- Email: [email protected]
A column method for deoxygenating human hemoglobin
AN.ILYTI(‘AL
BIOCHEMISTHY
A Column
Method
DOSALD FJVUI
15, -304
(1966)
for Deoxygenating
Human
Hemoglobin’
F. HOELZL WALLACH, MITCHELL AND CHARLES A. JANEWAY
H. GAIL,
(he Department of Biological and ProteirL Foundation, Received
Chemistry, Boston, October
Haward Medical Massachusetts
School
29, 1965
In studying the kinetics of the reaction of human hemoglobin with oxygen by the continuous flow method of Hartridge and Roughton ( 1) , it became necessary to prepare large volumes of deoxygenated hemoglobin. Conventional methods involving physical equilibration of hemoglobin solutions in oxygen-free atmospheres require prolonged periods of time for full deoxygenation and so risk the hazard of methemoglobin formation. For these reasons we have developed a new deoxygenation method. A column containing a mixed-bed ion-exchange resin is partly loaded with sodium dithionitc, a powerful reducing agent for oxygen (Na,S,( ), + 02 + H,O e NaHSO, + NaHSO,) (2). The dithionite, which is confined to a 4-6 cm layer on the column of resin and is localized primarily within bhe resin beads, maintains the partial pressure of oxygen (PO.‘) of the aqueous phase at less than I mm Hg. Oxyhemoglobin dissociates as it passes through this region of low ~0,. Oxidation products of sodium dithionite, which include sulfate, sulfite, and hydrogen ions, remain bound to the resin. The column also maintains the pH of the solution at about 7 and removes ionic constituents introduced in the original oxyhemoglobin solution. Thus a deoxygenated hemoglobin solution of low ionic strength is obtained. METHODS
Preparation
of the Hentoglobin
Solution
Forty milliliters of packed human red cells are suspended in an equal volume of 0.15 N NaC1 and sedimented by centrifugation at 12,800 X g (Lourdes Betafuge, 9RA rotor, 5°C). This washing procedure is repeated twice. The packed red cells (ca. 30 ml) are then lysed by adding them to 8 vol of 0.01 M phosphate buffer at pH 6. This suspension is brought to ‘Supported VSPHS.
by
grants
CA
0738-02,
lSOl-FR-5381-01, 300
and
5 T5
GM.5144
from
the
HEAMOGLOBISDEOXYGESAl’IoS
301
pH 5.8 to cause :~ggregation of the stro1lia. (,infficicnt tinic 1riust be allowed for aggregation; otherwise stro1lia will k1ggregate on the resin. Ten minutca at’ room teitiper:~ture was :ldequntc~ for our protein conccntrnti0nG.j Tl1e susperkon i.5 then spun ;It 28.700 X (/ for IO Inin. The ~supernatant ia derantctl. brought to pH 8 with 1.O 31 Nat )H ( rapid Fiwh he1~ioglot~in stoc*E; constirringj ! and immetiitttc~ly refrigen1tcd. taining about 2 meq of heme per liter i> prepnwd in this 1n;tnncr e\-er> day. Piepam fion of the c’olu wn Tlrc ~sin: Bio-Rad mixed-bed resin XC: 501-SS(U) is cn~ployetl. -ig 501-581 D) is a dye-containing mixed ionic resin n-11ich contains a basic resin ISG l-X8 OH-) with benzenc-K+-(,C’H::),s groups imbedded on a polystyrene matrix wit11 8% divinylbenzene cross linkages and an equivalent amount of acidic resin (AG 5OW-18 I+) with benzene--SO,groups imbedclecl on an iclenticnl resin atr11cturc. The resins :Irc analytical grade and are 20-50 mesh (Rio-Rx1 catalog number 47020). This misedbed resin allows rapid flop rates, does not lcnk dithionite, and yields an ion-free. neutral effluent. Its dye is a useful indicator of remaining columnbinding capacity. Glass columns I 2.5 cm id. X 30 cm lwight) are packed at t-he hott.om with a 1 cm Ixyer of glnss pool. TIN columns ztre filled with water. and 8 ml of resin is added with stirring to assure uniform settling of t,he beads. A reservoir containing distilled water feeds into the tops of t’lie columns, and the airtight tops also 11xve an inlet