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Characteristics of a ferrous iron complex of L-epinephrine
Life Sciences No . 10, pp . 509-513 ,1962 . Pergamon Press, Inc . Printed in the United States .
CHARACTERISTICS OF A FERROUS IRON COMPLEX OF L-EPINEPHRINE Gerald Litwack Biochemical Laboratory, Division of Cardiology, Philadelphia General Hospital and Department of Medicine, Graduate Sohool of Medicine, University of Pennsylvania, Philadelphia, Pa . (Received 24 August 1962) IN the course of studying the inhibition of the liver tyrosine oxidase system by L-epinephrine and analogs l the desirability of
prpearing a ferrous complex of this hormone became apparent . Although Although such a complex has been described empirically in stucües of the effects of cations and anions on .the the oxidation of 4 catecholamines2 and by Yogeler5 , no attempts have been reported to characterize the complex. The complex can be prepared as follows : molar amounts of Lepiaephrine and FeC12 (2 :1) are weighed into a beaker at 25°C,
about 30-60 ml distilled water are added per 10 mmoles FeC12 . The pH is rapidly adjusted to 7 .0 with a motor driven stirrer and the purple solution is stirred at room temperature for 5 min. Several
volumes of chilled acetone are added, the precipitate is filtered on a Huchner funnel with suction, and washings are made with cold acetone . The dark purple solid is dried in vacuo in a dessicator and is relatively stable in the dry form. Titratioas of L-epinephrine with various amounts of FeC12 are illustrated by Fig . l . Titratioas were carried out in aqueous solutions using the Leeds and Northrup line operated pH meter
(Model 7664) and a miniature glass silver chloride type electrode assembly . The titratioas were conducted in a nitrogen atmosphere at 25°C . The pH meter was standardized with Fisher buffers at pH 4 .0, 7 .0 and 9 .0 . Back titration of epinephrine revealed only very small changes in the titration curve so that decomposition of the hormone was minimized under these conditions . Comparison of curves 1 and 2 suggests that the secondary amino group of epinephrine is not involved is the complex so that the ionized phenolic group probably directs the positioning of the metal indicative of the structure on page 511 (structure I) . 509
510
CHARACTERISTICS OF A FERROUS IRON COMPLEX
No . 10
PH
FIG . 1 Titration curves of J .-epinephrine with increasing amounts of FeCl2 (under N2) . Curve 1 a 50 moles epinephrine ; curve 2 = 50 ~ moles epinephrine + 25 ~ moles FeC12 ; curve 3 a 50 p moles epinephrine + 50 p moles FeCl2 ; curve 4 = 50~ymoles epinephrine + 100 moles FeCl2 ; curve 5 = 50 fl moles epinephrine + 150 moles FeC12 .
r
r
FIG . 2
Result of a continuous variation experiment using 10 -3 M concentration of reactants, L-epinephrine and FeCl2 at 25oC . The initial pH a 7 .0 . Final pH values are as follows at the end of the experiment (for each point from left to right s 6 .85, 6 .80, 6 .75, 6 .75, 6 .75, 6 .6, 6 .5, 6 .4, 6 .35, 6 .25 . Inset shows the spectrum of the peak reaction .
No . 10
CHARACTERISTICS OF A FERROUS IRON COMPLEX
511
r
This configuration has been suggested previously for the borate complez6 althgqxgh moat hypotheses concerning complezea of epinephrine are based upon the ability of borate to complez with poly-
hydrosy compounds . The unusual naturß of curves 3-5 at higher ratios of Fe~2 opens the possibility for an additional structure in which the metal could be forced onto the side chain.
H ~To determine the ratios of reactants in the complez at physio~" logical pH* continuous variation experiments similar to that described by Williams and Neilanda? were performed . Oae of these experiments is illustrated in Fig . 2 . Four similar experiments yielded peaks at ~Fe+ 2, / jFé +2J + epinephrine s 0 .33 indicating a ratio of one atom Fe+ 2 to two molecules of the hormone coiacideat
with structure I . When this ezperiment is performed at pH 5 .0 using 2 .10-3 M reactants a second peak appears at CFe+ 2J/CFe +2 + epinephrine, a 0.5 is addition to a peak at 0 .33 . The structure I type complex can be formed also at acid pH as well as at neutrality . +2 undoubtedly occurs so that a certain concenSome ozidation of Fe tration of unstable Fe+ 3 complez map be formned . The interpretation of the 0 .5 peak is not an obvious one . The effect of pH upon the absorption spectrum (Zeitss PMQII apectrophot~ometer) of the complez formed using molecular ratios
512
CHARACTERISTICS OF A FERROUS IRON COMPLEX
No . 10
of 2 molecules L-epinephrine to 1 molecule FeC12 in aqueous solution is shown in Fig . 3 . The 560 m~ complex is formed at acid and neutral pH. At pH 9 .55 a shift occurs towards the U .V, and this is believed to be due to the formation of oxidation products like adrenochrome .
