Magnetochemical Structural Investigations with Particular Reference to Hemoglobin Equilibria and Compound Formation Citation Russell, Charles Daniel, Jr. (1940) Magnetochemical Structural Investigations with Particular Reference to Hemoglobin Equilibria and Compound Formation. Master's thesis, California Institute of Technology. doi:10.7907/6014-2d85. https://resolver.caltech.edu/CaltechTHESIS:01132025-221915071 Abstract No abstract. Item Type: Thesis (Master's thesis) Subject Keywords: (Chemistry) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Pauling, Linus Thesis Committee: Unknown, Unknown Defense Date: 30 August 1939 Record Number: CaltechTHESIS:01132025-221915071 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:01132025-221915071 DOI: 10.7907/6014-2d85 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 16951 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 13 Jan 2025 22:36 Last Modified: 13 Jan 2025 22:45 Thesis Files PDF - Final Version See Usage Policy. 20MB Repository Staff Only: item control page

MAGNETOCHEMICAL STRUCTURAL INVESTIGATIONS WITH PARTICULAR REFERENCE TO HEMOGLOBIN EQUILIBRIA AND COMPOUND FORMATION Thesis by CHARLES D. RUSSELL JR. In Partial Fulfillment of the Requirements for the Degree of Master of Science In Chemistry, California Institute of Technology Pasadena, California August 30, 1939 TABLE OF CONTENTS 1.. Acknowledgments P• l. 2. Introduotion P• 2. 3. Theoretical Discussion P• a. 4. Apparatus and Methods or Measurements P• 13. 5. Preparation and Standardization of Hemoglobin Solutions 6. Experimental Procedures, Results, Conclusions, and Remarks 7. P• 16. P• 19. Experimental Notes on Brief Investigations P• 41. 8. Summary P• 52. 9. Bibliography P• 53. -1- The wrtter wishes to tQ;ke ,111.; C?.ppo.r\ttnliriy to e,cpre,as hls Jn<J.ebtednees t,0 PretErs$.tr Linus Pauti.nth Jt, whose suggeitio.n thts r.:t.$e·~ reh was $tar-tecH ~d und$r w.~os$ ad.v4ee. enec:r11r;a gement, and ldnd1y o~f t:,.• cism 1 t was Q.a rried out. Deep gtati tt1.de ia ~xpressed to Dr"' t. fl. Davies .and Dr• C• D. aory,l.l fe1· \.h~it· m~uy h~lp.fuJ; S'llgg$S• tions offtn·ed dur-ing thl eourste. Qf this werk. The author $Xp~e,ssei.:, his appreeiation tC> Mr-. James P. Dev0n.s.h ire for his ktnd assit\anee 1n the ty-p!ng of the manuaeripi and i.n the preparlng of the f'1gur-es. The writer also Qffers hi$ most $1nc&r$ thanks a.n<l appr~(;l1a.t1on to his peu·~•u:i\$, wi t,hout whose many sa.ertt'lces thls study at the 0alif(!)rnia Institute could. not have been underttlk,n. - 2 - Introduction It can be stated, with a fair degree of historical accuracy, that the science ot magnetism began with ex... perimen.t al discoveries of the ancient peoples dealing with the attractive power of a mineral ore, lodestone, or magnetite, for iron. An old legend attributes the discovery of magnetite to a Cretan shepherd, who was strongly drawn to the earth by his iron-tacked sandals and iron-tipped crook, while traveling in Magnesia, a region in Asia Minor where magnetite 1s very plentiful. (1) However, even in the earliest books of the Hindus, · .•! the Vedas, a description of the lodestone may be found, and also in the ancient literature of the Chinese, Greeks, and Romans, references may be met bearing on the relationships of magnetic phenomena to earthly directions and celestial motion. It is interesting to note that the pivoted needle• or mariner*s compass, was established by the writings of Neok~m of st. Albans at about the time of the Crusades in the twelfth century.(2) The names of many scientists and philosophers im~ mediately present themselves to the research worker in the field of magnetism. Amoung the ancients, Thales, Socrates. Plato, Aristotle• and many others are all credited 11vi th the knowledge of the attraction of lode- stone for iron, but like most of the physical science -3- of the Greeks and Romans i ·t :was mostly qualitative and speculative. In the thirteenth century, there is found a man, Peter Pategrinua, of outstanding experimental ability, to whom is given the firs.t credit for the introduction of the term "magnetic polett.(1) William Gilbert of Oolohester (1540-1603) is per .... haps the first outstanding worker in magnetism. His famous treatise "De Magnetett reveals him to be a very outstanding personality with a great ability to coordinate experimental facts and to separate them from fiction. His greatest contributions were A. the discovery of the ~:· earth's magnetism, and B. J" the uncovering of the fact that iron ceases to be attracted when red-hot. The first quanti tativ:~ magnetic investigations were carried on by John Mi tchel.l (1724-1793) and John Robison (1739-1805)~ Together they brought forth the fundamen- tal law that ttthe attraction and repulsion power of mag, nets decreases __as the squares of the distances from the . . respective poles increase"(l) Mitchell's work was readily repeated and verified by Coulomb (1736-1806) who was the man responsible for the wellwknown Coulomb law of the mutual action between magnetic poles. The first application of the mathematical analysis to the clarification of physioo~chemical problems in the realm of magnetis-m and electrostatics was accomplished by Poisson, who used the famous "two-fluid theory'' as a - 4- means of deriving Coulomb's law. :Poisson•s tre,atment, refined and extended by Kelvin and Green, is the reeog~ nized basis of modern statistical theory of magnet_ism. (l) In the period shortly following, the field became crowded with many investigatbrs. Some of the more important ones were Oersted; Arago. Biot, savart, and Amp~re. Ampere seems to be the most. outstanding and is noted for his "moleeula, ... current" theory of magnetiam. Faraday has been called the f~undsr of magnetochem• istry by Bhatnagar and Mathur,(l) In 1831, he described his famous laws of electromagnetic induction before the Roya: · ·s ociety. ln 1e4s, he di~cove-red accidentally- the I, t diamagnetic qualities of ~~ss, and this. along with the Lt previous discovery by Brugm.