 0.10 4.23; 4.23; 4.04 4.17 ± 0.02 As the results obtained with bov.ine ferrihaemoglobin did not reveal any systematic difference in relaxation rates on addition of ethane diol, measu-rements were done with human ferri- and CO-haemoglobin only with 35 vol. 0/o ethane diol. J r. /s COHb ~ : a ..... . . ferri Hb . 0 10 20 30 40 5-0 .4 - 0 & & 3 -6 {J ... .. .2 -- fl • .. • fl .. • ... • 0 • • . 1-. • I I I -30 - 25 - 20 -1 5 -10 b .,, . ~ • ' 0 • 0 & ! ! • • li ,,. ~ •• ! ••.• i "i ••• .11. -30 -20 -10 0 10 . 0 .. .• COHb f erri Hb . . . .. 20 30 40 t/° C Fig. I. The temperature dependence of longitudinal proton magnetic relaxation times, T1 for aqueous solutions (0.1 M NaCl, pH = 6.4 ± 0.2) of human haemoglobin A without ethane dial (a), and with 35 v. 1/o ethane dial (b) . Circles and triangles refer to the solvents of the ferri- , and COHb solutions resp., which in turn are marked with the filled signs. The haemoglobin concentrations, mM per haem, were as follows: COHb - 5.1 (a) and 8.6 (b) ; ferri Hb - 5.1 (a) and 6.6 (bl 622 J. BRNJAS-KRALJEVIC E'T AL. The T1 data for human CO- and ferrihaemog1obin solutions and for the corresponding solvents are presented i:n Fig. 1. The relaxation rates normalized 1 per haem concentration, 1'1-, in dependence on reciprocal temperature are T1 given in Fig. 2. The finely drawn lines (either full or dotted for zero and 35 vol. 10/o added C2H 60 2, resp.) are for the overall, ferrihaemoglobin or COHb contribution to the relaxation rates of solvent protons while the thickly drawn lines represent their relaxation rates due only to the presence of the para-magnetic haem iron. 40 30 20 10 0 100 3.30 3.60 -10 -20 -30 ------3.90 1000 T/K tl'C 420 Fig. 2. The Arrhenius plot of the proton magnetic relaxation rates normalized per unit haem concentration, as derived from Fig. 1. The dashed lines refer to the solutions with 35 v. 0/o ethane diol and the others to those without ethane diol. The finely drawn curves depict this relationship for the indicated solutions while the thickly drawn ones are obtained by subtracting the COHb relaxation rates from the ferri-Hb data, thus obtaining the relaxation rates induced solely by the presence of the paramagnetic haem iron in ferri Rb-solutions . DISCUSSION In the following discussion neither of the two existing molecular models3 for the relaxation mechanism will be involved. Nor will the data be quanti-tatively evaluated by the existing theory, because the full intenpretation of thls kind of experiments is being treated elsewhere1•3•8·9• We are only interested here in the relative differences induced by the presence of ethane diol. It is thus sufficient to note that the low temperature relaxation rates as well as the difference between them and those at 40 °c are related to the distance from the haem iron and the relaxing proton(s) . Hence, any changes in haem environment caused by addition of ethane diol would be reflected in the pr.oton magnetic relaxation rates. Comparison of the data presented in Table I shows no consistent depen-dence -of the relaxation rates on ethane diol at constant frequericy; they are equally spread around a mean value. The same statement holds for the rates MAGNETIC RELAXATION IN HAEMOGLOBIN 623 measured at 0 °c and 40 °C as well as the difference between them. The mean values of the subtractions are 420 ± 90 s-1 moi-1 1 for 15 MHz and 480 ± 80 s-1 mo1-1 for 32 MHz. All the single measurements are within the region of •One a. The difference in values at the two frequencies is within one o as well. The T1 data on human haemoglobin (Fig. 2.) corroborate our conclusion that on the whole the haem surrounding is almost unaltered even in the presence of 35 vol. O/o of C2H 60 2 • In the formerly discussed case of bovine ferrihaemoglobin (Table I) we had n:o correction of the diamagnetic contribution to the relaxation rates measured in solutions of the paramagnetic ferrihaemo-globin. It is not large, but it does influence the shape of the temperature dependence of the relaxation rates ·as shown in Fig. 2. where we subtract the CO-curve from the ferri-curve. The difference .in the relaxati<0n rates between 40 °C and those at low temperatures is 400 ± 20 s-1 moi-1 1 for the H20 solution and 540 ± 10 s-1 mo1-1 for the solution with 35 vol. O/o of C2Hp2• If this dif-ference truly reflects some change in the haem environment (otherwise un-detected by visible spectrophotometry) it is a slight one, because the distance of the interacting spins, r, is related to these experimental parameters by r-6 • The two curves for CO-Rb, i. e. with and without C2H6 0 2 are different. These relaxatiion rates are due to the interaction ,of solvent protons with those mainly at the surface of the diamagnetic protein (COHb), and therefore, the larger the rates the greater the interaction of solvent molecules with the protein surface. Hence, the interaction of solvent protons with the protein surface is more pronounced in the solution with 35 vol. O/o C2H60 2 than in the one without it, so that it seems that ethane diol influences the structure of the hydration layer around haemoglobin and, perhaps thvough 'it, to a small degree the conformation of the haem pocket, but th1s conclusion has to be verified by deuterium and oxygen-17 relaxation measurements. The activation energies (for the relaxation rates), closely related to the dynamics of the haem pocket conformation, are another test upon the simi-larity of the haem environment in the haemoglobin solutions with