Absorption spectra of complex formation in aqueous solutions of 2 molecules L-epinephrine : 1 molecule FeC12 . Hoth Lepinephrien and the ferrous salt show neglible absorbancy above 320 m~ . Paper electrophoresis using a closed strip method with CC14 as insulator$ and 0 .05 M acetate buffer at pH 5 .5 (3 .5 Y/cm and 1 .4 mA/cm width at 20°C) resulted in movement of the complex towards the cathode while epin®phrine hardly migrated from the origin. This confirmed the freedom of the secondary amine under these conditions and constitutes additional evidence for structure I . This worlt was supported by Research Grant A-4187 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U .S .P .H .S . The author is indebted to Prof . J .B . Neilands, University of California, for his aid in the interpretation of titration curves . References l . G . LITWACX and V . WOJCIECHOWSSI, Nature . Lond . ~, 1675 (1.959) . 2 . P . CHAIX, J . CHAWET and J . JEZEQUEL, Hioch~ i~ Biophys . Acta 4, 471 (1950) "
No . 10
CHARACTERISTICS OF A FERROUS IRON COMPLEX
513
3 . P . CHAIR, G.-A . MORIN and J . JEZEQUEL, Biochim . Biophys . Acta 472 (1950 4 . P . CHAIR and C . PELLAGET, Biochim. Hiophye . Acta ~, 462 (1953) " 5 . G . YOGELER, Arch . E, xp-Path . Phar=. 1~4, 281 (1940) . 6 . E .M. TRAUTNER and M . MESSER, Nature, Lond . ~, 31 (1952) . 7 . Y.R. WILLIAMS and J .B . NEILANDS, Arch . Biochim . Biophys . ~, 56 (1954) . 8 . R. MARSHAM and J .D . SMITH, Hiochem. J . ~2, 552 (1952) "
CHARACTERISTICS OF A FERROUS IRON COMPLEX OF L-EPINEPHRINE Gerald Litwack Biochemical Laboratory, Division of Cardiology, Philadelphia General Hospital and Department of Medicine, Graduate Sohool of Medicine, University of Pennsylvania, Philadelphia, Pa . (Received 24 August 1962) IN the course of studying the inhibition of the liver tyrosine oxidase system by L-epinephrine and analogs l the desirability of
prpearing a ferrous complex of this hormone became apparent . Although Although such a complex has been described empirically in stucües of the effects of cations and anions on .the the oxidation of 4 catecholamines2 and by Yogeler5 , no attempts have been reported to characterize the complex. The complex can be prepared as follows : molar amounts of Lepiaephrine and FeC12 (2 :1) are weighed into a beaker at 25°C,
about 30-60 ml distilled water are added per 10 mmoles FeC12 . The pH is rapidly adjusted to 7 .0 with a motor driven stirrer and the purple solution is stirred at room temperature for 5 min. Several
volumes of chilled acetone are added, the precipitate is filtered on a Huchner funnel with suction, and washings are made with cold acetone . The dark purple solid is dried in vacuo in a dessicator and is relatively stable in the dry form. Titratioas of L-epinephrine with various amounts of FeC12 are illustrated by Fig . l . Titratioas were carried out in aqueous solutions using the Leeds and Northrup line operated pH meter
(Model 7664) and a miniature glass silver chloride type electrode assembly . The titratioas were conducted in a nitrogen atmosphere at 25°C . The pH meter was standardized with Fisher buffers at pH 4 .0, 7 .0 and 9 .0 . Back titration of epinephrine revealed only very small changes in the titration curve so that decomposition of the hormone was minimized under these conditions . Comparison of curves 1 and 2 suggests that the secondary amino group of epinephrine is not involved is the complex so that the ionized phenolic group probably directs the positioning of the metal indicative of the structure on page 511 (structure I) . 509
510
CHARACTERISTICS OF A FERROUS IRON COMPLEX
No . 10
PH
FIG . 1 Titration curves of J .-epinephrine with increasing amounts of FeCl2 (under N2) . Curve 1 a 50 moles epinephrine ; curve 2 = 50 ~ moles epinephrine + 25 ~ moles FeC12 ; curve 3 a 50 p moles epinephrine + 50 p moles FeCl2 ; curve 4 = 50~ymoles epinephrine + 100 moles FeCl2 ; curve 5 = 50 fl moles epinephrine + 150 moles FeC12 .