~ s of tLe diamagnetism of ;~ bismuth, attraoted the a.tt;~tion of cherr,ists to the study of magnetic phenomena. Fa~~.:da.y. }:\ in turn, examined large numbers of substances for their magnetic properties in different. states and classified them aoe,o rdingly. Following Faraday. it is found that the number of research workers in magnet.oohemistry has undergone another abrupt increase and up until the present day the number has been steadily growing due to the interesting proble~s presenting themselves in the field. Some of the more important persons in the period following Faraday were Tyndall; Weber. Ewing, and Evershed. The -5 - last three all proposed theories of magnetism. The theories of Ewing and Evershed being pictorial descrip• tions appeal* to those chemists who are not mathema• tioally inclined. One of the most outstanding magnetic workers was Pierre Curie• who devised in 1895 the most successful magnetic balance. His experimental researches on the magnetic properties of substances in relation to temperature(l895)(l), Honda's work on the elements, and Pascal's work on organic compounds constitute the pillars on which the modern theory of magnetism has been raised. some of the later workers in magnetochemistry have made really outatand_ing contr1 but ions to the field. AmoneL them are Langevin, Weiss, Gans, Oxley, stoner, Van Vleck, and Pauling. Actually. struotur~l magnetochem• . istry began about 1931 with the independent researches of Bose• Stoner, and Pauling. Paulings's first paper on the nature of the chemical bond* gives many examples of deductions regarding the atomic arrangement, bond angles, apd other properties of molecules and complex ions from magnetic data. with the aid of oalculations involving bond eigenfunctions. The magnetQ.chemist:ry of hemoglobin started with Faraday over ninety years ago, on November 8, 1845. * L. Pauling- Jour. Amer Chem. Soc., 53, 1367 (1931) - 6- He investigated the magnetic properties of a cake of dried blood and made a note 11 muat try recent fluid blood•"• If he had carried out his suggestion and de- termined the large difference in tne susceptibilities of arterial and venous blood, the discovery would have excited a great interest in magneto-hemoglobin-chemistry. The course of blood and hemoglobin research would thus have been very definitely influenced. A. Gam,,ee#% and one or t i.rvo more recent investigators have raported blood to be about as diamagnetic as water. The difference between arterial and ven.o.u s blo~d however was not discovered until later, Felix Haurowitz and Herbert Kittel in 1933 stated"* that their magnetic meas·urements indicated that the tf~atoms ot the free rad- teal 1n porphyrins were arranged diagonally opposite. Hisayuki Kudo in 1934 annQunced~HHr that human and ani"" ma.l bloods were diamagnetic with a susceptibility $lightly greater than water .• wriit~n The first important paperi~~~H'# wasA by 1.1.nus Paul- tng and c. D• Coryell in 1936 at the California Institute. They $howed by tll~.ir magnetic measurements that oxyhemoglobln and ear'bomoxyhernoglobin possessed no unpaired -.~-··........ ' Ci ½~* ~.t ~J* ~ ~~\~~{-4'/ ri1' . ,,. L. Pauling and a. D. Coryell .. Proc. Nat.• Aead. Sci.,., 22, 210 1 (1936) ea, 503 (1901) A. Gamgee .. Proo. Roy. so~., London, £"' . Haurovvitz and H.. Ki;tle .... Ber. t 66B, 1046 (1933) H., Kudo ..,. Aota Med. Sea.no,.,, Bl• 511 (1934) L. Pa:fl:ing and c. D. Co.ryell .. Proc. Nat. Aead. Sci., 22, 210• (1936) - 7- electrona and that :f'errohemoglobin contained t9ur .BB.· paired electrons per heme. This proved that the bonds from iron to the surrounding atoms were ionic in hemo ... globin, and covalent in oxyhemoglobin and carbonmonoxy... hemoglobin. Much work has been carried out since this first or1ginal paper. In 1937, C.Q ryell ., Sti t,t t and Paul- ing* worked out the magnetic properties and struoture of methemoglobin and some of its oompouno.s. The great im- portance of thi s work was in QXplain1ng the sigmoid.al character of th~ ,:,. variation of the magnetic susceptibility of ferrihemogJ.obin solutions with pH. In 1938, Taylor and Coryell made a very intensive study of the magnetic susceptibility of iron in ferrohemoglobin.~} ln 1939, Stitt and Coryell studied magnetically the equ1librium between ferrohemoglob1n, oyan1de iron, and cyanide ferrohemoglo.- :_ bin.{~ Much work is still being done 1n the magnetoehemist:ry of hemoglobin and it is hoped by the author that the work done in this dissertation might be a worthy contributio~ to the fi~ld. -tt Coryell, ,r,o. ,fl'tH} Stitt, and Pauling ... Jour. Am. Chem. soc., 59, 633, (1937) - T~ylor and Cpryell ... Jour. Arn. Chem. Soc., GO, 1177, (l 938) Stitt and Coryell• 12€>3, Jour. Am, Chem. soc.• _a, (1939) Theoretical Discussion In a classical paper in 1905, Langevin(l) gave a new outlook on the facts of para- and diamagnetism. He applied the concepts of the electronic hypothesis to formulate an exact matheniatieal theory which pro .. vided a very satisfactory explanation of both paraand diamagnetism. Since Langevin's treatment was not based on any specific assumption, it could be modified to accord with the principles of the later quantum me. chan1cs. D1amagnet1sm .,._.,. Substances are said to be diamagne\io when they are repelled by a magnet. There is produced in every substance• upon the application of a magnetic field, a magnetic polarization arising from the 11 Larmor prec.essiontt of the electron orbits, which is an accelerating influence of the field on the elee~ trons.(4) Th.is polarization 1s negat,ive in direction, thus giving rise to a field which 1s opposed to the ap ... plied field. Since the "Larmor precession" is indepen- dent of any thermal variation of the kinetic energy of the atoms. it follows that the diamagnetism should be independent of the temperature. However, this is not universally true, as there have been numerous diamagnetic substances found whioh are influenced by temperature. Also the diamagnetic polarization of a substance is almost independent of structure; it can be oalculated(4) - 9- with reasonable accuracy as the sum of the terms corresponding to the atoms composing the substance, with perhaps some small corrections for the bond•types involved. Paramagnetism - - A substance ia said to be paramagnetic when it is polarized in a direction opposite to its diamagnetic polarization. that is, with the field. In this condition, the substance is attracted by a magnet. This paramagnetic polariza.t1on(4), often of large magnitude, results from the presence in the sub• stance of atoms, ions, ot mol ecules with permanent mag .. netic dipol e moments, which in ~eneral are due · in part to the spin magnetic momentsof µnpaired electrons and in part to tl1e magnetic moments associated with the or- bital motion of the electrons. The orienting tendency of the permanent magnet dipoles in .a magnetic field is opposed by the effect of te:nperature, and, aoc:ordingly, paramagnetic polarization is temperature dependent. For ideal· systems• having amall interaction between the dipoles, it is inversely proportional to the absolute temperature.(4 ) Molal susoeptibilitl - ~ Let ;(molal be the molal susceptibility of a substance; N, the Avogadro :number; k, the Boltzmann constant; T, the absolute tenif" perature; tr, the molecular diamagnet1c suS-:O.e pti bili ty (a negative quant~:ty); and ff• the permanent magnetic. dipole moment. It is then possible to relate all these quantities by the means of the Langevin-Pa:uli equation. (4) 'Xmol al = NtK + NµB 3kT - (I) In the extreme right-hand form of the equation, C is called the Curie constant and is equal to 13kT (II) The dipole magnetic moment, , is ...usually expressed in Bohr magnetons, with magnitude eh 47Tmo (III) and in this unit, it is related to 9 by the equatjon:(,,,\) (IV) If it is assumed that the · orbital contribution to the magnetic dipole moment is zerot or if a correotion can be applied, the magnetic dipole moment, is related to the number of unpaired electron spins, n, by the following equation.