70 BIOCHIMICA ET BIOPHVS1CA ACIA BBA 35167 IRON-TRANSFERRIN COMPLEX FORMATION JOHN ROSS, SHAUL KOCHWA AND LOUIS ROBERT WASSERMAN Department of Hematology, The l~lount Sinai School of 2VIedicine and The Mount Sinai Hospital, New York, N.Y. (U.S.A.) (Received June i2th, 1967) (Revised manuscript received September 25th, 1967) SUMMARY The rate of formation of the chelated Fe(III)-transferrin complex was examined using Fe 3+ and Fe 2+. Fe 2+ salts show smooth stoichiometric reaction above pH 6.5 and the rate increases with rise in pH. The reaction rate is unaffected by the presence of reducing agents such as hydrazine salts but is increased greatly by the action of oxidizing agents such as equivalent amounts of KaFe(CN)~ at pH 5.4. and above. Fe 3+ salts show initial high rates of complex formation at pH 5.o and above but the yield of complex is less than theoretical and tends to decrease with rise in pH value, probably due to competitive hydrolysis of the Fe a+ salt. Because of this difference in behavior of Fe 3+ and Fe 2+ salts, titration at constant pH of the reaction mixture was reexamined. The formation of the chelated complex is accompanied by liberation of approx. 3 protons per Fe atom bound when Fe 2+ salts are used. The same number of protons was titrated with Fe 3+ salts but since only a fraction of the complex was formed correlation could not be made. Parallel results were obtained with conalbumin. INTRODUCTION The combination of Fe with transferrin (siderophilin) and also with conalbuminl, 2 has been studied by many investigators 3. The salmon pink colored compound, ab- sorption maximum 47 ° m#, contains 2 Fe atoms per molecule of protein. Studies of the electron spin resonance spectra a, the electrophoretic mobility and proton relaxation rate 5 have indicated that the Fe in combination with transferrin is chelated in the ferric state at two equivalent and independent sites. Although the complex that is formed from Fe 2+ is not different chemically or spectrometrically from that prepared from Fe a+ salts 6-s, there is a conflict of opinion as to which form of Fe is more efficient regarding rate and completeness of reaction. In the present study the yield and rate of formation of the colored complex, Biochim. B,iophys. Ac!a, 154 (1968) 70-77
Transcript
70 BIOCHIMICA ET BIOPHVS1CA ACIA
BBA 35167
I R O N - T R A N S F E R R I N C O M P L E X F O R M A T I O N
JOHN ROSS, SHAUL KOCHWA AND LOUIS ROBERT WASSERMAN Department of Hematology, The l~lount Sinai School of 2VIedicine and The Mount Sinai Hospital, New York, N . Y . (U.S.A.)
(Received June i2th, 1967) (Revised manuscript received September 25th, 1967)
SUMMARY
The ra te of format ion of the chela ted F e ( I I I ) - t r a n s f e r r i n complex was examined using Fe 3+ and Fe 2+.
Fe 2+ sal ts show smooth s to ichiometr ic react ion above p H 6.5 and the ra te increases wi th rise in pH. The react ion ra te is unaffected b y the presence of reducing agents such as hydraz ine sal ts bu t is increased g rea t ly b y the ac t ion of oxidizing agents such as equiva lent amounts of KaFe(CN)~ at p H 5.4. and above.
Fe 3+ sal ts show ini t ia l high ra tes of complex format ion a t p H 5.o and above bu t the yield of complex is less than theore t ica l and tends to decrease wi th rise in p H value, p robab ly due to compet i t ive hydro lys i s of the Fe a+ salt .
Because of this difference in behav ior of Fe 3+ and Fe 2+ salts, t i t r a t ion at cons tant p H of the react ion mix tu re was reexamined. The format ion of the chela ted complex is accompanied b y l ibera t ion of approx. 3 protons per Fe a tom bound when Fe 2+ sal ts are used. The same number of pro tons was t i t r a t e d with Fe 3+ salts bu t since only a f ract ion of the complex was formed correlat ion could not be made.
