LIST OF SYMBOLS the observed absorbance in the aqueous phase, corrected for dilution resulting from addition of titrant ti = the molar absorptivity of species i in the aqueous phase, measured in a 1.000-cm cell ti' = the absorbance of a 1M solution of species i in the aqueous phase, measured in the titration cell yi = the molar ionic activity coefficient of species i K ' ~ , B= the conditional basicity constant of base B in the aqueous phase K D ,=~the non-electrolyte sorption coefficient of species i K I E ,=~ the ion exchange distribution coefficient of species i Ks,= ~ the electrolyte sorption coefficient of species i Kw = the autoprotolysis constant of water Aobsd =
the number of moles of base B present, in all of its forms n, = the number of moles of titrant acid added V,, = the volume of the aqueous phase in liters WR = the mass of resin phase in kilograms RB =
RECEIVEDfor review December 18, 1973. Accepted April 12, 1974. Taken from the Ph.D. Thesis of F.F.C. submitted to The University of Iowa, December 1972. Presented at the 167th ACS Meeting in Los Angeles, Calif., April 1974. This work was supported by an American Chemical Society, Division of Analytical Chemistry Fellowship, sponsored by the Procter and Gamble Company. F.C.C. was an Analytical Division Fellowship (Procter and Gamble) Holder, 1971-72.
Chemistry of Bilirubin and Biliverdin in N,N-Oimethylformamide John D. Van Norman and Robert Szentirmay' Department of Chemistry, Youngstown State University, Youngstown, Ohio 44503
The oxidation of bilirubin and biliverdin is examined In N , K dimethylformamide at platinum and vitreous carbon electrodes. In acid or neutral solution, bilirubin Is oxidized at 4-0.7 volts vs. the Ag/AgCI (satd), NaCl (satd) reference electrode. Addition of base results in the neutralization of two acidic hydrogens with the formation of a basic form which oxidizes at +0.4 volt. Quantitative formation of a complex of bilirubin with zinc Ion results under basic condltlons and this complex oxidizes at 0.0 volt. The complex formed has two zinc ions per bilirubin molecule. Conclusions are drawn Concerning the formation of bilirubin-metal ion complexes. The behavior of biliverdin is also described.
I
M
Present address, Department of Chemistry, Ohio State University, Columbus, Ohio 43210. (1) T. K. With, "Bile Pigments, Chemical, Biological and Clinical Aspects," Academic Press, New York, N.Y., 1968. (2) I. A. D. Bouchier and 6 . H. Billing, "Bilirubin Metabolism,'' Biackwell Scientific Publications, Oxford and Edinburgh, 1967. (3) T. Hargreaves, "The Liver and Bile Metabolism," Appleton-CenturyCrofts, New York, N.Y., 1968.
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Bilirubin is a yellow linear tetrapyrrole belonging to a class of compounds found in the human body called bile pigments. Approximately 300 milligrams of bilirubin are formed daily from the enzymatic catabolism of hemoglobin, taking place primarily in the reticuloendothelial cells in the liver, bone marrow, and the spleen. Because of the importance of bile pigments in the physiological system, a great deal of attention has already been given to the study of bilirubin and other bile pigments. Excellent reviews of these studies covering aspects through 1967 have been made by With ( I ) , Bouchier and Billing ( 2 ) ,and Hargreaves ( 3 ) .All studies indicate that much is still uncertain concerning the chemistry of bilirubin. Investigations have been severely limited by the lack of stability and solubility of bilirubin in aqueous solutions including physiological solutions. This has led a number of investigators to the utilization of nonaqueous solvents for the study of the chemistry of bilirubin and its analogs. Emphasis has been placed upon the determination of structure since it is believed to play
V
V H
M H
P H
P H
M H
m Flgure 1. Some possible forms of bilirubin
an important role in the chemistry of bilirubin. These studies have been primarily spectroscopic in nature. The exact tautomeric structure for bilirubin (BR) has been a subject of debate ever since its discovery. Figure 1 shows three possible structures; the bis-lactim (I), the monolactam-monolactim (11), and the bis-lactam (111).Not included in these structures are the possible inter- and intra-molecular hydrogen bonds between propionic acid hydrogens and hydrogens on pyrrole nitrogens with pyrrole nitrogens or carboxyl oxygens. Velapoldi and Menis ( 4 ) have proposed a mono-lactam-mono-lactim form to explain the ability of bilirubin to form metal complexes. Bilirubin and other bile pigments have been shown to form complexes with a large number of transition and rare earth elements (1, 4, 5 ) with simultaneous oxidation of the bilirubin possible. The literature available on the studies of bile pigments presents only five investigations which deal with direct electroanalytical studies of bilirubinoid systems (6-10). (4) R. A Velapoldi and 0. Menis, Clin. Chem., 17, 1165 (1971) (5) J Fog and B. Bugge-Asperheim,Nature, 203, 756 (1964). (6) B. Tvaroha, Coiiecf. Czech. Chem. Commun., 26, 2271 (1961).