(4) Bohr Magnetons The :foll.owing short table shows the relationship be ... tween the magnitude of the dipole moment and the num ... bar of the unpaired electrons. - 11 - - No. of Unpaired Electrons Magnetic Dipole Moment l l.73 2.83 3.88 4.90 6.92 2 3 4 5 Calculation of the molal ma~nette s,uso~ptibility from exp¢)rimental _d~t,! ....... In the ~o.uy nH!thod(l) for determining magnetic susceptibilities, described in the sect.i on of this thesis on "Apparatu$ and Methods of Me asurement", an equation relating the foroe Jtx, or measured change in weight , w, with the field f;ltrength, H, and the oross.-seotional area of the tube, A, can be written ae follows : Fx (VI) where K1 and R2 represent the volume susceptib;Llities of the material in the cylinder and the medium respectively. Then, if three measurements are taken; solu• tion versus water, solvent versus wa:t,er. and air versus water; it is possible to write dQvm three equ~tions re• speotively. L\Wsolution-water =- -.iHKsolution·Kwater>A\i 2 • ) AH2 L'\J Vf solvent-water --- .J..2 (• Ksolvent·Kwater . L\ Wair.water = i(Kair""Y-water)AH2 - 1 2- After combining the above three equations, the following two equations are obtained. Ksolu•Kwater = ( ilWsol ution-water> (Kair - Kwater) ( (VIII-a) Wair.water) Kso iv•K wa t. er~·-----................................... ~< ~wsolvent-water)(Kair~Kwj,~k@t) _ ___,,....,..-"' - - (VIII-b) ( l)Wa.ir-,Nater> However, Ksolute-::::: Ksolution - Kaol vent Therefore, by subtracting (VIII-b) from (VIII-a), an equation for the volume susceptibility of the solute is obtained,. But, (IX) _ susceptibility of solute K~solute : · cm3 Hence, equation (IX) must be multiplied by lQQQ. C whe:re C is the molar concentration of the solute, to give the expression for molal susceptibility oft.he solute. 'Xmolal=-(_~_w_s_o_lu_-_w_a_t_e_r_•_J_ ,v_1s_o_l_v_-_w_a_t_e_r_)_(_K_a_i_r_•K_w_a_t_e_r_)(_1_0_0_0_) ( L\l~lair-water) (C) (X) 'I'his expression is used in the thesis to calculate molal magnetic susceptibilities .. Apparatutt and Methods .££ Measurements All magnetic measurements in this reasearch have been made using the Gouy method(2), which involves measurement of the change 1.n weight, L\W, of a vertical cylinder of a substance when one end is placed in a field of strength Hand the other in zero field. A large half-ring v.rater ... cooled magnet was used, the flat surfaces of the pole pieces being 38 mm. in diameter and 22.5 mm. apart. The fields were between 7000 and 9000 oersteds, but it was not necessary to know the absolute field strength since all measurements were made relative to water at a known temper ... ature {20-25°c). Glass tubes of oonatant diameter were separated into two compartments by a glass septum. and provided \Vi th ground glass caps for the ends and suitable sup- ports for supens1on from the arm of a Becker disoohainomat1c balance. The tubes were plaoed in a thermostated, asbestos-lined compartment, with the septum of the tube between. the pole pieces. The sub- stances to be compared occupied the two compartments; solid measurements were made relative to air and solution measurements were made relative to the sol- vent (water). Solids were introduced in small portions and well tamped in the upper compartment. The solid tube had an internal diameter of 6 mm., -1 4 - whereas the solution tubes had internal diameters of approximately 18 mm. The compartments were about 15 cm. long. The tube constants measured were water versu water, to correct for the magnetic properties of the tube. and air versus water, our primary standard in susceptibility. In all LH'V measurements, four readings were taken; one at zero field, one at an ammeter setting of 9, one at an ammeter setting of 14. ru1d one again at zero. The W's were obtained by s-u.btract- ing the average of the zero readings from those at 9 and 14, to give AWg and L1 W14, respectively. L\.W14 was converted to w9 by dividing it by a factor of 1.347, and it. was then averageJ. with the first ~v9 to give the value repor·t ed in the tables. In ~agnetio titrations, reagents were added by means of a lee. glass hypodermic syringe and needle through a rubber stopper placed at the top of the solution compartment. The syringe was graduated into hundredths of a cubio oentimeter and cotud be very easily read. l[easurements of' pH values were made with a Beckman glass-electrode pH meter. As a standard, 0.05 M potassium hydrogen phthalate solution, pH 3.98, was used . -15- I f d tn thi magn t t e ch t th o if .mi Int t t of T ohnology Th - J.6 - Preparation.!!!£ Standardization 2L Hemoglob!!!, Solutions {a) Preparation of Ferrohemoglobin Solutions: Bovine blood, defibrinated by rapid stirring, was centrifuged onoe to remove the serum. and the corpuscles were washed with 0.14 M potassium chloride and centrifuged at least four times. After each period of centrifuging, the upper layer of potassium chloride solution was removed with an aspirator. The blood cells were hemolyzed with peroxide-free ether. the stromata emulsion was centrifugeJ off, and the diasol ved ether was through the solutions. removed by bubbling air The solutions were etored as oxyhemoglobin at approximately o0 c. in an electric refrigerator until used. ( b) Preparation of Ferri.hemoglobin Sol11tions: 1 N Lactic aoid was addeJ to the solutions of oxyhemoglobin to bring the pH down to about 4.6-4.9 and the systems were left to auto-oxidize over a period of forty-eight hours at room temperature. Auto-oxidation is complete after this period. The pH was then brought upto 7.0 by adding either NaOH or Na3Po4 aolu.tions with rapid stirring. 'rhe systems were centrifuged at this pH to remove ·the small -1 7 - amount of denatured protein formed during the acidification. The solutions, brought to a desired pH, were then stored as farrihemoglobin or ferrihemoglobin hydroxide at o0 c. in an electric refrigerator until used. (c) standardization of Hemoglobin Solutions: In the case of oxyhemoglobin, a known volume of solution (20.0-25.0 oc.) was reduced to ferrohemoglobin with 0.3-0.6 g of Na2Sz04 and the ~W9 was measured. The system was then saturated with CO and another A, Wg was talren, to correct for the diamagnetism of the protein and the r .e agents. The change 1n AWg corresponds to a change in molal sus- ceptibility (per heme) of 12,290 X 10• 6 c.g.s.u. at 2s 0c., the effGctive magnetic moment of ferroharnoglo• bin per heme being taken as 5 •. 