Para l le l results were ob ta ined with conalbumin.
INTRODUCTION
The combina t ion of Fe wi th t ransferr in (siderophilin) and also wi th conalbuminl , 2 has been s tud ied b y m a n y inves t iga tors 3. The sa lmon p ink colored compound, ab- sorpt ion m a x i m u m 47 ° m#, conta ins 2 Fe a toms per molecule of protein. Studies of the electron spin resonance spec t ra a, the e lec t rophoret ic mob i l i t y and pro ton re laxa t ion ra te 5 have ind ica ted t ha t the Fe in combina t ion wi th t ransfer r in is che la ted in the ferric s ta te a t two equiva lent and independen t sites. Al though the complex t ha t is formed from Fe 2+ is not different chemical ly or spec t romet r ica l ly from tha t p repared from Fe a+ salts 6-s, there is a conflict of opinion as to which form of Fe is more efficient regarding ra te and completeness of reaction.
In the present s tudy the yield and ra te of format ion of the colored complex,
Biochim. B,iophys. Ac!a, 154 (1968) 70-77
IRON-TRANSFERRIN COMPLEX FORMATION 71
as measured by absorption at 47 ° m/~, was examined using Fe 3+ and Fe 2+ salts with transferrin and conalbumin over a range of pH in the presence of buffer solutions. The liberation of H+ when Fe 2+ and Fe 3+ salts combined with these proteins was measured by titration at constant pH.
EXPERIMENTAL PROCEDURE
Materials Transferrin was prepared from human plasma by procedures previously de-
scribed 9,1°. With large batch preparations, transferrin in yields of up to 80% was obtained and the homogeneity was examined by starch gel, acrylamide gel, and immunoelectrophoresis. Some preparations showed the presence of about 5% of v-G globulin of/3 mobility (as determined by immunoelectrophoresis and Hyland (Hyland Laboratories, Los Angeles, Calif.) immunoplates) which was disregarded in the present study. Fe was removed from these preparations by acidifying to pH 4.0-4.5 with I.O M sodium citrate-citric acid buffer and immediately passing over Amberlite IRA 4Ol ion exchange resin in the acetate form. The Fe free eluate was adjusted with o. i MNaOH to pH 7.2, dialyzed to remove sodium acetate and equilibrated against the buffer to be finally used.
Conalbumin was crystalline material obtained commercially (Nutritional Biochemicals Corp., Cleveland, Ohio).
All buffers and reagents were prepared using boiled out, deionized water cooled in a current of N 2 t o eliminate dissolved 02.
(NH4)zFe(SOa)2"6H20 and NH4Fe(SOa) 2 • I2H20 were used for standard Fe(II) or Fe(III) solutions. These were prepared immediately before use and stabilized with I ml of I M HCI per IOO ml of reagent. Fe(NO3) 3 and F e S Q solutions for unbuffered titration experiments were prepared from selected crystals and used immediately.
NaHCQ, Tris-bicarbonate and KHCO 3 solutions were prepared from o.I M NaHCO3, o.I M Tris and o.I M K2CO 3 by passing CO 2 gas into the aqueous solutions at 25 ° until the desired pH was obtained.
All reagents were of analytical grade.
Methods
Rate measurements. The rate of binding of Fe(II) to apotransferrin was measured by recording at room temperature the rate of change in absorbance at 47 ° m# with a Cary spectrophotometer Model 14 (Applied Physics Corporation, Monrovia, Calif.) as depicted in Fig. I. A solution of protein (io 50 rag) in 2.5 ml ofo . I M NaC1 (pH 7.2) was mixed with 0.5 ml of o.I M NaHCO 3 (pH 7.2) in a IO mm cuvette, and placed in the cell compartment at 25 °. A small volume (20 #1) containing 4 #g of iron was placed on a polyethylene spatula, added with mixing, and a stopwatch was started (A). Recording was started within 5 sec of addition (B) and by noting I-rain intervals (C) from the time of mixing, zero time was obtained by extrapolation. Recording was continued for 5 min or until no further change in absorbance showed the reaction to be completed. To obtain a measure of the total apotransferrin initially present, further additions of Fe were made such as at A 'B 'C ' until excess was present as shown by the absence of any further increment in absorbance.