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1 9 7 4
Van Norman (9) has shown the feasibility of using N , N dimethylformamide (DMF) as a solvent for electrochemical and spectral studies of bilirubin. This investigation is an attempt to utilize spectrophotometric and electroanalytical techniques for the study of the chemistry of bilirubin and biliverdin, an oxidation product of bilirubin, and their metal complexes in DMF. Slifstein and Ariel (IO) have applied cyclic voltammetry, controlled potential coulometry, thin layer cell techniques, and Kalousek commutator methods to the study of electroreduction and electrooxidation of bilirubin in dimethylsulfoxide (DMSO).
EXPERIMENTAL Apparatus. The electrochemical cell employed was a Metrohm Model EA-874 titration vessel which had five openings in the upper portion to permit insertion of the appropriate electrodes, gas bubblers, and addition or sampling pipets. Reference electrodes used were either AglAgN03(0.01M), (DMF) or an Ag/ AgCl(satd), NaCl(satd), (DMF). The latter reference electrode was the nonaqueous electrode furnished by Metrohm, EA 425. Solution contact for the latter was made through a 5-mm 0.d. glass tube closed a t the lower end by a fine porosity frit. This electrode was used in most of the electrochemical studies. The electrodes for voltammetry were either two Sargent-Welch platinum electrodes of approximately 2 cm2 area or a vitreous carbon electrode serving as the indicator electrode and the Pt electrode serving as the counter electrode. The vitreous carbon electrode was made by pressure fitting a vitreous carbon crucible (Beckwith Carbon, ?*-in. 0.d. X '&in. high into a hollow Teflon tube. A mercury pool inside the tube made electrical contact with a copper lead wire. Controlled potential coulometric measurements were made with a large platinum foil as the working electrode and a platinum counter electrode isolated in a fritted glass compartment. Potentiometric measurements were made using a Coleman glass electrode in conjuction with one of the above-mentioned reference electrodes. For voltammetric and controlled potential studies, the DMF was 0.1M in NaC104 as the supporting electrolyte. All voltammetric and controlled potential measurements were obtained with either a National Instrument Laboratory "Electrolab" in conjunction with a Valtec Model 1024 X-Y recorder or with a Princeton Applied Research Model 170 Electrochemical System ( I 1). Potentiometric measurements were obtained with an Orion Model 801 digital p H meter. Near UV and visible spectra were obtained using either a Beckman DB or Cary 14 spectrophotometer. Weighings were performed on a Mettler H20 semimicro balance. Reagents. All chemicals used were of reagent grade. The N,Ndimethylformamide was purified following storage over molecular sieves by a distillation (25-40 "C) under reduced pressure (2-10 mm Hg) in the presence of anhydrous CuSO4 which served as a drying agent and for the removal of amine impurities arising from the decomposition of the DMF (12). Distillation was performed under an inert atmosphere (low pressure) of argon or nitrogen retaining the middle fraction (") for use. Fresh solvent was prepared as a matter of necessity on a biweekly basis. The purified DMF was stored under the inert atmosphere in a laboratory freezer. The argon used for the distillation or for an inert atmosphere for the chemical studies was purified by passage over copper chips at 600 "C to remove traces of oxygen. The bilirubin used was obtained from Sigma Chemical Company with a quoted purity of 99%. Solutions of this bilirubin in chloroform had measured molar absorptivities of 60,000 f 600 well within the acceptable range (13). Biliverdin dihydrochloride (approximately 80% biliverdin) was also obtained from Sigma Chemical Company. Procedure. Bilirubin and biliverdin solutions were prepared directly by weighing or by diluting a more concentrated stock solution. Concentrations were checked spectrophotometrically. Solutions were 0.1M in NaC104 for voltammetric and coulometric studies. Zinc(I1) solutions were prepared electrochemically with a zinc (7) (8) (9) (10) (11) (12)
B. Tvaroha, Naturwissenschaften,48, 99 (1961). 6.Tvaroha, Casopis Lekaru Ceskych, 100, 569 (1961). J. D.
Van Norman, Anal. Chem., 45, 173 (1973).
Ch. Slifstein and M. Ariel, J. Electroanal. Chem., 48, 447 (1973). G. W. Ewing. J. Chem. Educ., 46, A717 (1969). A. J. Bard, Ed., "Electroanalytical Chemistry," Vol. 3, Marcel Dekker Inc., New York, N.Y., 1969, pp 75-90.
(13) W.