43 Bohr magnetous. ~lIn the case of ferrihemoglobin a knovm volume of solution was made alkaline id th NaOH to a pH .of a'bou.t 10.5, at which point there is only methemoglobin hydroxide present, and a ~W9 was taken. The system was then reduced with 0.3-0 •.6 g. of Na2.S z04, saturated wit,h CO, and another LIW9 measured. This change in Aw9 corresponds to a change 111 molal susoepti bility (per heme) of 8350 c.g.s.u. at 25°c., for solutions {t-D. s. Taylor and c .. D. Coryell, Jour • .Arn. Chem. so. 1177 (1938) soc., - 1 8- of low ionic strength.~ It is possible then to make an accurate calculation of the oonoentration, c. in heme-iron, of the hemoglobin solutions by means of the following formula:· C = (AW9-.t1W9cOHb)(0.748 X 10- 6 )(103) (XI) (AW9 air-water>;)(, molal wheret i1W9 is the t1 Wg for the solution; .6 W9coHb is the LIWg f.,r ", th~ ~ol ution saturated with CO; L1 W9air-water i s the LI. W9 :for ai r versus water;(all .LlWg's are oorreot- ed for the L1W9 of the tube, water versus water) ~molal is the molal susoepti bil 1 ty of the species in solution, i.e., 12,290 for ferrohemoglobin and 8360 • -6 for methemoglobin hydroxide; and the factor o.748xl0 is the value accepted for the volume susceptibility of air versus wat9r at room temperature and oan be ealoulated from values obtained in the International Critical Tables, volume VI. D. Coryell, F. Stitt, and L. Pauling, Jour. Am. Chem. SOQ., .§1, 633 (1937) ffc. -19- Exp~rirnental .Procedure :Results, Cone·lul!!ions • an.d Remarks -2 0- Ferrohemoglobin Ethyl Isocyanide Warburg, Negelein, and Christian have reported a compound of ferrohemoglobin and mfthyl isocyanide• having a molar ratio of isocyanide to iron equal to one.* It was hoped by the author that a magnetic study of this type of complex might prove valuable in determining the structure of suoh compounds. Since ethyl isocyanide is more soluble in water and has a higher boiling point than ' the methyl derivative, the ~quilibrum of ferrohemoglobin with ethyl 1$ocyanide was investigated. Preliminary qualitative ex~ periments revealed the compound to be diamagnetic and also to have an absorption speotru.m with maxima at approximately 5540 j and 5250 i. 20 cc. of cow hemoglobin, the cenoentration of which was 0.0160 moles per liter in heme-iron• was placed in a differential tube and the L1W9 was measured at 20°c. Known volumes of a solution of ethyl iso- cyanide, of a known coneentration, were added suocessi vely to the system, the .6W 9 being measured after each addidion. The addition of the reagent was accomplished by the means of a ~lass syringe, gradua.t ed in hundredths of a · e#bio centimeter. The titration was repeat~d onee with a new >k o. Warburg, E. Negelein, and w. Christian, Biechem. z... &a4:.,,,- 26. (1929) :.i -21 - standard solution of ethyl isoayanide . Great care was taken in the second run to eliminate all noticeable sources of error. No apparent change in the AW9 with time was noticed. The results of the two experiments are tabulated in tables I and II. The li Wg's• corrected for the L'.'l.W9 of the tube itself and for the volume changes, are plotted as a function of the volume of the isooyan1 de solution added. The intersection of the two straight lines in each graphical plot is taken as the endpoint of the reaction. Calculation .Q.f. the m,olJ!.f ratio 2.! iaooyanide !.2, heme-iron : (a) Titration No. l : ~ End- point-• o . 560 cc . of isocyanide solution (O.Ol60)~20.0) -,.....: moles , of heme-iron • (1000 • ,,(_O . 670H O. 5601 {:J.000) - mole·s of isoo. yanide ~olar ratio = moles of isocyan,ide - 1:.17 Ans. moles of heme-iron - • • ( b) Titration No 2:~ End.point•• 2. 18 co. of isocyanide solution (O.OlGO) (1000 ~zo,oo~ . • -to,147)C2,1a) • (1000) = mo· 1 es of heme-. iron - ;'2 rrtoles of 1soe¥~1. .:..e. molar ratio = moles of heme .. j.r•on - 1 oo Ans moles of lsooyanide- • -• -22- Conclusions . and Remarks: . . . . . - . ' Speetre$CQpie and magnetic evidence ot the torma·• tion of a compound ot terrohtmogle>l;}in with ethyl 1se• ~yanide has bei,n obtained.. Magnetic data have been pre.... sented which demont;JtFf.lt.e that within the experim.e ntal error the molar ratio of isooyanld.~ te hemt•iron is one~ It ha$ also been shown .that the eompl$X is a very stable one, exhtb:1t1ng very little fliEJ$0ciation under th$ oonditions of the experiment. If the complex were a.ppr•• c!ably diseoeiated, there should exiet a noticeable cur- v.atur«- near the end-point of the graph. No such curva... turt is apJaient. It is cont;:luded that terrohemoglobin ethyl tao• oyanide 1$ diamagnetic, is V'Jry $table with respect to 4i$soo1•t1on into terrohemoglob1n and ethyl isoeyanide, and has essentially oovalent. d2sp3 ootahedral coordina... tion abo'l,l.t the iron. It• coordination bond.type is tbus similar to that of oxyhemoglobin, carbonmon~yhemoglob1n, and eyanid& terrohemoglobin. The addition of n-l>butyl iaoeyanide t.o tei-rohemoglob1n solutions lC>wered th• susoept.1bi11 ty. However. stnoe ciena.turatt.on wa.$ obs.-rved, no attempt was macte to interpret the da.t a obtained. The signifi ... cane~ of the data. was futtheur obscured by the high in• $Ol ubil 1ty ot n... butyl isocyanide in aqueous fer:rohem.oglo .. bin solution1:J. -23 - TABLE I •- FERROHEMOGLOBIN ... ETHYL ISOCYANIDE TITRATION NO. .l Tot<l.l Volul'\'\<1 of tsocy<H'ltc(e lo"t<tl Votu.me Sofn. Added ~-stern of 6.W9 L1W9 6W9 CO(", for Tube for Tuloe L\ \}J 9 ~Ot'"- torTuloe UV\c\ \fol~v>ie ~no.vi.«je mg. 1. o.oo oo. 20.0000. 10.34 mg. -0.24 mg. 10.58 mg. 10.ea 9.05 20.05 9. 131 2. o.os -0.24 9.29 20.15 7.54 7.78 7.83 3. 0.15 -0.24 20.25 5;54 5.23 5.47 -0.24 4. 0.25 3.:;2 · 20.35 -0.24 3.02 s .26 5. o•.35 le40 20.45 1.13 -0.24 1.37 6. o.45 ,..Q.52 -0.53 7. o.55 20.55 -0.76 -0.24 -0.76 a. 0.64 20.64 -0.98 -0.74 -0.24 9. • o.75 .0.24 -o.,a ...o.94 20.75 -1.1a 20.85 10.0.86 ·-0.24 -o.aa -0.92 -1.12 o!i'0.86 20.95 -1.06 -0.24 ·- 0.82 11.0.95 .0.91 12.1.00 21.00 -0.24 -0.95 -1.15 ., Original eone. hemoglobin sQln.: o.0160 moles Fe/ lit. Original cone. 1sooe3atiide soln. ':. o.670 moles / 11 t. Temperature -- 20.0 C• ]'ABLE !I - FERROHEPJIOGLOBIN • ETHYL ISOOYANIDl!j TITRATION NO. 2 Total Volu,me of f,s.o~a:-0,,'cle 5c>ln , dd.ed Tota.I \/o lu.~e of SJsTum L1W9 ~W 9 for ~W9 (!Ot'", Tu.Pe -forTu.loe A vJ9 coc -fov- Tu \oe, o.nc.\ Vo\u..me. chan~e 1. o.oo co. 20.00 cc. l0.92 mg. -.0.24 mg. 11.16 mg. 11.16 mg. a;.42 7.98 e.22 to.so 2. 0.50 -0.24 21.00 ..0.24 s.21 6.47 4.97 3. 1.00 1.99 2.40 4. 1.50 21 ~;0 2.23 -0.24 s. 2.00 22~00 -.0.24 -0.23 -o.45 -0.21 6. 2.50 ...1.1a -1.06 22.50 -0.24 -0.94 -1.oa 7. a.co 23.00 -0.94 -0.24 •l.18 e. 3.50 ...1.22 23.50 -0.98 -0~24 -1.15 ...1.13 -1.18 9. 4.00 24~00 -0.24 -0.94 -1.33 24.50 -1.09 10.4.50 -l.33 -0.24 Original cone. hemoglobin soln. : (;'>.• 01 60 moles Fe / 11 t. Original cone. isoeianide soln.:::: 6.147 moles/ lit. Temperature -- 20.0 .c. -24J; i I •; + .. +· .; " + ~ + -+ :t::: +- ➔. i -~ H . rt ~ ,m -- -1--+ t+ .W.W... 1 J£~1.t trl: ~ ++ ·a I+ -++ '. f-\tt .... >-+' , :.... +1 t-+ '--l :EE t-', Ii + fj <-++-+~ 4 -"-, .. • ... Et+ J: -~ tt H- !