Plotting the log of the ratio of concentrations of uncombined reactants (of
Biochim. Biophys. Acta, i54 (I968) 70-77
72 ,1- ROSS, S. KOCHWA, L. R. \VASSERMAN
7 2 21 ? [ ................
~,~
s :
t : ig. I. Fe t r a n s f c r r i n corn )lex f o r m a t i o n t r a c i n g , re m g a p o t r a n s f e r r i n in 2.5 ml o . i M Tris HC1 buf fe r + o. 5 m l o . I M Tr i s b i c a r b o n a t e ( p l t 7.2). A d d i t i o n of 4 / t g Fee+ at A; recording started a t B; t - m i n v a l u e a t C. T i m e scale i d iv . == 5 sec. A b s o r b a n c e recording at 47 ° m # on ordinato~ (full scale = o. too a b s o r b a n c e uni t s ) .
apotransferrin (Tr) and free Fe) expressed in mmoles/1 against t ime in sec gave an approximate ly straight line relation for most of the reaction. This suggested that for the overall reaction Tr + 2 Fe -~ TrF% an arbitrary second order constant Kt could be calculated using the relationship
2.3o 3 b(a .r) K t log
t ( za - b) a(b 2.r)
where a and b are initial concentrations of the reactants and x is the concentration of product formed at t ime t, the rate being expressed in mmoles/1 per sec. For calculations the molecular weight was taken as 9 ° ooo for transferrin n and 76 600 for conalbumin 12, and ext inct ion values at 47o m # o f E 12';~l 0.57 ° (ref. II ) and EI12L = 0.620 (ref. I3), respectively, at saturation.
Titrations of reaction mixture. Radiometer equipment including a TTT1 titrator with P H A 63oT scale expander SBR2c Titigraph and S B U I a syringe buret was used for H÷ titration. The reaction was carried out at 25 ° in 0.2 M KC1 for apoconalbumin and o.I M KC1 for apotransferrin. The protein (30-40 mg) in 6 ml of unbuffered KCI solution containing IOO ,ul of 5 mM solution of KHCOz was brought to various pH values between 5.5 and 8.5 by addition of o .o i M acid or base. To this solution were added IOO/~1 of 2.5 mM Fe solution, and the vo lume of o .o i M K O H required to maintain the pH at the original value was measured. A small stream of N~ gas was passed over the solution while the latter was stirred magnetical ly. After 3 min a sample was removed from the cell and the increase in absorption at 47 ° m # measured with a Beckman D U spectrophotometer.
Biochim. Biophys. Acta, t54 (1968) 70 77
IRON-TRANSFERRIN COMPLEX FORMATION 73
RESULTS
Complex formation with Fe(II) Apot rans fe r r in in o.I M NaC1 at p H 7 and above in the presence of excess
NaHCO 3 combined with Fe( I I ) to give the theore t ica l yield of complex in less than IO rain as measured b y the absorbance increase at 47 ° mff. When sodium ace ta te or Tris-HC1 was subs t i tu t ed for NaC1, theore t ica l yields were ob ta ined wi th react ion ra tes of s imilar value for a given p H with all three buffers. Accordingly , the sodium ace ta te and Tris-HC1 buffers were used for ra te measurements over the range p H 5-8.
Unde r condi t ions of cons tan t p H and buffer s t rength the react ion ra te of color fo rmat ion with Fe( I I ) rises sharp ly as the molar ra t io of b ica rbona te to Fe( I I ) is increased to a 5o-fold molar excess and for higher ra t ios remains ahnost at a level value (Fig. 2A). A molar excess of approx. 4oo-fold was used dur ing subsequent exper iments to suppress ra te va r i a t ion due to b icarbonate .