Meinke, Anal. Chem.. 43(6), 28A (1971).
metal strip as the anode and a platinum cathode in an isolated fritted compartment or by direct weighing of zinc iodide. Copper(I1) solutions were prepared by weighing out previously dried copper perchlorate. The copper perchlorate was dried under vacuum for 2 hours a t 100 OC. Concentrations of the copper(I1) solutions were calculated from weights and checked independently by an iodometric method. Solutions were introduced into the electrochemical cell (25-40 ml) which had been preassembled with the appropriate electrodes, gas bubblers, etc., and deoxygenated by passage of an inert gas, argon, or nitrogen, for one-half hour. Appropriate electrochemical measurements were then made directly on these solutions while spectra of the solution species were obtained by removing samples when needed (100-500 ~ 1 with ) subsequent dilution to give maximum absorbance values of 0.1-1.0. Additions of reagents could be accomplished in a similar manner. Bilirubin solutions so handled did not undergo noticeable photodecomposition or chemical reaction over a period of several days as evidenced by no change in the spectrum or electrochemical behavior. Platinum electrodes were cleaned in hot concentrated nitric acid, washed with distilled water, then DMF, and allowed to come to equilibrium with the solution being studied. Sometimes the platinum electrodes would have to be removed and wiped clean or treated again with nitric acid as the reaction products would adsorb upon the electrode surface. This problem generally occurred in the controlled potential experiments and was not as great when a vitreous carbon electrode was used.
RESULTS AND DISCUSSION Preliminary Investigation-The Reactivity of Bilirubin. The reactivity of bilirubin dissolved in DMF was investigated in a qualitative manner by adding mixtures or solutions of various materials to bilirubin solutions, all in DMF. The results are summarized in Table I. The term insoluble, as used in the table, refers to the observed fact that no salt crystal of the compound dissolved and the DMF remained uncolored. The Gmelin series referred to is the oxidation series (9,14): bilirubin ( ye1 l o w )
-
biliverdin (meen)
-
purpurin
---t
(purple)
c holetelin (ye1l o w )
All solutions in DMF appeared stable except the solutions of KMn04 which repeatedly changed to the brown color of MnO2 after standing for a half hour. Addition of 10F (aqueous) HCIOI resulted in a gradual change of the bilirubin yellow color to the green verdinoid (biliverdin) color. This change seemed dependent upon the amount of acid used but was slow enough in a relatively concentrated solution of acid in the DMF to be followed spectrophotometrically on the Cary 14. The verdinoid peak at 380 nm appeared simultaneously with the diminution of the bilirubin peak at 450 nm. This is a unique situation since bilirubin is insoluble in aqueous acidic solution and cannot be observed, and addition of HC104 to chloroform solutions of bilirubin results in the extremely rapid oxidation of the bilirubin. Another interesting situation was the discovery that iodine and bromine in solution oxidizes bilirubin to the biliverdin and then on to the further oxidation products. One could essentially do a photometric titration of bilirubin with the halogen. Solvent System. Since bilirubin possesses six potentially active hydrogens, two propionic acid protons and 2, 3, or 4 hydrogens on the pyrrole nitrogens depending upon its structure (Figure 1) and since bilirubin is soluble in DMF under both acidic and basic conditions, it was deemed necessary to investigate the potentiometric behavior of bilirubin. Before this could be accomplished, the solvent system (14) B. Zak. N . Moss, A. (1954).
Boyle, and A. Zlatkis, Anal. Chem., 26, 1220
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
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Figure 2. Potentiometric titration of picric acid with TMG in DMF
Figure 3. Potentiometric titration of bilirubin with HCI in DMF
Table I. Reactivity of Bilirubin in DMF Compound
KIOI Potassium biphthalate NazS205 Ferrocene K2Cr207 K2Cr207(acidic) Tetramethylguanidine EDTA Ethylenediamine Triethylenetetramine Cu(C104) ’ 6H20 FeC13 ZnC12 ‘XHZO Zn(CH3C00)2.6H20 Co(N03)2.6HzO FeSOI.XH20 K4Fe(CN)6 3H20 NiC12 6H20 Fe (NHc) (Sol)z . ~ H z O
high miscible yes, clear solution yes, clear solution highly soluble, but unstable highly soluble, yellow highly soluble, red insoluble in neutral or acidic solution insoluble in neutral or acidic solution insoluble insoluble highly soluble, yellow soluble, yellow soluble, yellow high, colorless insoluble or slightly soluble highly miscible, colorless highly miscible, colorless highly soluble, blue solution high, yellow color high, colorless high, colorless high, bluish red insoluble insoluble or only slight soluble high, green blue insoluble
and the acids and bases used had to be investigated. Kolthoff et al. ( 1 5 ) have reported the use of an aqueous-conditioned glass electrode as an indicator electrode in acid-base potentiometric titrations in DMF. They reported that the glass electrode is well behaved, yielding an ideal Nernst slope of 59 mV. A Coleman glass electrode was used in our study. (15) I. M. Kolthoff, M. K. Chantooni, Jr., and H. Smagowski, Anal. Chem.,42, 1622 (1970).