for introducing hemoglobin or clithionitc. The column:: arc conipletcly filled with wntct and re*in so that, there are no gas bubbles. The effluent, is collected in an osggcn-free container. C’ltnrgin~~ of the col~mt~ with. clithionite: The calculated sodium dithionite requirement is 0.114 gm/liter of H,O in equilibrium with ambient air and 0.94 gin/liter of 2 mcq (in respect to heme groups) stock hemoglobin eolut.ion. ;Z twofold esccss of the day’s calculated dithionite requirement is int1,oduced onto the column, and 1 liter of water passed (20 ml/min) through the column to sweep the dithionite onto the resin and deoxygcnnte the entire coluiim; 500 ml of H,O is sufficient to apply t.he new dithionite charge on subsequent days providing the system remains closed to xir. OPERATION
Operation
OF
THE
COLUMS
of the Colrrmn
Normally two colu~zmsare employed, one for deosygenating water and one for drosygenating hemoglobin. Two liters of deosygenated water and 60 1111 of 2 meq Ihcnle b wo11psJstock lie1noglobin solution are neces-
302
WALLACH,
GAIL,
Ah-D
JANEWAY
sarv for our rapid flop experiments Dithionite (0.5 gin) is dissolved in 20 ml of water and this solution added to the hemoglobin column. ,\fter washing with I liter of water (20 ml/min) , 60 ml of stock hemoglol)in solution is int’roduced and followed by natcr i20 ml,/min) until all the deoxyliemoglobin is elutctl. The hemoglobin is collected in an oxygen-free container. The color of the effluent is deep purple (in contradistinction to the initial oxyhemoglobin solutionj, and the effluent ~0, as meusured on an Inst’rumentation Laboratories model 125A oxygen electrode system is les;sthan 1 1nm Hg. Dcoxygenated water is prepared similarI;\- on the other rolumn. For our purposes the capacit?- of the hemoglobin col~mm suffices for t8hree deoxygcnation, q and that of the water column for folir deoxygcnatione. RESULTS C’onductivity and pH of the deoxyyenated efluent: =In Arthur H. Thomas Co. model RCM 15Bl conductivity meter was used, and the conductivity of the effluent, was found to be 4.9 X lo-‘; Inho-cn-‘. The same instrument yielded I-alues of 2.6 X IO-‘: mho-en-l for water l~~cd over a Bar&cad model BD1 deionizing column and 1.44 X 10 ’ mhoCl11 ’ for a 0.01 31 KC1 solution (all readings at 20°C). The effluent thcrcfore has an ionic strengtlt of less t.hwn 5 X lo-“. The effluent pH W:W between 6.8 and 7.2 iInstrumentat,ion T,aboratory Inc. model 135.4 plI met8erwith catalog number 14040 glass electrode). These findings taken tog&her establish that the effluent contains neither significant quantities of ionic contaminant:: introduced in the stock osyltemoglobin solution no1 ditliionite or oxidation I)roduct. q of dithionite (,including sulfate, siilfite, and associated hydrogen ions). The protein: The main objection to the UPCof dithionite for deosygcnating hemoglobin has heen that it may denature human hemoglobin and produrc other hrllle piglllents (3). WC thcrcforr tried to detect abnonnnl pigments or alterations in the processed hemoglobin yljcctrophotoinctrically. ( 1) (~‘ompnriron of stock or!/hemoglobin with reosygentrted processed hemo~globin. A sample of deoxygenated human hemoglobin was reosy-
genat,cd by diluting with a large volume of 0.15 1V NaCl solution wltich had hccn ljreviously equilibrated with room air. The spectrum of the solution was th(bn collq~trcd with that of a similarly diluted solution of stock oxyht~moglohin. this control solution never haying been deoxygenated. The absorption spectra of the two samples as recorded on a Perkin-Elmer model 202 spectrophotometer were identical in t,he region 350-740 m/r. and had absorption maxima characteristic of oxyhemoglobin at 415, 543, and 577 mu.