1.5 f A
wO.5o
I (I: 0(~ I O0 400 800
MOLAR RATIO C02:Fe
~1,5 t B
0 5b */" [ o 0 0.1 0,2 0.3 0,6 0.5 0,6 0.7 0.8
M TRIS-HCL
/ / ,/
/ ®
i o L ~ I I I ~ _ _ _ _ ~ ~I
70 75
pH
Fig. 2A. Effect of change in mola r ra t io of CO2:Fe2~ on the reac t ion rate. 4 fig Fee+ added to i o mg apo t rans fe r r in in o. i ~/[ Tris HC1 troffer wi th increas ing a m o u n t s of o. i M Tr i s -b i ca rbona te . Tota l vol. 3 ml; p H 7.26-7.3o. B. Effect of va r i a t i on of buffer concen t r a t ion on the reac t ion rate. 4 fig Fe2+ added to IO mg apo t rans fe r r in in t o t a l vol. of 3 ml (o. 4 1hi o. l M Tris b i ca rbona te + 2. 5 ml Tr i s -HCI o.o 5 i .o M) p H 7.26-7.28. , apocona lbumin .
Fig. 3. Effect of pH on the reac t ion rate. 4 f i g Fe2+ added to mg apo t rans fe r r in in 2. 5 ml o. i l~l Tris-HC1 buffer a t p H 7.o, 7.i3, 7.i8, 7.37 con ta in ing o. 5 ml o.E M Tris b icarbona te . apo t r ans fe r r in ; , a p o c o n a l b u m i n , [~, o.I )/I NaC1; 0 , o. i 51 sod ium ace t a t e ; /~, o . i 1~'I imidazole hydroch lor ide s u b s t i t u t e d for Tris w i th apot ransfer r in .
Varia t ion in buffer concent ra t ion of Tris-HC1 showed tha t the ra te of color fo rmat ion with apot ransfe r r in at p H 7.26 exh ib i ted a shallow min imum at 0.2 M buffer concent ra t ion (Fig. 2B). Apocona lbumin showed a sl ight ra te increase wi th rise in buffer concent ra t ion . In subsequent measurements o.I M Tris-HC1 was used with apo t rans fe r r in and 0.2 M Tris HC1 wi th apoconalbmnin .
In Table I A d a t a are given of one ra te exper iment of the react ion of Fe ( I I ) wi th apo t rans fe r r in in the presence of excess ca rbona te and buffer. The values of K for a series of points show some var ia t ion and poss ibly a slight rise as the react ion proceeds towards complet ion. When enough apot ransfe r r in was used to pe rmi t successive
Biochim. Biophys. Acta, 154 (1968) 70-77
74 j . ROS%, S. KOCH~NA, L. R. \VASSERMAN
TABLE IA
R A T E O F R E A C T I O N O F F e ( [ I ) \ V I T I I A P O T R A N S F E R R I N
A solut ion of 8 .oFg Fe(l l ) as (NH~),aFe (SO~)., was added to 6.349 mg apotransferr in (Tr) dis- so lved in o.I M Tris HC1 buffer till 7.2 w h i c h was 0.05 M in Tr i s - carbonate . F inal v o l u m e was "2.991 ml.
t A at TrFe 2 Tr Fe I f (sec) 47 ° m~ filrmed ( ~ M ) ( ~ M ) (mmoIes/l
additions of (NH4)oFe(SO4) 2 solution (4/~g Fe), the calculated reaction rate constant also showed some irregular variation over the range of I to IO increments of Fe (Table IB). However, these variations in K are small compared with the large and consistent effect of the change of the pH of the substrate as shown in Fig. 3- The value of K is quite low at pH 6.5 and rises sharply over the range pH 7.o-7.5.