r
r
FIG . 2
Result of a continuous variation experiment using 10 -3 M concentration of reactants, L-epinephrine and FeCl2 at 25oC . The initial pH a 7 .0 . Final pH values are as follows at the end of the experiment (for each point from left to right s 6 .85, 6 .80, 6 .75, 6 .75, 6 .75, 6 .6, 6 .5, 6 .4, 6 .35, 6 .25 . Inset shows the spectrum of the peak reaction .
No . 10
CHARACTERISTICS OF A FERROUS IRON COMPLEX
511
r
This configuration has been suggested previously for the borate complez6 althgqxgh moat hypotheses concerning complezea of epinephrine are based upon the ability of borate to complez with poly-
hydrosy compounds . The unusual naturß of curves 3-5 at higher ratios of Fe~2 opens the possibility for an additional structure in which the metal could be forced onto the side chain.
H ~To determine the ratios of reactants in the complez at physio~" logical pH* continuous variation experiments similar to that described by Williams and Neilanda? were performed . Oae of these experiments is illustrated in Fig . 2 . Four similar experiments yielded peaks at ~Fe+ 2, / jFé +2J + epinephrine s 0 .33 indicating a ratio of one atom Fe+ 2 to two molecules of the hormone coiacideat
with structure I . When this ezperiment is performed at pH 5 .0 using 2 .10-3 M reactants a second peak appears at CFe+ 2J/CFe +2 + epinephrine, a 0.5 is addition to a peak at 0 .33 . The structure I type complex can be formed also at acid pH as well as at neutrality . +2 undoubtedly occurs so that a certain concenSome ozidation of Fe tration of unstable Fe+ 3 complez map be formned . The interpretation of the 0 .5 peak is not an obvious one . The effect of pH upon the absorption spectrum (Zeitss PMQII apectrophot~ometer) of the complez formed using molecular ratios
512
CHARACTERISTICS OF A FERROUS IRON COMPLEX
No . 10
of 2 molecules L-epinephrine to 1 molecule FeC12 in aqueous solution is shown in Fig . 3 . The 560 m~ complex is formed at acid and neutral pH. At pH 9 .55 a shift occurs towards the U .V, and this is believed to be due to the formation of oxidation products like adrenochrome .
Absorption spectra of complex formation in aqueous solutions of 2 molecules L-epinephrine : 1 molecule FeC12 . Hoth Lepinephrien and the ferrous salt show neglible absorbancy above 320 m~ . Paper electrophoresis using a closed strip method with CC14 as insulator$ and 0 .05 M acetate buffer at pH 5 .5 (3 .5 Y/cm and 1 .4 mA/cm width at 20°C) resulted in movement of the complex towards the cathode while epin®phrine hardly migrated from the origin. This confirmed the freedom of the secondary amine under these conditions and constitutes additional evidence for structure I . This worlt was supported by Research Grant A-4187 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U .S .P .H .S . The author is indebted to Prof . J .B . Neilands, University of California, for his aid in the interpretation of titration curves . References l . G . LITWACX and V . WOJCIECHOWSSI, Nature . Lond . ~, 1675 (1.959) . 2 . P . CHAIX, J . CHAWET and J . JEZEQUEL, Hioch~ i~ Biophys . Acta 4, 471 (1950) "
No . 10
CHARACTERISTICS OF A FERROUS IRON COMPLEX
513
3 . P . CHAIR, G.-A . MORIN and J . JEZEQUEL, Biochim . Biophys . Acta 472 (1950 4 . P . CHAIR and C . PELLAGET, Biochim. Hiophye . Acta ~, 462 (1953) " 5 . G . YOGELER, Arch . E, xp-Path . Phar=. 1~4, 281 (1940) . 6 . E .M. TRAUTNER and M . MESSER, Nature, Lond . ~, 31 (1952) . 7 . Y.R. WILLIAMS and J .B . NEILANDS, Arch . Biochim . Biophys . ~, 56 (1954) . 8 . R. MARSHAM and J .D . SMITH, Hiochem. J . ~2, 552 (1952) "