1458
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Reaction with BR
Solubility in DMF
oxidation to verdinoid green oxidation through Gmelin series oxidation through Gmelin series oxidation through Gmelin series rapid oxidation to verdinoid green rapid oxidation t o verdinoid green none none none none none none oxidation to verdinoid green none none none none rapid lightening of color rapid shift to green-yellow shift to pink color , shift to red color rapid shift to amber color none reacts on shaking, weaker color rapid shift to yellow green none
Initially, 1 X 10-2M solutions of picric acid and tetramethylguanidine were prepared by weighing into 100 ml of DMF. These were potentiometrically titrated against each other and the expected end point was found within i2%. Figure 2 shows the titration of 28.0 ml of picric acid (2.86 X 10-sA4) with TMG (9.9 X lO-3M) using a glass indicator electrode and an Ag/AgNOa (0.01M), DMF reference electrode. The end point is indicated on the plot. Titrations of this type were also performed using HCl solutions instead
ANALYTiCAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
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Figure 4 A and 46. Change in the oxidative voltammetry of bilirubin in DMF with the addition of TMG
of picric acid solution. Titrations were performed either adding acid to base or the base to acid. In acid solutions, the millivolt readings were stable, with little drift; but, under basic conditions, it took 4-5 minutes for the readings to stabilize. After 10 minutes, the readings would again start drifting; so under basic conditions, readings were taken 5 minutes after addition of base or acid. If unpurified DMF was titrated with picric acid solutions, there was an initial change of 200 to 400 mV with an indication of to mole per liter of basic impurities. This was attributed to amine impurities arising from the decomposition of the DMF. The distillation from anhydrous CuS04 removed most of this impurity as shown potentiometrically. The concentration of the basic impurity (as estimated spectrophotometrically at 430 mn as the p nitrophenolate ion following addition of 10-4M p-nitrophenol) was less than This was the method used by Kolthoff (15). Plots of log concentration us. potential of the glass electrode for solutions of picric acid, HC1, and TMG gave straight lines with slopes between 60 and 70 mV. This straight line behavior was noted for both types of nonaqueous reference electrodes used in this study. Bilirubin Acid-Base Behavior. Addition of bilirubin to DMF gave a change of +125 mV, toward acid behavior. Titration of bilirubin with either HCI or picric acid in DMF gave little indication that bilirubin could accept a proton as shown in Figure 3. Curve A is the titration of 25.0 ml of DMF with HCl (5.9 X 10-4M) and curve B is the same volume of DMF made 2.29 X 10-4M in bilirubin. It was noted that use of HC1 solution which was several months old resulted in oxidation of the bilirubin, but this did not occur in freshly prepared acid solutions. There is apparently an acid catalyzed decomposition of DMF. Titration of bilirubin solutions with TMG gave titration curves with two breaks in it a t 1:l and 2:1, TMG:BR molar ratios. The details of this titration will be reported elsewhere (16). The spectrum of bilirubin changes upon the addition of the TMG. With the addition of the first equivalent, there is a slight decrease in molar absorptivity and a slight broadening of the peak. With the addition of the sec(16) J. D. Van Norman and R . Szentirmay. Bioinorg. Chern., in press.