Above pH 6.5 the rate of color formation is not inhibited or decreased by the presence of molecular excess of such reducing ager~ts as salts of hydrazine or hydroxyl- amine. The prior addition of an equivalent amount of Fe(CN)6 3- gives immediate and complete reaction at pH 5.4 and above.
Rate of reaction with Fe a~ salts Fe 3+ salts combine with apotransferrin or apoconalbumin over a wide pH range
(4.8-1o.o). Although the initial rate of complex formation is much faster than with Fe 2+ salts, the reaction stops at approx. 70-85% yield. No satisfactory rate constants
TABLE 1B
REACTION RATES FOR SUCCESSIVE ADDITIONS OF Fe ~+ TO APOTRANSFERRIN E a c h addi t ion of Fe 2+ was sufficient to s a t u r a t e IO% of the ini t ia l b inding c a p a c i t y of trans- ferrm (mol. wt. 9o ooo), Tris HC1 buffer, p H 7.15. Addi t ions were m a d e at i o - m i n intervals .
Fe e+ added Apo t rans~rr in K~5 (lt21I) calculated (mmoles/ l
Fig. 4- Comporison of reac t ions of Fe 2+ and Fe 3+ on apo t rans fe r r in by change of absorbance a t 47 ° mu wi th t ime when (a) 4/*g Fe2+ added to io mg apo t rans fe r r in in so lu t ion in 2. 5 ml o t o. i M NaCI pH 7.2 + 0. 5 ml of o. i ~/[ NaHCO 3 ;(b) 4/*g Fe3* added to io mg apo t rans fe r r in in so lu t ion in 2. 5 nil o fo . t M NaC1 pH 7.z + o. 5 ml o f o . [ M NaHCO.~ .
were calculated. The reaction curves are compared in Fig. 4 where equal amounts of Fe(III ) and Fe(II) are mixed with aliquots of excess apotransferrin in o.I M NaCI at pH 7.2 and the change in absorbance is shown for the first 2 min of reaction. Complete reaction would give an absorbance of o.o6, and with Fe 3+ salts this value is not reached even after 6o min reaction t ime.
T A B L E I I
C O M P A R I S O N OF A L K A L I T I T R A T I O N A N D Y I E L D OF I R O N C O M P L E X D U R I N G I N T E R A C T I O N OF ~ ' e 3+
A N D F& + SALTS Vv'ITH T R A N S F E R R I N A N D C O N A L B U M I N ( B O T H I R O N F R E E )
Reaction mixture
IOo/zl Fe(NOa) a in 6 ml 0.2 M KC1 (pH 7-5) ioo / ,1 FeSO 4 in 6 ml 0.2 M KC1 (pH 7.5) ioo ,u l Fe(NO~)3 in 6 m] o.i M KC1 (pH 5-5) i o o # l FeSO~ in 6 ml o. i M KGI (pH 5..5)
Titration Yield from ( equiv absor(~ance
o~ KOH) (/o)
3.2 2 . 2
3.6 o
IOO/*l Fe(NO~)~ in 3 ml o.2 M KC1 neu t ra l i zed to pH 7-5 + cona lbumin in 3 ml 0.2 M KCl (pH 7.5)
i o o / A FeSO 4 in 3 ml o.2 M I(C1 neu t ra l i zed to pH 7.5 + cona lbumin in 3 ml 0.2 M KC1 (pH 7.5)
i o o u l Fe(NO3) a in 3 ml o . i M KC1 neu t ra l i zed to pH 7..5 + t ransfer r in ~n 3 ml o.I M KCI (pH 7.5)
1oo/ , l FeSO, in 3 ml o.1 M NC1 neu t ra l i zed to pH 7.,5 + t r ans fe r r in in 3 ml o . i M KC] (pH 7-5)
Cona lbumin in 6 ml o.2 M KC1 (pH 7.59) + ioo ltl Fe (NO3) 3 Cona lbumin in 6 ml o.2 M KCI (pH 8.o[) ~ ioo ul FeSO,. Cona lbumin in 6 ml o.2 M KC1 (pH 8.o) + ioo fil 2. 5 mM
K3Fe(CN~ . + ioo [d FeSO 4
ml o.I M KC1 (pH 7.97) + IOO/~1 Fe(NO3) a ml o.I M KCI (pH 7-97) + i o o / , l FeSO 4 ml o. i M KCl (pH 7.97) + lOO/Zl K3Fe(CN)G +
Transfe r r in in 6 Trans fe r r in in 6 Trans fe r r in in 6
ioo/~1 FeSO 4
ml o. i M KCI (pH 5.5) + ioo/*1 Fe(NO3) ~ ml o. i M KC1 (pH 5.5) + ioo F1 2.5 mM ioo ul FeSO,
Trans fe r r in in 6 Trans fe r r in in 6
K~Fe(CN)~ +
o I I
o IO
o 5
o 4
3 .2 47 2.8 9,5
4.1 too
3.4 51 3 .1 95
4.0 98
3.0 48
3.3 86
Biochim. Biophys. Acta, 154 (t968) 70-77
7 0 J. ROSS, S. KOCHWA, L. R. WASSERMAN