HERIOGLOBIX
303
DEOXYGENATIOX
(2) Spectrum of the deo.ryhernogZobi~~ solution. l>eosygenated hemoglobin solution w:~s collected in an air-free flow cell, and the absorbance in tdw Poret region was determined on a Beckman nK q~ectropl~otometcr. This >olution was found to haw the spectral characteristic!: of cleosyhemoglobin as reported by Bcnesch et cd. (,-I). Tllere ww no “shouldet~” in the 408-412 m+ region, indicating t,hat methemoglobin was not present, in quantities exceeding 5% of the total heme pigment. This conclusion is confirmed by the fact that, upon reoxygenwtion of t#he hemoglobin the alwrption maximum in the Soret region remained at 415 m,~. Thr following table contains our obeerred and literature values for h,,,;,,. valuebtar XI,>%,. Osyhen~oglobin (control) Deosyhemoglobin Reoxygc~nated h~rnoglol~in
Olwrwd.
415 430 415
,,I+
13erNwh.
I,,$4
415 430
13t Biochemiccrl tests of the protein. The deoxyhemoglobin prepared 1~~ this method was used to demonstrate the Bohr effect and to study osygcnation kinetic+, and it seemed to be functioning properly. To demonstrate the Bohr effect, the difference in H+ binding by deoxyhcmoglobin and oxyhemoglobin was determined using the llwthod of hntonini et trl. (5). Our results agree with those of hntonini et rrl.. yic~ltling :I pH change of --0.20 pH unit on oxygenation of a 1.0 gm s deoxyhemoglobin solution containing 0.3 M N:iCl at 20°C. Preliminary StuclieSof oxygenation kinetics using t.he continuous flow method of Hartridge :ind Boughton (1 ) yield I-nlues greater than 2 X I@;W’ wc ’ for the o~chr-all :woc.i:~tioii coii.~t;~iit (k’j at. 20°C anal p1-T7.0. ;\n estiniatc of k’ lw(v1 on the disso&t.ion const,ant, (k) as tletcrmined by Dal&l ant1 ( )‘Brivn (6) aml the equilil)rium constant ah clctcl,niinccl by Lystclr ( 7 1 is 5 X 10, W’ st’c I. The fact that 0111‘ob~trvcd k’ is consi&nt with the theoretical estimate sugg(‘t: that our deos~henloglol)ill preparation is not clwat,urcd or nltcwd and that, the efllucnt cloc>bnot wntain ~lol~gll tlitllionitc~ to :tltcLr osygcwltion kinctiw. DISCUSSIOK
Thih bystem ha:: many advantages. It is easy to set up and to use, and i* lwtll l,apid and reliable. The duration and extent of contact of the hcnwglobin n-it11 the dithionite is minimal, since most. of the dithionite ir bouml inside the resin beads and is in a narrow band on the column. The :~ole of dit,hionite is to provide an oxygen-poor aqueous phase in n-hicll the oxyhemoglobin dissociates. Furthermore, because the concentration of hemoglobin on the column is high (ea. 0.5 mM). and because no oxygen is available in the column for peroxide formation, there is little ridl< of forming abnormal hence pigments (3). The effluent is also free of
304
WALLACH:
dithionite. oxidation l)rotlucts duced with the oxyheiiioglobin
GAIL,
Ah-D
JAKEWAP
of dithionite, and ionic contaminants stock solution.
intro-
SUMMARY
The mixed-bed resin column charged with dithionite is shown to be an efficient deoxygenator. Deoxyhcmoglobin prepnrcd on this column from stock oxyhemoglobin in solution has normal spectral characteristics, and upon reoxygenation the oxyhemoglobin spectrum is identical with that of the original stock oxyhemoglobin solution. The deoxyhelnoglobin solution has a ~0, of less t,han 1 mu Hg and an ionic strength of less than .5 X lo-“. and exhibits a Bohr effect and deoxygenation kinetirs consistent with litcrature values Dcoxygenated water is also prepared by this nlcthod. Dit.hionite, oxidation products of dithionite, and ionic contaluinante in t,hc elluent remain bound to the resin. REFERENCES 1. H.IRTRIDGE, H., AND R~UGHTON, F. J. W., Proc. Roy. fkx. London, Series -\ 104, 376 (1923). 2. Joua~, R., J. Chim. Phys., 56, 327 (1959). 3. DALZIEL, K., .~ND O’BRIEN, J. R. P., Biochmn. J. 49, slvii (1951). 4. BENESCH, R., BEXESCH, R., AND M.ICDUFF, G., &?.ence 144, 69 (1964). 5. ANTONINI, E., WYMAN, J., BRUNORI, M., BUCCI, E., FROXTICF,LLI, C., ,~XD RawFANELLI, A., J. Biol. Chem. 238, 2950 (1963). 6. DALZIEL, K., .~ND O’BRIEN, J. R. P., Biochem. J. 78, 236 (1961). 7. LYSTER, R., Ph.D. Thesis, Cambridge, England, 1955, which is referrtvl IO 1)~ Antonini, E., Rossi-Fanelli, A., and Caputo, A., Adam. Prolein. ChrYnc. 19, 167 (1964).