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Figure 5. Oxidation of biliverdin.2HCI under basic conditions in DMF
ond equivalent of TMG, a progressive increase in the molar absorptivity occurs maximizing at the second equivalence point with a concomitant bathochromic shift in the maximum from 453 to 462 nm. The hyperchromic effect is approximately 10%. Oxidative Voltammetry of Bilirubin under Basic Conditions. The oxidative voltammetry of bilirubin a t the platinum electrode in DMF under neutral conditions has been elucidated by Van Norman (9). He showed quantitatively that bilirubin is oxidized to biliverdin at +0.6 V and biliverdin is further oxidized a t +00.8 V us. the SCE. In the present studies, under prolonged usage, the saturated calomel electrode (SCE) proved unsatisfactory, as the contact orifice eventually plugs up, probably because of precipitation of KCI. Using the Ag/AgCl (satd), NaCl (satd), (DMF) reference electrode, the bilirubin was oxidized at f 0 . 7 V and the biliverdin at +0.9 V. The small prewave that occurred a t +0.4 V in the work reported by Van Norman depended upon the age of the bilirubin solution. He did not
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 11, SEPTEMBER 1974
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WAVE LENGTH
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Figure 6. Spectral and voltammetric changes in a bilirubin solution during electrolysis at 4-0.5V in the presence of TMG
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0
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Figure 7. Plots of absorbance (right) vs. coulombs passed
at 460 nm (left) and peak current
purify the DMF. Solutions of bilirubin in purified DMF did not exhibit this small wave suggesting that the basic impurities in DMF react with bilirubin. The nature of the prewave was affirmatively resolved by observing the behavior of bilirubin under basic conditions. Anodic voltammograms were obtained a t a platinum indicator electrode us. a Ag/AgCl (satd), NaCl (satd), DMF reference electrode. Figures 4A and 4B show the changes resulting from the stepwise addition of equal amounts of TMG to a 4.7 X 10-4M solution of bilirubin in DMF; the scanning rate was 1 V/ min. Curve A is bilirubin with no added base, curve E has a TMG:BR ratio of 1:l while curve 1 has a TMG:BR ratio of 2:1. The anodic wave a t +0.7 V is quantitatively converted into the wave a t +0.45 V while the biliverdin wave remains unchanged. Under higher ratios of TMG:BV, the biliverdin wave is altered and merges with that of bilirubin. This is shown in Figure 5. Curve A is the oxidation of biliverdin. DHCl (1.75 X 10d4M)in DMF. Curve B is with the two HCl's neutralized, curve F is for a 2:l ratio of excess base to biliverdin, and curve G for an 8:l excess. The dotted line is background for DMF with excess base added (note same amount of base as for curve G). Again, the scan rate was 1 V/min. 1460
Controlled Potential Coulometry of Bilirubin under Basic Conditions. Under the basic condition of 2:1, TMG:BR, controlled potential electrolysis of BR at +0.5 V us. the AglAgC1 (satd), NaCl (satd) electrode with current integration resulted in the progressive increase in biliverdin and diminution in bilirubin. Figure 6 shows the observed changes in spectra and voltammograms as a function of coulombs passed. Curves A are with no coulombs passed. Curves B through G are for various amounts of coulombs passed as plotted in Figure 7 us. absorbance or peak current. For a two-electron process, the quantity of bilirumole) should theoretically require bin taken (7.08 X 1.37 coulombs for complete oxidation. However, a correction of 0.09 coulomb must be made to account for losses in sampling for the spectral measurements, yielding a theoretical value of 1.28 coulombs needed. The plots give values of 1.20 coulombs and 1.25 coulombs compared to the 1.28 coulombs calculated, good agreement. Complexation of Bilirubin with Zn(I1) Ions. The preliminary investigation showed that a relatively stable zincbilirubin complex could be reversibly formed, and many of the references in the literature referred to stable zinc-bilirubin complexes (4, 5, 17-19). It was found necessary to electrochemically generate the Zn(I1) solutions as the zinc. acetate used in the preliminary experiments proved unsatisfactory. It was also qualitatively found that the Zn-BR complex occurred better under basic conditions. To resolve the conditions under which the complex forms, a systematic study was undertaken to vary the molar ratio of base, TMG, and BR. The details of this spectrophotometric study will be published elsewhere, but it was clearly shown that complexation occurred best under the molar ratios of BR:ZN:TMG of 1:2:8 (16). The spectrum of the zinc-bilirubin complex has a maximum a t 530 nm. A similar study was performed voltammetrically using a vitreous carbon indicator electrode and scan rates of 2.5 ( 1 7 ) A. W. Nichol and D. E. Morell, Biochim. Biophys. Acta. 177, 599 (1969). (16) P. O'Carra, Nature, 195, 699 (1962). (19) C. H. Gray, A. L. Kulczycka. D. C. Nicholson, and Z . Petryka, J. Chem. Soc.,London, 1961, 2268.
ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 1 1 , SEPTEMBER 1 9 7 4
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Figure 8. Effect of addition of Zn(ll) to bilirubin solutions under basic conditions
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Figure 9. Spectral changes occurring during the controlled potential electrolysis of zinc-bilirubin solutions
V/min. Figure 8 shows the shift of the anodic wave of BR under conditions of BR:TMG of 1%from +0.4 V to 0.0 V as a function of added Zn(I1). Curve A is for 9.7 X lO-9M bilirubin. Curves B through F are the addition of successive amounts of Zn(I1); curve B BR:Zn, k0.4; curve C, 1:0.8; curve D, k1.2, curve E, L1.6; curve F, 1:2. As the wave a t 0.0 volt increases, the wave at +0.4 V decreases and disappears when the BR:Zn ratio reaches 1:2 (curve F). A wave does appear a t approximately +0.2 volts which has been identified as the oxidation of the zinc-biliverdin complex as shown by the voltammetry of biliverdin solutions. Controlled Potential Coulometry of the Zinc-Bilirubin Complex. Solutions in which the BR:Zn:TMG molar ratios were fixed at 1:2:8 were electrolyzed a t 0.0 V with current integration. Figure 9 shows the spectral changes as
a function of coulombs passed. Curve A is for bilirubin-zinc with no coulombs passed. Curve B is for 8% of theoretical; curve C is 16%; D is 33%; E is 50%, F is 63%, G is 75%, H is 87%, and I is 98% of theoretical. Figure 10 shows the same solutions with 2,4-pentanedione added to decomplex the bilirubin. The curves are labeled similarly to Figure 9. Note that the spectrum of bilirubin decomplexed reverts to the basic form at 462 nm. A 100% oxidation to the biliverdin form could not be accomplished at 0.0 V and shifting the potential more positive resulted in mixed products as the biliverdin complex with zinc could then be oxidized. Potentiometry of Zn(1I) in DMF. In a n effort to clarify the 1:2:8 ratio of BR:Zn:TMG, it was necessary to titrate Zn(I1) ion with TMG to see if the zinc ion was acidic in nature. The resulting potentiometric titration is shown in Figure 11, in which 20.0 ml of ZnI2 in DMF (4.7 X lO-4M) is titrated with TMG (2.08 X 10d2M).The theoretical end point based on a ratio of 2:1, TMG:Zn(II) is 9.0 ml; the experimental value is 8.8 ml. It is quite clear that for every
ANALYTICAL CHEMISTRY. VOL. 46, NO. 1 1 , SEPTEMBER 1974
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Figure 12. Spectral changes occurring upon the addition of TMG to a solution containing biliverdin-2HCIand Zn(l1)
W A V E LENGTH (nm)
Figure 13. Spectral changes occurring upon the addition of Cu(ll) to bilirubin solutions under basic conditions in DMF
zinc ion, two TMG molecules are needed to neutralize the solution. Complexation of Biliverdin with Zinc(I1). It was found that zinc(I1) would complex best with biliverdin under basic conditions but that only one zinc ion would complex with a biliverdin molecule. Figure 12 shows the spectral changes resulting in the addition of TMG to a solution containing equal amounts of zinc(I1) and biliverdin. Curve A is the spectrum of biliverdineZHC1 (6.11 X 10-4M); 1462
curve B, biliverdin2HCl plus Zn(I1) equal amounts. Curve C has l:l:l, BVs2HCl:Zn:TMG ratio; curve D is 1:1:2, curve E, 1:1:3, curve F, 1:1:4, curve G, 1:1:5, curve H, 1:1:6, and curve I, 1:1:8. The verdinoid peak a t 380 nm diminishes as the complex peak a t 420 nm increases. Complexation maximizes at a BV:Zn:TMG ratio of 1:1:5. Complexation of Bilirubin and Biliverdin with Cu(I1). A short study of the reaction of Cu(I1) with bilirubin and biliverdin was undertaken as the qualitative study indicated that its reaction with the bile pigments was irreversible. Addition of up to five moles of Cu(I1) ion per mole of bilirubin in neutral DMF showed that only a fraction of the bilirubin reacted as determined spectrophotometrically (about 10%). In the presence of base such as TMG, the reaction appeared to proceed quantitatively. Initial addition of Cu(I1) in the form of the perchlorate salt resulted in a red color which changed into a yellow-green color within 3-4 seconds. The red color is believed to be the Cu-BR complex which is then rapidly oxidized. Figure 13 shows the quantitative conversion of bilirubin with a maximum a t 460 into a Cu-oxidation product complex with its maximum at approximately 435 nm with a shoulder at 360 nm (TMG:BR is 8:l). Spectrum A is bilirubin only. Curves B, C, D, E, F, and G represent 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 moles of Cu(I1) per mole of bilirubin. The product could not be decomplexed with 2,4-pentanedione or with diethylaminetetramine. Neutralization, however, resulted in the development of a spectrum (spectrum H) associated with that of the purpurin. Addition of Cu(I1) to biliverdin solutions under basic conditions resulted in the same product as the bilirubin solutions as shown in Figure 14. Curve A is biliverdin in basic solution. Curves B through F represent 1:0.4, L0.8, 1:1.2, 1:1.6, and 1:2 ratio of BV:Cu. The isosbestic points indicate the existence of two species. Discussion. Bilirubin has been found to be sufficiently soluble in DMF so that spectrophotometric and electro-