Titrations of reaction at constant pH (Table II) At pH 7.5 Fe(OH)3 or Fe(OH)2 does not combine with apotransferrin or apo-
conalbumin since the iinmediate addition of solutions of these proteins at pH 7.5 to previously neutralized ferrous or ferric iron solutions shows no change in pH and very slight increase in absorbance.
When Fe a+ or Fe 2 ~ salts are added to solutions of a IO molar excess of protein at pH 7.5 between 2 and 3 equiv of alkali are titrated per mole of Fe, which is in agree- ment with values reported~,~2, ~5. However, the yield of complex in 3 nfin as measured by absorbance increase was approx. 5o~)o with the F ~+ salt and 95% with the Fe 2+ salt.
At pH 5.5 Fe 3+ salts with the protein solution give three titratable equivalents but a complex formation yield of less than theoretical as measured by absorbance i n c r e a s e .
The Fe 2+ salt at pH 5.5 in the presence of apotransferrin shows no indication of reaction by color formation or pH change. When Fe 2+ salt is added to the protein solution containing an equivalent of KaFe(CN)6 at pH 5.5 and 8.o, there is rapid production of 85% and lOO% of colored complex with titration of 3.3 and 4.o equiv, respectively. Since the reduction of K3Fe(CN) 6 to K4Fe(CN)6 requires I equiv of alkali, protein complex formation yields 2.3 and 3.o equiv H+ per mole, respectively.
DISCUSSION
Purified solutions of apotransferrin and apoconalbmnin combine with Fe 2+ following approximately a second order reaction form in the presence of excess bicar- bonate over the pH range 6.5-8.o. Since the presence of excess of such reducing agents as hydrazine does not interfere with the rate of color complex formation, the conversion from the ferrous to the ferric state is not necessarily dependent on autoxidizing agents other than those bound to or part of the transferrin molecule.
The pH dependent autoxidation reaction 9 is probably a limiting factor in the stepwise formation of the chelated complex from Fe 2+ since other observations indicate that Fe 3+ forms the complex at very high reaction rates over a wide pH range. It has been shown that apotransferrin and apoconalbumin form stable complex with divalent metals copper and zinc 11,14. Under anaerobic conditions SCHADE 15 noted that when ferrous salt was added to transferrin with bicarbonate present, an absorption peak at 430 m# was observed, suggesting the formation of a yellow ferrous complexes. Similar be- havior was not obtained with conalbumin. Although no conclusive evidence is available that divalent iron is bound to apotransferrin and then autoxidized to the trivalent state, a number of observations are consistent with this view. Histidine promotes the autoxidation of Fe 2+ (ref. 19) and imidazole behaves similarly above pH 6.5, which is the pH range over which purified apotransferrin exhibits the autoxidation reaction in combining with ferrous iron. Involvement of two imidazole groups at each chelating site is indicated by difference between titration curves of apotransferrin and Fe a+ saturated transferrin in the pH range 8.0 to 5.5 (ref. 16) and also by electron spin resonance spectra 4. It would therefore appear that the proteins transferrin and conal- bumin, may have an internal structure of imidazole groups adjacent to tyrosine residues facilitating autoxidation of Fe 2+ at pH ranges above 6.5. Imidazole or histidine present in the reaction solution as buffers do not affect the reaction rate to any marked degree (Fig. 3).