-+++ t-l-+ +++ t:t ~ -1- I ~ l:t-t -1- ++ +-+ E +· + + ~ JJ f j± ii ~ ! , .1 --, .,- + ;.t.j l.U 1--'- ±J ,;r. LT 7 -~ -H LU -l IT' T --j + + :r. -+ +- -H h- -ill.1 ,+ H j_ -+ µ... +- ~ l l.1 ' H ... .J --t t+ en :1--. ·-t--1- + + • • T l--+ ±t la' r 1= <-Ll +-' rt - H-j ~ +-' ' + H- · H, t.1,J ~)I t+tij H •· .. l,c.. ± T. :-i ::p: Hi rrm· tt j 1--tw..;· ,_, T H + + .t.;...i +-+ l-1- ...... tt :t;..:I l-1- t+1 p: ·t + EitiElllffillittEUEt111 •• -4 + LL:J LLLLL L1 ..L f-J:l--1: .. I lt.~ : fk m +- ·- . _j.. ~ -1- ± ~;t: -~± ;:t:·wf _·..;:_:_~:I -+ l±..tt->++H--1--++-+ ..L...! .. ,t= I-+-!- + ~. ......... ~t It H F '-'-'-'- .LL.. L ....... 1..4- t:t1 •.J:tl:1 ... :i::i !=f -25' : .t -+ + -L, ITT l -i-+ ! ~- .,i. ~l t W- t ! :_ _, . -'- :t .. t, ; I ; _j: t i+ ' ~· + t : ± ±-1::+ 1t- !+ +- ,,:,_ + it L:: -,,_,_ + -· ~ + ,+ I + :q: -~ tt _, + + t· + + f + ~ 1f t .. r - Tr -r- t --++ ++- t- -t-,+ t - t 1-, tt 1-+ -j-r rttt _j: 'ti 1ttt :t :t: ffi -:-:-- ~- + ~rtt +l-1--'el-+-1+.,:j__!>-l-l-l+ .j._ -+,- t H- .. TT 8= Et+ ! + If ffi sl ~- f .;:: ► 44-- 1-1- H- H- itt rr Ht+ .. 1-1- L -,+ -t , ' ,- ·+-;· .± :tt It H~ +- +t- +-+--t n };t - >-e-+ i: +- ;:+-.-1= ::;:. ·!• ff :t i + t-t -\ t •: -, tH IT i - t -+-+ ft -t I++ m >+ • rt+1 1 :i--: '. rt- t- - _J.b:: -- _7 + t ITT i:I tti -t '-+ 1-1- a·~ c+-- t rt + '+ I± I tt +- _, t j f m tt ++11-. +-H-1-t-H-+H+-++H-++-tt-+'rt-H-tttt+tt ~ .+11· i +- -'- ,+ . +-'+ - -+ t ftn+ ._. -+I l t Li TT HI ':-t: ~ _>-j- -r t'-- i::il=1-: t '-+± .... I- 1-· ; + u -,, ,-+ -\. -+-• -+-+ 1-;:;-ti -,+ I-++,- Cl -t-t ~ fl ,, ++ =+t ft, +.' +-+ +!+ t::: t tt j:: -+ ti__;_~ .. ,--t!- =!r.., H- + 1-t-1-r- H ; 1.1-. + - i- J.l+ _J. F t ~ t,--+ ,_ I++ I--·++ -+-1----1,..f- ~- I+ t-t T rt- r++-;: +~ if:tl t ~ -t~ ~+; TT_++-'-+-+H I- ·•-t IT H-1 -::• .:i mm T -,- ::t, -+ \ + il +J± .,- t t i:;, I + . -!-f -t++- ttt- ,+ 1=-,t t -I ++ ± + +t- fi= r-r ·Fff E ...u f+ -1-1-+ -· H- -1-1-1-+ +t + + · ' -26- Ferrihemoglobin Imidazole A qualitative experiment indicated a decrease 1n the susceptibility of a ferrihemoglobin solution upon the addition of imidazole. Therefore it seemed desir- able that a series of magnetic titrations should be made at different pH values, namely, 6.86, 8.20, and l~.30, in order to study the equilibrium of this sys.. tern ·~ ' . In all titrations, 20 oc. of bovine methemoglobin, at a known pH and a known concentration, was placed in a differential magnetic tube and a AW 9 measured. Known portions of imidazole solution, made up by weight, were then successively added, the AW 9 being :m easured after each addition. In the run at pH 6.86 the im!dazole solution was brought to this pH before adding. In all othertitrations the irnidazole solutions at their orig• ' • inal pH values (about 9) were added directly to the ferrillemoglobin systems and the pH of the hemoglobin solutions was ,measured be.fore and after each titration. The ferri- hemoglob:i.n solutions at pH 6.86 and 10.30 contained phosphate buffers• those at pH .a.20 contained borate. Following each experiment the system was reduced with then saturated with CO, and a .4w 9 measured to oorrect for the diamagnetism of all reagents. Na.2Sz04 • After several trial experiments five reasonably -2 7 - satisfactory runs were made. pH of 6.86. on ,1w The most, precise was at a The effect of the addition of imidazole up- 9 was practically instantaneous. with time was noticed. No c.hange in AW g The experimental results are tabulated in tables III~V, and the best curves for the three different pH values are plotted in gr aphs 3•5, in which the fraction of Hbim to the total Hb present is plotted as a function of the 11 free 11 imida.zole concentra .. tion (Im). Let us assume the following reaction: Hbim + ~-Rb+ + Im The dissociation constant is then (Hb+)(Im2 (Hbim +) ::::. K This constant has been evaluated at tlle three different pH values taking into account any largeJpH during the reaction. (Hb +) was calculated from the total (Hb+ +HbOH) in solution using the pK ,8.15 reported by Coryellt Stitt, and Pauling% for the fen·ihemoglobin: ferrihemoglobin•hy.. droxide equilibrium. The imidazole was corrected at the pH in question using the acid pK of 1midazole. reported by Kirby and .Neuberger** to be 6.95. The method used to calculate the di::::isociation con- stant of Hbim is moderately simple. If AW g is plot·ted c. D. Coryell, F. St1ttt and L. Pa-qling - Sour. Arn. Chem. Soc., 59, 633, (1937) A.H. M. Kirby and A. Neuberger - B.C.J • .§_g, 1146(1938) -2 8 - against the volume of imidazole solution added, it is found that the curve approaches a limiting value of AW9 at high c<>ncentrations of imidazole. This asymptotic value is taken as the AW9 for the Hbl~ complex. Hb + is a function also of pH. W9 for Thus• if the pH ohangea du.ring the course of the reaction by an amount LfpH, a correction must aooordingly be applied to the AW9 of the solution. LE:lt x be the amount ot ferrihemoglobin present, (1-x) the amount of ferrihemoglobin imidazole. Liw1 the _4 w9 for ferrihemoglobin at the original pH, AWi the AWg for ferrihemoglobin after pH change with the reaetion. A w11 the .4Wg for ferrihemoglo:b in imidazole ( the limiting JW 9 ). 'l'hen sinceLH!f 98 b changes with a change in pH, we have and LlW9Hb =- Ll~r': for x :::: 0 ' This gives LlW9Hb= LJWr. +(LlW:r- ~ t )x Also we have tiW 9Hbim =-bWII for (l•x) =l (11) Then, combining: ( ~•i i and (II), we obtain LlWg = /jWl x +(AW 1 - LJw 1• )x +LYNI:i"(l-x) This relationship can then be plotted and values read -29- directly from the graph. We also have _ or (Hb-+-) = X l t 10 8.15 l 08, 15-pH J 108.15-pH-t-1) If we assume that pH varies linearly with the reaction, we obtain the relation Then (III) and (IV) oan be substituted into the equilibrium expression along with the determined val ue s of x to give the following: x K= 1-i f108.15-pH- ,1pH(l ...x) j · (Im)f108.l5-pH- LlpH(l ... x)+1) The concentration of "free" imidazole, (Im), is calculated from the experimental data in a straightforward manner. Great difficulty was experienced in evaluating the limiting value for AWg beeause of the . wide distribution of points at high concentrationsof imidazole, which may have been due to irreversible conditions in this region. The dissociation constants may be in error due to the fact that the asymptotes were chosen so as . -30- to make the least possible variation in the ealculated constant. conelu8ions -- There is fair agreement• within experi-. mental ~rror, between the two constants at pH 6.86 and 8.20 but these values are about eight times as great as the constants determined at pH 10.3. The experimental data also fit the theoret1Qal curves better at the lower pH values. Pe_rhaps at. pH l0.3 denatw·ation had oocured to an appreciable extent making it diftioult