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 11, SEPTEMBER 1974
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8(
Flgure 14. Spectral changes occurring upon the addition of Cu(ll) to biliverdin solutions under basic conditions in DMF
chemical analysis can be combined as investigative tools to follow its behavior under acidic, basic, and neutral conditions. This is in contrast to the solubility difficulties encountered in handling bilirubin in neutral or acidic aqueous solution. Nevertheless, the neutral form of bilirubin in DMF appears to be the same fully protonated molecule that exists under neutral aqueous conditions ( I ) . However, the relatively low solubility of bilirubin in DMF (6.7 X 10-4M) requires a solvent of high purity since it reacts readily with basic impurities as a dibasic acid resulting in a considerable change in the chemical properties. Under slightly acidic conditions, no significant electrochemical or spectral changes have been observed. This “lack” of reactivity with acids is fully consistent with the acidic nature of bilirubin, its failure to form acid salts, and its insolubility under acid aqueous conditions (19). The quantitative behavior of bilirubin as a diprotic acid has been followed potentiometrically, spectrophotometrically, and voltammetrically. The results indicate that, under neutral conditions in DMF, bilirubin exists as the neutral molecule maintaining intra-molecular hydrogen bonding. This neutral form appears inert to complexation with Zn(I1) or Cu(I1) and maintains a relatively high oxidation potential of +0.7 V us. Ag/AgCl (satd), NaCl (satd), (DMF), or +0.6 V us. SCE. With the quantitative removal of two protons, presumed to be the carboxylic acid protons, the oxidation potential drops to approximately +0.04 V (+0.3 V us. SCE). However, the point of oxidation has been shown to still persist a t the central methene bridge which oxidizes in a two-electron process to form biliverdin as does the neutral molecule. The removal of the two protons apparently disrupts intramolecular hydrogen bonding and leads to steric changes or inductive effects which leave the methene bridge more prone to oxidation, hence the lower oxidation potential. Removal of the two protons also leads to the formation of a basic form which reacts with Zn(I1) and Cu(I1). The preliminary investigations show that bilirubin will also react in DMF with Fe(III), Ni(II), and
Co(I1). I t is believed that quantitative reactions of these metal ions with bilirubin will also be dependent upon the presence of base and the production of the basic form of bilirubin. The complexation of Zn(I1) with bilirubin maximizes a t a BR:Zn:TMG ratio of 1:2:8 indicating that two zinc(I1) ions are in the chelated form of bilirubin in a manner similar to that for samarium(I1) reported by Kuenzle et al. (20). The complexation of Zn(I1) with biliverdin dihydrochloride occurs at BV:ZN:TMG ratio of 1:1:5 suggesting that complexation follows after removal of the 2 hydrochloride protons, the 2 propionic acid protons, and 1 hydrogen from a pyrrole nitrogen. Zn2+ + BV’2HCl
+
5TMG ZnBV-
+
5HTMG’
+
2C1-
This is quite reasonable as biliverdin has 2 fewer hydrogens on the pyrrole nitrogens. The spectra and relative ease of zinc-biliverdin complex formation are fully consistent with previous observations by Gray et al. in ethanolic zinc acetate (19). The oxidative potential of the Zn2BR complex is approximately 0.0 V us. Ag/AgCl (satd), NaCl (satd), DMF (-0.1 V us. the SCE) and shows a considerable change from that for the neutral and basic forms of BR. The experimental results suggest that oxidation proceeds by the same two-electron mechanism which leads to verdinoid formation as in the other cases. However, this conjecture has still not been firmly established. This progressive ease in oxidation of the neutral, basic, and complex forms of BR is fully consistent with the chemical reactivity observed for the above species in various solvents and is analogous to their reactivity in water. Copper(I1) appears to complex with bilirubin under basic conditions and either catalyze or cause an immediate (20) C. C. Kuenzle, R. R. Pelloni. M. H. Weibel, and P. Hemmerich, Biochern. J. 130, 1147 (1972).
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 11, SEPTEMBER 1 9 7 4
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oxidation to form a complex of Cu(I1) with the purpurin formed from oxidation of bilirubin. Cu(I1) is well known as an oxidant in nonaqueous solution (21). The behavior observed spectrophotometrically in DMF is analogous to the observations of Velapoldi and Menis for the reaction of Cu(I1) in chloroform-methanol (1:l) mixture ( 4 ) .Their ex: planation for this type of oxidation relates to the formation of a square planar bilirubin complex which results in a strain a t the already labile central methene bridge. The argument may be qualitatively extended to explain the relative ease of oxidation of the ZnZBR complex compared to the neutral or basic form of bilirubin.