Biochim. Biophys. Acta, 154 (1968) 70-77
I R O N - T R A N S F E R R I N C O M P L E X F O R M A T I O N 77
Fe 3+ salts combine with solutions of apotransferrin and apoconalbumin at rates that are initially higher than with the F d + and over a much wider pH range. However, the reaction is not very reproducible and the variable yield of colored complex was less than theoretical, probably because of the partial formation of non-reactive Fe(OH)a formed by the competitive hydrolysis of the Fe a+ salt.
Addition of an equivalent amount of a mild oxidizing agent such as Fe(CN)6 a before or after the introduction of the Fe 2+ to the protein solution gives an almost immediate theoretical yield of complex over the range of pH 5-8. In this procedure hydrolysis of Fe 3÷ is avoided.
It has been reported that titrations at constant pH show that between 2 and 3 protons are liberated per atom of iron when the transferrin or conalbumin colored complex is formed4,12,15. The yields of complex after 3 to io min, as measured by absorbance increase, permit reasonable correlation of titration values with binding to the protein using Fe SO 4 at pH 8 and also using ferrous sulfate in the presence of Fe(CN)~ a- at pH 5.5- When Fe(NO3) a or NH 4 Fe(SO4) 2 is used, the results are ambigu- ous because the color development is much less than theoretical. There is no evidence that Fe(OH)3 which may be formed will combine with the protein in the time required for titration.
A C K N O W L E D G E M E N T S
The careful technical assistance of Miss MARILYN BROWNELL and Mrs. ELLEN WOLF during this investigation is gratefully appreciated.
This study was supported in part by U.S. Public Health Service Grants AM 04434 from the National Institute of Arthritis and Metabolic Diseases and Grant CA 04457 from the National Cancer Institute, and the Albert A. List, Frederick Maehlin and Anna Ruth Lowenberg Funds.
R E F E R E N C E S
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203. .5 P . AISEN, A. LEIBMAN AND H . A. REICH, J. Biol. Chem., 241 (19661 1666. 6 G. E. CARTWRIGHT AND M. M. WINTROBE, J. Clin. Invest., 28 (19491 86. 7 C. E . RATH AND C. A. F I N C H J. Clin. Invest., 28 (19491 79. 8 W . N. M. RAMSAY, Clin. Chim. Aeta, 2 (19571 221. 9 B. A. KOECHLIN, J. Am. Chem. Soc., 74 (10521 2749 .
i o i . L. NAGLER, S. I(OCH~,VA AND L. R . WASSERMAN, Proc. Soc. Exptl. Biol. Ned., 8 (19621 746. I I D. M. SURGENOR, B. A. KOECHLIN AND L. E. STRONG, J. Clin. I~west., 28 (19491 73- 12 R . C. WARNER ANn I. WEBER, J. Am. Chem. Sot., 75 (19531 5094 . 13 R . C. \ gARNER ANn I. \ATEBER, J. Biol. Chem., 191 (19511 173. 14 C. G. HOLMBERG AND C. B. LAURELL, Acta Chim. Scand., i (1947) 944. 15 A. L. SCHAI)E, R . W . REINHART AND g . LEVY, Arch. Biochem., 20 (19491 17o. 16 E. E. HAZEN, JR. , P h . D. Thes i s , H a r v a r d U n i v e r s i t y , 1962. 17 J . Z. HEARON, D. BURK AND A. g . SCHADE, J. Natl. Cancer Inst., 9 (1948) 337-