to control experimental conditions. This would give rise to errors in the .4.w 9 measurements. If the molal susceptibility for Hblm is oalculated at ...three different pH values using the chosen asymptotes, it is noticed that there 1s a large decrease from 2,940 c.g.s.u. at pH 6.86 to 1,290 o.g.s.u. at pH 10.3. This perhaps may be due to the tact that acid groups on the globin coordinated with the iron atoms of the hemes ionize i:appreciably at the higher pH values. This eon• olusion is highly speculative due to the lack of further experimental data. Experimentally it is noticed that the formation of compounds of ferrihemoglobin hydroxide with F-. oN·*• and imidazole there 1s an increase in pH of the system, indicating a liberatiQh of ~H•• whereas in the addition of NH 3 and CzH50H* to methemoglobin hydroxide no D. Coryell, F. Stitt, and L. Pauling• Jour. Am. Chern. Soc., 59, 633 (1937) ~ C. D. Coryell• Unpublished experiments. • C,. -31- increase in pH is obserVJed. Dr. Linus Pauling has offer- ed an explanation of this phenomenon as follows. He as ... sumes that there :t:,;it~ groups on the globin coordinated with the iron atoms of the hemes which are acid groups whose energies of dissociation are sensitive to changes in the state of af:ia;, ·~ t·,:·,r,s about the coordination center. . ' .... ·': '. .. • • ,- In the addition of donor molecules to Hb • these molecules ocouPY the same positions on the iron atoms as do the OH"" ions in HbOH. Replacement of hydroxyl by other anions such as F"" • CN .... xm-. has very 11 ttle effect on the strength of the acid groups on the globin and no increase in ionization occurs. Therefore any addition of one of the above anions would cause only the liberation of OH-, thus increasing the pH of the system. When anions coordinated with the iron atoms of the hemes are replaced by neµtral molecules an increase in the acid strength of the acid groups occurs, because of the loss of the attraction of the negative charges of the anions for the protons. Increased ionization of the globin acid groups results, Therefore when neutral molecules substitute for the oa· ions in ferrihemoglobin hydroxide the hydroxyl ions liberated are neutralized by the hydrogen ions from the globin acid groups to form water. This would account for constancy of pH in the experiments with the neutral molecules. In the case of imidazole there are two resonating -32- structure which oontribute to the valence state of the molecule, namely, H H ~6, N?" 'N-H l l - /c~ + N N-H I_I H-c====c-H H-C - - C - H (I) (II) The contribution of {II) may be large enough to give the left nitrogen atom a charge of minus one-half. Since this nitrogen atom is presumably bonded to the iron atom, this would make the imidazole substitution for the hydroxyl ion in ferrihemoglobin hydroxide a oase intermediate between the substitution of anions with a charge of minus one and neutral molecules of zero charge. If the acid group on the globin were very little ionized in acid solution anti appreciably ionized in strongly alkaline solution, the apparent dissociation constant of Hb!m would be greater at a pH of 6.86 than at a pH of 10.3. From the data relating the experimen• tal, apparent dissociation oonstant as 4 ;t.'µJ;lP,~.l:!!>~: of .pH, thedissooiation constant of the globin acid group is calculated to be about 10•9.5. In the absence of further experimental data we may assume the pK of the globin r'i. !.Oid group to be about 9, 5. Table III . - ... Methemoglobin,,,.Imidazole System ... P!!_ a. 86 Volum.e !m Soln.,Added Exp. Cor. L::,.119 Uncorr. .6W9 (x.) ~tl!?Hb) moles liter 1. o.oo ee. 1.30 mg. 8.,50 mg. 0.000 0.00000 2. 0.40 4.SO 6.,11 0.00159 3. 5. 0.60 0.;80 1.00 6. 1.20 3.20 2.95 2.21 5.16 4.61 4.41 3.74 o.S5'1 o.499 0.,580 o.610 1. 1.,40 l.60 1.80 2.00 4. a. 9. 10. 11. 12., 13:. 14. 15. 16. 17. 18. 19. 3.ao 1.,93 1.,77 1.,72 0,.01010 2.,4 2. '74 0,.860 2.64 2.2 2.4 2.,42 0.,81'5 o.9o'7 2.39 o.911 0.11440 0,.01750 0.,02320 0.02860 1.09 4.00 0.,92 0 .. 58 o.• 42 o.41 a.oo 0.15 0.13 9.00 10.00 0,.116 0.,778 2.,54 2.,66 2.,46 o.ss 7.,00 o.oose6 - - - -·- ·-,.., ... ---------- ,.........,,._.,, ...,___.., ,,,_ ._.._..._... 2.01 2.74 0.11 .... .,_.,.__.,~........,.. --_______ ------- = 2.5 ! o.z x 10.e Cone.. Hb Soln. = 0.,.00974 moles pe:r lit .. in heme-iron Mean :K Cono. Im Soln. ::::: 0,. 355 .b. w.. oor. asymptote 9 Temperature 2.,4 0.01120 2.50 6.00 2.1 2.4 2.5 3.1 o.a1s 1.44 5.oo ..-... 2.2 2,.4 2.1 3.42 3.30 3.29 3.03 s.oo o.no o. 758 0.00253 0.00369 0.00503 0.00011 0.00732 K x 103 = 25°c. moles per lit. :; 1. 80 mg. -Y., molal 2.s 2.,7 ---·-----....., ._ --- -34Table IY•A ... - Methemoglobin I midazole System"" pH lm Exp. Soln. Added AW9 Oor, o.oo oo. 8.83 mg Volume 1. 2. 3. 4. 5. 6. 7~ 0.30 0.50 0.77 1.00 1.25 1.50 8~ l.75 9. 10~ 2.00 2.25 2~50 2~75 ll~ 12. 13. 14. 15. s.oo 4~00 s.oo Mean 7.66 mg 5.41 4.30 3.12 2~ 61 2~03 1 .. 79 •· l.50 HbJm ) ..6.W9 ( Total Hb 6.61 5.59 4.44 3~95 1~14 3.57 3.15 ~.88 2~53 2.86 1~13 2~54 1.20 0.72 0.78 o.69 2.64 1.1, 2~12 2.26 2.24 (Im) moles llter 0.0000 0.00000 0.287 0.00249 0.433 0.00466 0.592 0.00784 0.662 0.0113 0.745 0.0141 0.776 0.0187 0.813 0.0224 0.863 0.0261 .0.817 0 ..0305 0~862 0.0340 ,0 .848 o..0378 o.925 0~0411 o. 9,0 4 0~0554 0.906 0.0685 8.2 K X 103 2.9 2.7 2.3 2f. 4 2.0 2.2 2.0 1~6 2.7 2.1 2.6 l.3 2~3 2.7 K = 2.3±:0.,3 x 10-3 Cone. Hb soln. c=: 0.0118 moles per 11 t. in heme-il"on Cone. Im soln. = 0.408 moles per 11 ter ..6.W9 cor. asymptote =l.62 mg. ~molal=- 2,180 X 10• 6 ,.o.g.$.U • .Temperature = 2s 0 o , L1 pH= o. 23 · (assume linear with reaction) Table IV-B - • MethemoglQbin Imidazole SYst$m .. pH 8.2 1. ,a. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12 13. 14. 15. Volume Im Soln. Added Exp. Cor. \V9 W9 o.oo cc. 7.43 mg 5.73 4.16 3.36 2.86 0.20 0.40 o.eo 0.80 1.00 1.20 1.40 1.60 1~80 2.00 2.50 3~00 4.00 5.00 Hblm Total Hb 8.60 mg 8:. 96 2~32 5.42 4.64 4.16 3.63 1.93 1.69 1.41 1.43 1.38 1.10 0.93 0.77 0.46 3.01 ?~72 2.77 2.73 2~46 2~31 2~17 1.86 3."24 Im moles lit!t 0.000 0.00000 0.248 0.00111 0.457 0.00252 0.566 0.00505 0.602 . 0.00801 0.707 0.01070 0.763 0~01370 o.796 0~01680 0.838 0.01970 o.~30 0.02310 o.836 0~02640 o.~74 0.03380 0.895 0.04150 , 0.916 0.05500 0.963 0.06780 K X 103 l.6 1.3 l.6 1.9 1.8 1.7 1.7 1~5 1.9 2,.0 1.9 1.9 1.9 1.0 ,. Cone. Hb solr'l. = o. 0118 moles per. liter in heme-iron :: Con~. Im soln~ ::::0,408 ,moles per: liter .6..Wg : cor:. asyrnptote-=l.62 mg ~m~lal-=2;180 x 10,; Tempera;ture : 26°c ApN = o.23 (assume linear wit.h reaction), 6 c.g.s.u. -36- 1. a. 3. 4. , 5 •.. 6~ .,, •· 6. •• :io. 11. Volume .Im E~p. SQln. Added L}.Wg o.oo cc. 1.00 2.00 $.00 4~00 s.oo ,.oo ,.oo 8~00 t.oo 10.00 Mean 5~39 mg 4~00 2.91 2~23 1~53 1;06 Q.,37 ~ .. 94 014, 0.32 0.53 6,53 mg 5~43 0:.000 . 0.199 0.364 0.483 4.41 3.86 ~.32 ().5$1 o.0691 •0; \36 ·, 0:51 ', 0.21 o.va1 '. <f,.39, ·(h749 Q-. 