and without ethane diol irrespective of which molecular model is used to explain them. Changes in activation energies with addition of ethane diol would indicate differences in the conformational states of the haem environment, although nothing definite could be said about their nature. The mean value for all the activation enevgies data presented in Table II, for bovine ferrihaemoglobin, which are not corrected for the diamagnetic contribution is 4.2 ± 0.2 kcal moi-1 (1 kcal == 4.184 kJ). The value is frequency independent which is to be exipected and shows no tendency of changing with ethane diol addition. All the obtained values are within the experimental error except the data for samples with no ethane diol at 15 MHz and with 10 v. 0/o ethane diol, at 32 MHz. Their deviation could be due to random error as the mean does not change with their rejection. The two curves for human ferrihaemogloibin corrected for the diamagnetic (COHb) contribution have Ea = 6.2 kcal mo1-1 (no C2H r, 0 2) and Ea = 5.2 kcal mo1-1 (with 35 vol. 0 /o C2H 60 2). The comparison of activation energy data supports ·the conclusion that ethane diol has no effect on the conformational state of the haem pocket which would result in changes of the relaxation process. 624 J. BRNJAS-KRALJEVIC ET AL. The low temperature part in Fig. 2. merits comment. The COHb cor-rection becomes important at these temperatures and is in fact ·about more than half the value of the relaxation rates for the (uncorrected) ferriHb solution. With this correction, the paramagnetically induced relaxation rates are practically constant in this range of temperatures. As pointed out in ref. 1, no unique stereochemical parameter can be derived from the low temperature data because of the shape of the haem pocket. The relaxation rates at low temperatures are an overall measure of the accessibility of the haem pocket for solvent molecules and so Fig. 2. suggests that the conformational state of the haem pocket does not change from about - 10 °c down to at least - 30 °c. This means that for all practical purposes, when working without C2H00 2 , the measurement close to zero centigrades should be corrected by the corresponding v·alue of the COHb - relaxation rate. This corrected low temperature value can then be subtracted directly from the uncorrected relaxation rate for the ferriHb solution at 35-40 °c where the COHb correction is of the same magnitude (- 70/o) as the experimental eror in T1 determination. However, because the COHb correction changes Ea and because the minimum for the ferriHb curves is not always well defined above zero centigrade the most accurate way of doing all these measurements is to extend them below 0 °c and correct fully for COHb. Even if it involves some departure from the »standard« native state of the haemoglobin molecule in solution, this procedure may eliminate other systematic errors. As for the possible overall perturbation of the native Hb conformation on addition of ethane diol our results are in agreement with the spectro-photometric4 and other studies5. The proton magnetic relaxation measurements are complementary to the difference spectrophotometry4 in that they yield information about the gross conformational state of the haem surrounding. Acknowledgement. This work was done under the PL-480 contract No. 02-004-1 between the N. I. H . (U.S. A.) and The Institute of Biology, University of Zagreb, with the partial support of the Research Fund of the S. R. of Croatia (contract IV/3). S. V.-P. thanks the latter Fund and The Institute of Biology for a postgraduate scholarship. The authors are indebted to Mrs. V. Bracika for her excellent technical cooperation. REFERENCES 1. G. Pi fat, S. Mari c i c, and S. Gr a n d j a, Biopolymers 12 (1973) 905. 2. K. D. Barte, B. J. D a I e, D. W. Jones, and S. Mari c i c, J. Magn. Res. 12 (1973) 286. 3. S. Vu k - Pav Io v i c, B. Benko, and S. M ari c i c, submitted for publication. 4. Th. T. Herskovic s and J. Green b I at t, Arch. Biochem. Biophys. 130 (1969) 30. 5. P. Dou z o u, Biochimie 53 (1971) 77. 6. B. F. Cameron and Ph. George, Biochim. Biophys. Acta 194 (1969) 16. 7. E. Antonini and M. Bruno r i, Hemoglobin and Myoglobin in Their Reactions With Ligands, North-Holland Publ. Co., Amsterdam, 1971. 8. G. Pi fat and S . Mari c i c, Biophys. Chem. 3 (1973) 112. 9. G. Lah a j n a r, I. Z up an c i c, R. BI inc, G. Pi fat, and S. Mari c i c, Biopolymers 13 (1974) in press. MAGNETIC RELAXATION IN HAEMOGLOBIN 625 IZVOD Protonska magnetska relaksacija hemoglobina u vodenim otopinama s etan diolom J. Brnjas-Kraljevic, S. Maricic i S. Vuk-Pavlovic Etandiol je bio dodavan vodenim otopinama govedeg i ljudskog met(Fem)- i CO-hemoglobina da bi se podrucje mjerenja nukleai:ine magnetske relaksacije pro-sirilo na temperature ispod tocke smrzavanja. Nije bilo zmacajnije razlike u brzinama relaksacije uz dodatak etandiola osim u slucaju ljudskog met- i CO-hemoglobina, sto bi se moglo pripisati razlikama u hidrntacijskoj ovojnici. Energije aktivacije dobi-vene iz Arrheniusovih grafova za brzi-ne relaksacije govedeg Hb takoder su (unutar ± 5°/o) nezavisne o etandiolu. Zakljucuje se da se opce konformacijsko stanje dfopa hema ne mijenja dodatkom etandiola, tako da se mote mjeriti i do - 30 °c. INSTITUT ZA BIOLOGIJU I INSTITUT ZA FIZIKU SVEUCILISTA U ZAGREBU 41000 ZAGREB Primljeno 28 . svibnja 1973. 

Proton Magnetic Relaxation in Ethane Diol-Water Solutions of Haemoglobin

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