SUMMARY Bilirubin exists in solution in DMF in a variety of forms depending upon solution conditions. A neutral form h$ been identified having a maximum in its spectrum at 453 nm and an oxidation potential at f0.7 V us. the Ag/AgCl (satd), NaCl (satd), DMF reference electrode. The basic (21) B. Kratochvil, D. A. Zatko. and R. Markuszewski, Anal. Chem., 38, 770 (1966).
form of bilirubin with two protons removed from the proprionic acid side chains has a spectral maximum at 462 nm and an oxidation potential of +0.45 V. Addition of Zn(I1) ion and more base in a ratio of BR:Zn:TMG of 1:2:8 results in the quantitative formation of a zinc-bilirubin complex which has a spectral maximum a t 530 nm and is oxidized at 0.0 V. This complex has two zinc ions per bilirubin molecule. Biliverdin also forms a complex with zinc(I1) changing the spectral maximum from 380 nm to 420 nm and the oxidation potential to a lower value. Copper(I1) can react with both bilirubin and biliverdin to oxidize them to one of the oxidation products in the Gmelin sequence and to complex this product. The chemistry of bilirubin is very complex and any oxidation-reduction studies of metal complexation studies must be accompanied by consideration of the acid-base behavior of this biologically important compound.
RECEIVEDfor review January 31, 1974. Accepted April 24, 1974.
Evaluation of a Method for Real-Time Deconvolution Arend den Harder' and Leo de Galan2 Laboratorium mor lnstrumentele Analyse, Technische Hogeschool, Delft. Nederland
This paper presents an evaluation of a procedure for the deconvolution of two-dimensional profiles, such as spectra, chromatograms, etc. It is based upon a proposal by Hardy and Young that has apparently never been tested on real spectra. It is shown that by using only the first and second derivative to the experimental spectrum, the original band system can be recovered if the width of the broadening function is less than or equal to the width of the nondistorted bands. The method can be applied to a variety of broadening functions and provides for simple and effective noise suppression. In comparison with other methods for deconvolution, the proposed procedure offers the advantage that a spectrum can be deconvoluted while it is being scanned. This makes it especially attractive for small on-line computers.
Experimentally observed spectra, chromatograms, and the like are generally subject to several, independent broadening processes. Atomic spectral lines are subject to Doppler broadening and collisional broadening ( 1 ); atomic and molecular spectra are distorted by the finite resolving power of the monochromator (2-5); electronic filtering inPresent address, Hoogovens-Estel, I J m u i d e n , T h e N e t h e r lands. A u t h o r t o whom correspondence s h o u l d b e directed. (1) A. C. G. Mitchell and M . W. Zemansky. "Resonance Radiation and Excited Atoms," University Press, Cambridge, England, 1961 (2) R. N. Jones, R . Venkataraghavan, and J. W. Hopkins, Specfrochim. Acta, Part A, 23, 925, 941 (1967) (3) D. A . Ramsay, J. Amer. Chem. Soc., 74, 72 (1952). (4) J. R . Morrey, Anal. Chem., 41, 719 (1969) (5) L. de Galan and J . D. Winefordner, SpeCtroChh. Acta, Part B, 23, 277 (1968)
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troduces asymmetry (6, 7); X-ray diffraction profiles are influenced by structural effects in the specimen (8), etc. All such interactions are mathematically described by the convolution integral
T ( y )= l ; t ( x i B(y
- x ) dx
(1)
where t ( x ) is the nondistorted line profile, B ( y - x ) is the broadening function, and T ( y )is the recorded line profile. The mathematical properties of this integral have been described by Hsu (9) and Shapiro ( I O ) . Whereas the calculation of TCy) from known t ( x ) and B ( y - x )is simple and straightforward, the reverse procedure of calculating t ( x ) from known T ( y ) and B ( y - x ) is rather more difficult. This procedure is known as deconvolution. Two methods of deconvolution appear to be widely used. The first method is of long standing (11-15) and has been designated by Jones ( 2 ) as pseudo-deconvolution. Here the observed profile T ( x ) is first convoluted once more with the broadening function B ( x ) to give a convolution result C(x). The true profile is then calculated as t ( x ) = T ( x ) * / C ( x ) This . is an approximation since the interI. G. McWilliam and H . C. Bolton, Anal. Chem., 41, 1755, 1762 (1969). E. Grushka, Anal. Chem., 44, 1733 (1972) B. E. Warren and B. L. Averbach. J. Appl. Phys., 21, 595 (1950); 23, 497 (1952). H. D. Hsu, "Outline of Fourier Analysis including Problems with Step by Step Solutions," Uniteck, New York, N.Y., 1967. K. S. Shapiro. "Smoothing and Approximation of Functions," Van Nostrand, New York, N.Y., 1969. H . C. Burger and P. H. van Cittert, Z. Phys., 79, 722 (1932) H. C van Hulst. Bull. Astron. lnst. Neth., 9, 225 (1941). A . L. Khidir and J . C. Decius, Spectrochim. Acta, 18, 1629 (1962). S. Ergun, J. Appl. Crystallogr., 1, 19 (1968). J . SzBke, Chem. Phys. Lett., 15, 404 (1972).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