1.22 ().133 0.6?4 0 •.l.44 ·o.4i 0~676 0.637 ,, 0.,0535 0,48 0~36 @.0975 o~.111 0,656 2~79 s.0.1 e•.so ,, 0~·0836 a;90 2~49 2~39 0 .• 0000°· 0.01~1 0.;0i66 0.29 ·o.2s ~o.aa 'K = O. 34 :t 0,.06 x· l(f"' 3 Cone. Hb soln.::: o._0 123· moles pe·r lit.er 1n heme..,tron Cone. ·tm soln. =o.450 • mole$ per 11 te.r ~W9 ~or. asymptote =1.:00 mg. )lmolal = l,29~ x 10~ Temper:atute = 2400 6 ¢.g.s.~. -3 7 - Table V-B - - M2.;.~llemoglobin Imide.zole System - pH Volume Im Exp. Soln. Added AW9 Cor. L\W9 Hblm ( Total Hb) -l. 2. 3e 4. 5 •. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15. 16. o.oo cc. 5.36 mg 6.50 mg 0.50 1.00 1.50 2.00 2.50 3~00 3.50 4.00 4~50 5.oo 6.00 7.00 8~00 9.00 10.00 Mean 4.32 3.57 3.00 2.78 2.32 1.90 1~40 1.23 0.95 0~85 0.84 0,.43 0 . 48 0.43 0.45 5. 61 4.96 4.48 4.42 4.04 3.55 3.05 2.92 2.65 2.58 2.69 2.26 2~42 2.46 2.58 0.000 0.162 0.280 0.367, 0.378 0.448 0 .5o6C 0.62'( o. 651, .o. 700 o.11~ o.695 0~ 771. 0.742. 0~735 0.713 · ;· (Im) moles 1.9~~ K X 103 liter 0.00000 0.00902 0.0181 0.0272 0.0367 0.0453 0.05~0 0.06Q4 0.0683 0.0755 0,.0828 0.0974 0.110 0~122 0.133 0.144 0.29 0.29 0.29 0.37 0.35 0.28 0.22 0.23 0.20 0.21 0.27 0.20 0.26. 0.30 0.36 K -= 0. 27 -t_ 0. 04 x l 0 -3 Cone. Hb soln.= 0.0123 moles per lit e r in heme-iron Cone. Im soln. =0.450 moles per liter 6 I\/_ L1. w9 eor asymptote=- l.OO mg I'--'.: . ~ 1,290 x 10~ c. g .s.u. mo1 a1 . Temperature= 24°c L'.lpH : 0.10 (change ne glected) - 38- ..,. ~ gj ci .c z ~ 0 ►~ • .c ,. .;, z~ ,; ci .c 7. u ~ ,.,a:., -:S "< "' ~B ~ 4/:l ;j X l::l'l ,.,., ::, 0 -3 9- Sulfur Nitride ~ The following possible structure for ! !4 S4 might be suggested. ,This structure would necessitate a rather large mag ... netio moment due to the paramagnetic contribution of four unpaired electron spins. one electron on each of the sulfur atoms. This compound was measured magnetic- ally both in crystalline state and in a solution of carbon disulfide and was found to be diaznagnetio in each case. On the basis of this _ev;1-de.nae, the above structure and any other structl.U"e involving unpad.red electrons can be ruled o~t. A. J. stosJok has suggested that the structure of N4s4 might be . kiat of an eight-membered. ring.~ be represented as follows: It can This structure would be diamagnetic. very etable, and a cup-wise configuration. such as f'N=N/Y 5 '-N=N/ s would allow the nitrogen bond angles to assume the ~A. J. stosiok • Doctorate PrGpositions. California Institute of Technology (1939) - 40- usual value. Conclusion: The magnetic data show the compound, N4S4, to be diamagnetic; Thia supports the structure proposed by Stosick, and in the absence of X-ray and ElectronDiffraction data, it seems to be the best possible structure to write for the present. - 41- Experiment.al. Notes on Brief Investigat1ong -42- Note op. ~ Stab.it~ ~J:.: Qf oxihemo~lobin Solut~one- (a) 30 cc. of oxyhemoglobin solution (oono. =0.0160 moles per liter in heme ... iron), prepared by c. D. Russell and c., D. Coryell on November 18; 1938, and l;;:ept at 0-4oc • until Deeember 21, 1938, and th~n .kapt at room temper- ature from ll:OO A. IvI. on, was placed in a stoppered glass magnetic tube and the t.w9 was measured as a fune- tio11 of the time. After a period of 1336 hou.rs a $pee• ·troseopic measurement was made indicating the preaenee of methemoglobin. The results are tabulated. in table I and the AW9 •it are plotted against the time in graph no. 1. (b) 25 co. of oxyhemoglobin solution• same as mentioned above, but kept at 0-4°c, until February 16; 1939; and then kept at room temperature -from 9:00 A. M. on, was placed in another stoppered glass magnetic tube and the ~w9 was measured as a funct!on of the time. The change in .4W9 was not followed as long a time in this run but after a period of about 400 hours, speotrosoopio observations showed the presenoe of ferrohemog;Lobin. 'the re• sul 't.$ are tabulated !n table II and the L::t w9 •s are plotted against the time in graph no.2,. -43- The ehang e in b.W9 is . almost li11ear up to approx ...· imately 276 hours in experiment (a) and tmti1 215 hours :i.n (b ). Both axper:l.ments then show a very rapid. in... crease i n A Wg with the time , run: ( t>) exh:i. biting t,.he aharperat increase. After the aharp rise in A W9 , the points 1n (a) continue to increase linearly• with about the same rate as before, r-eaohing a limiting value• ti w9 = ll. 55 mg., after 1336 hours; while in ( b) the points then follow very closely a borixontal s~ra.ight lint$ as long as the reaction :ls followed, .6. W9 = 11 . 67 ..ll.31 mg. It ia rather difficult to interpret these result$ . However. if we aa$ume that the react.ion ia that of autox,4- idation of HbOg to Hb • as in 'the following reaction: 4Hb02 + 4H ~ 4Hb + + 302 + HgO ·the data observed might indicate an auto-catalysis ot· thi s reaction. Perhaps the red·ueing a.Qtion of bacteria. or other living org~isms; in using up the oxygen, cau.ses reduction of Hb0 2 to Hb;·- which in turn oxidizes slowly + . to Hb in the presence of the oxygen left over . These experimental data furnishe-E! very interesting facts concerning the stability of ~xyhemoglobin solu..- tions and a study of the kinetics of this reaction at different pH's• temperatur~, and other conditions might prove valua ble to hemoglobin ohemistry. - 44- + fabl e I - - ( Hb02 ~ Flb ) Sy$~ No . Of r eading Time i n Hours Exp. A W9 in mg . A W9 for th~ tube in mg . Exp, .1 W9 oorreeted for tube in mg. ~~ 1. 2~ 3~ 4. 6. 6. 1$,.0 o. o 1 . 61 t . 84 43 . 5 2. 36 68 . 5 11$. 5 137. 0 7. a. 162. 0 187 . 5 9. 10. 209 . 5 232. 0 11 . 12 260.0 308. 0 327 . 5 14. 350. 0 15. 16. 399~0 l3 . 17. 18. 19. 20 . 21 . 376.0 449 . 0 475.5 519.5 593 . 0 1336. 5 2 ~'12 &. 99 i . 48 3. 71 4.08 4 ~11 4.62 4.75 5. 96 a.oo 9. 87 10.48 1o~se 11 . 16 11~28 ll.71 12.38 13.25 1. 70 1.10 1.70 1.70 1•.70 l.70 1 ~70 1 . 70 1~·70 1~70 l . 70 •0. 09 0. 14 0 ~s, l . 0$ 1. 29 l . 78 2 ~01 2. 38 2 . 4). 2. e:a 3 . 05 l . 70 l.70 l . 70 l . 70 1~70 4. 2& l . 70 l . 70 9 . 46 9.58 6 . 30 8.17 8~7.8 8~96 1 . 70 10. 01 1. 70 10. 68 1,70 ll . 55 - 45 - Table No. of reading II - - (Hb0 2 - H'b .) System Time in Exp. t1 W9 hO)tt'S in mg. t1 W9 for the tube Exp.Ll W9 in mg. eorre~rted fo-r tube -0.48 -0.48 ... 0.48 -0.12 0.20 -0~48 0.75 0~92 1.16 1~48 l. o,o 2. 3. 4. 5. 7. 24.0 48.0 72.0 96.0 120~0 144.0 8~ 168~0 9. 10. 12. 2~62 11~19 ... Q.48 ll. 192.0 216~0 240.0 264,.0 11.06 13. 288.0 312.0 330.0 ll.31 11.44 6. 14 . 15. -0.60 -0.28 0~03 0.27 0~44 0~68 l~OO l.03 1.42 -0.48 -0.48 -0~48 o.5l .. 0~48 l.6l -0.48 ...Q.48 1.90 3.10 11.67 11.09 .. Q.48 -0~48 11.54 11.57 10.83 10.96 ...Q.48 ... 0.48 -46- k. r:H ·-t: , j rt· : ,- j_ n -~ td t I ,. +-'+ .... -+ · + I+·+ ~: t i lb I -•I - ft .,.: ; +· t!t t-• 1,-- i I 1:-l• .. -• I f ·- -- "i ;: ·<-- t 't L .:. +H +W ,,_ H-1 ,: r!: ti ·i l ~.t-1 ++-t ct- ":4 .j.. • . ·ttt r+> +; +-~ )+ !"11; --II+++ -H· j.j..j..., 1 + ·t-1-t t+ , tt+ c,- + ... -~ tl .... f·+ +rt ,.. ti ....... H- +r ' ,t .. ,+- ., •+ :t: .. . ~ ' t-,+ +· ,,. H-+- J:t r:;l i=·:: I+ +r+t+ ::+ -- : I ; ·+ .. t: -t ·- 1·.• •t r_+ j ~1+ . ··; F l- f~- .. ,. +:4-i ·r··r I .:1, t±t'+ + H-< +t->; 1:±. ' ,... j ti . + -t-t-t- ill i :t H++J..i..,_.,_,·t~+- j::; .. 1-1 .I. :t ·~ -[:f H -~ tr l :t -L.j' H- t- H 1--h- r -i -- :t ~ I ~ IF H I .,. '.+ +-++ t:: .j .. + .u.+ .,-j. + _j. +t +. '+.. .µt :b. J.j.. + .L..W... + .. tt i H U1-- .J.. ~ I--- .. -++++ -+++ +- H "i-+ -++ t- m. + , ++, µ.. •·; " ·· ++ .... t :t). tt ~ I-+ ...;- H- .. , . : IT .I +t h- l=l: ~- ·J ', "...U..W-++..W..ll-l-+..1 T I\ i t ++. .a ~ .j.... H+l c 1-+· P."'" t·i.. ~-t-, tttT TT I-+-• 1tr ! f+W ... ~ .i:~ ~ Ht+ -+- + +- + • +t- :t:+ h" +" ., n:·i=J.. a ' 1~~ t:t J: + E tti't't·..,t1r-t1f j:H++H~-<-< tt ~ .~ B\ i: ~ .., H ·t-H H+-1-+!......,-H,t rn '.I. + J: [ t-t -H- .,~l-jf-4+µ..+µ..+f->t-~~+~+++--➔-~ ' tJj '.: H-+.,._ Jj.. .., t:, ·+ •+- l-+- 1 tttt_.t-+..w.. ++ 1-lli .... -47t I i -I+ t:: :::+ s+ ! f4 +j- -t- r H-- + + lt I+ ± ·:.1.t + ... + .... ; . 1t ~ - ;-1- ±t '" t":I h- ' m ~ t± ++ . t+ H~I .... .L +- f-L --t- -1- 1+ .. ,•-- ...L 1 tt -I +++ .. H--<'-J ,,.:-J itr.:::. + ·+ + j. +- =l ~ .. -tt! ' ·t- H·+ J --1+ c +I+ '., Jh ft :l rt +-++........+w.+w.H- H--t ':::L >-: i+ ·+ + ,-, It • If -l- ' ..·r H-t ... P+ .:- ~+ ·•·I:. >++'i r~ i L ! rir f-t" ,. IT · t .j- 1.1. .,.. I++ · h- ,.w. J + . .. LI. , ,;.;..:.ti ·:f -1- ' l w µ -r u, ,(:~ .. -++1 1 .-+- ., ++H !I..J h· w + J-1- l.J.. h- ·; -t- ·-+ ft t-:~ + + 1 ; : .-1- +'..J. '.:,.. ., .j. f+t fflW--1--1.-1-UU J ltt Ii •.,..,.. l=t ,. t +- tft l:tt .... ti:+ tr' rt ± lf_ t E tF i :i:; l:t L + t +;. ... l- ;+ L t ·➔ _, + -1 + t--+---r 1-t . -J ++ ·+ -1 ·"- t,-W ;T r it 1t+'H-- ± + + ..).. =1 ':E+ffil+mmmsrtmm1+1t-•~ .tt I,-!.',+__ + -~ 1 -U + + :++fL. 1--i'. :r: •t-t-i I+! t+t - ' L 'L ~ ) , p:i:..l.± ,.-,... tJe+±li::tJjf-1-jl--J:ti .lj_ It L.....-, H'+I--H-,i-+-+--f-++:µh "T . 1.'"'..+.._.+w.-1-HI..J+H++-++-+++-I-L++t+,j-_J~' ..Wt±b ' ..I... . ii· f-+-- m t --l :t. +-t-·· ! • + ..l..l. ,L H - I+ J± t1 M Ti -1 11 . + :+ i:.,+t l± e--1--1 + u l::i: .. + ·'-- •➔ 7 7 ill..J lt-t • ;:-: 41--1- L ~ • + :j l+.u... . .l Ull.J ....... t:J:+ H, rL t !:I L j- - , i r h +-H ffi1 1--rl It!+~ ... .., . 11: .,.. tr .... , UIJ , 14 'u ~ .!. ""h .,- .t-- -48- Note _S?!!. the Metbemoglobin H;ydroxide-Me~yl Amine Hz_drochloride System Upon the addition of methyl amine hydrocnloride tQ methemoglobin hydroxide at pH = 10.15, only a very slight ohangs is noticed in the absorption spectrum of the system . 'f'nere is , however t a very marked deorease in the susoept1b111ty of the solution. Th~ experimental values are reported in table III in terms of LlWg , uncorrected for the diamagnetism of the reagent added but corrected for the tube. After the add.i tion of nearly 3 g . of CHgNH 2HCt; the pH had fallen to a value Qf 9.oo. Since the weights of CHiNHeHCl added were observed only roughly, and since there was such a large change in pH (•l.15 units ) during the course of the experiment. no attempt was made to interpret the result$ obtained . There is, however, rqµgh qualitative agreement with the unpublished experiments of C. D. Coryell or1 the HbOH ... mt5 systsm . His reaul ts show also a decrease 1n the auscept1'Qility of the solutions with increasing eonoen.tration of liJH 4OH , but onl y a ve~y slight change in. the absorbtion spectrum. ( The addition of n-butyl amine to HbOH; at pH 10.2, caused denaturation and no further data were obtained.) - 119 - Table III -- 1, 2. 3. - ---·------- HbOH • CH 3N"clzHC1 System HbOH soln. at pH 10.15 (25 cc.) Addn. of o.a20 g. OH~2HCl 9f 4. }1 5. ft ,,. o.793 g. 0.364 g. n 0~988 g .. " " n tt it ll 11 ,, II u 3.41 mg. 1.77 mg. 1.07 mg. 0.27 mg. o.o5 mg. - 50- ~ £E_ t~1e Magnetic Susoept-1 bil 1t~ of Europium Salts An attempt was made to determine the magnetic susceptibility of bivalent europium carbonate {EuC03) but, no matter how small the cross .. section of the glass mag .... netio tube, the salt was so strongly paramagnetic that the tube waa drawn to the pole pieces. If a very low field were used it might be possible to obtain an aoourata ll W. An attempt was then made to determine the magnetic . . susceptibility o! the bivalent europi um ion (Eu ~t ) by dissolving the europium carbonate in hydrochloric aoid solution and measuring the AW9 • .1 W9 for solution 4: 12. 28 mg. {uncorrected) (unoorraot,ed) L\ W9 for air-water ::: 49,05 n Ll Wg for water.water= 1 .. 35 11 .Ll W9 for HCl soln. :::. 1 .06 n (uncorrected) This value is a little more than a third of the val• ue reported by Selwood.* Undoubtedly the bivalent ion was largely oxidized by tlle air to Eu t-+-t- • 1 cc. of 4.5 N H010 4 was then added to the syatem to oxidize the Eu t-+ and air was passed through to insure * f>. Selwood. - Jour. Am. Chem. soc., .§.§., 4869 (1933) - 51- complete oxidation. A ~w 9 was then measured • .6 Wg for solution = 6.60 mg. (uncorrected) L\W9 for air;..water - 49.05 mg. (uncorrected) L1W9 for water-water-= 1.35 mg. LlW9 for HCl soln. == 1.06 mg. (uncorrected) This value if corrected for the dillmagnetism of the Eu agrees within experimental error with that reported by "" Selwood foi· the tr1 val ant europium ion. - 52 - Summary l. The reaction between ferrohemoglobin and ethyl isocyanide has been studied magnetically and the stability of the complex toward dissociation has been demonstrated. The molar ratio of isocyanide to heme- iron has been calculated. A covalent d2sp3 octahedral configuration for the atomic groups surrounding the iron atom has been postulated. 2. The ~ quilibrium between ferrihemoglobi n and i midazole J~s been investigated at differen t pH values. Apparent dissociation constants and molal susce ptibilities of the complex have been reported as a function of the pH. The pK of the acid groups on the globin co- ordinated wi t h the iron atoms of the hemes has been evaluated. 3. A structure involving no unpaired electrons has been suggested for sulfur nitride on the basis of magnetic data. 4. The auto-oxidation reaction of oxyhemoglobin solution at room temperature has been briefly inves• ti gated. 5. The magnetic properties of the ferrihemoglobin- methylamine hydrochloride system have been qualitatively studied. 6. A value for the molal susc eptibility for the trivalent europium ion has been reported. - 53- Bibliograp11r (1) s. s. Bhatnagar and K. N. Mathur ... "Physical Principles and Applications of Magnetoahemistry~, MacMillan, (1935). ( 2) E . C, Stoner - nn1agnetism and Matter", Methuen. (1934). (3) w. Klemm- 11 I:1Iagnetochemie 0 , Verlagsgasellsohaft Akademisch$ M. B.H . (1936). ( 4) L. Pauling ... "The Nature of the Chemical Bend"• Cornall Uni vers 1 ty Press, ( 1939). (5) J . H. Van Vleok .. "The Theory of .Electric and Magnetic Susceptibilities" , Press, (1932). Oxford