BIOCHEMISTHY
A Column
Method
DOSALD FJVUI
15, -304
(1966)
for Deoxygenating
Human
Hemoglobin’
F. HOELZL WALLACH, MITCHELL AND CHARLES A. JANEWAY
H. GAIL,
(he Department of Biological and ProteirL Foundation, Received
Chemistry, Boston, October
Haward Medical Massachusetts
School
29, 1965
In studying the kinetics of the reaction of human hemoglobin with oxygen by the continuous flow method of Hartridge and Roughton ( 1) , it became necessary to prepare large volumes of deoxygenated hemoglobin. Conventional methods involving physical equilibration of hemoglobin solutions in oxygen-free atmospheres require prolonged periods of time for full deoxygenation and so risk the hazard of methemoglobin formation. For these reasons we have developed a new deoxygenation method. A column containing a mixed-bed ion-exchange resin is partly loaded with sodium dithionitc, a powerful reducing agent for oxygen (Na,S,( ), + 02 + H,O e NaHSO, + NaHSO,) (2). The dithionite, which is confined to a 4-6 cm layer on the column of resin and is localized primarily within bhe resin beads, maintains the partial pressure of oxygen (PO.‘) of the aqueous phase at less than I mm Hg. Oxyhemoglobin dissociates as it passes through this region of low ~0,. Oxidation products of sodium dithionite, which include sulfate, sulfite, and hydrogen ions, remain bound to the resin. The column also maintains the pH of the solution at about 7 and removes ionic constituents introduced in the original oxyhemoglobin solution. Thus a deoxygenated hemoglobin solution of low ionic strength is obtained. METHODS
Preparation
of the Hentoglobin
Solution
Forty milliliters of packed human red cells are suspended in an equal volume of 0.15 N NaC1 and sedimented by centrifugation at 12,800 X g (Lourdes Betafuge, 9RA rotor, 5°C). This washing procedure is repeated twice. The packed red cells (ca. 30 ml) are then lysed by adding them to 8 vol of 0.01 M phosphate buffer at pH 6. This suspension is brought to ‘Supported VSPHS.
by
grants
CA
0738-02,
lSOl-FR-5381-01, 300
and
5 T5
GM.5144
from
the
HEAMOGLOBISDEOXYGESAl’IoS
301
pH 5.8 to cause :~ggregation of the stro1lia. (,infficicnt tinic 1riust be allowed for aggregation; otherwise stro1lia will k1ggregate on the resin. Ten minutca at’ room teitiper:~ture was :ldequntc~ for our protein conccntrnti0nG.j Tl1e susperkon i.5 then spun ;It 28.700 X (/ for IO Inin. The ~supernatant ia derantctl. brought to pH 8 with 1.O 31 Nat )H ( rapid Fiwh he1~ioglot~in stoc*E; constirringj ! and immetiitttc~ly refrigen1tcd. taining about 2 meq of heme per liter i> prepnwd in this 1n;tnncr e\-er> day. Piepam fion of the c’olu wn Tlrc ~sin: Bio-Rad mixed-bed resin XC: 501-SS(U) is cn~ployetl. -ig 501-581 D) is a dye-containing mixed ionic resin n-11ich contains a basic resin ISG l-X8 OH-) with benzenc-K+-(,C’H::),s groups imbedded on a polystyrene matrix wit11 8% divinylbenzene cross linkages and an equivalent amount of acidic resin (AG 5OW-18 I+) with benzene--SO,groups imbedclecl on an iclenticnl resin atr11cturc. The resins :Irc analytical grade and are 20-50 mesh (Rio-Rx1 catalog number 47020). This misedbed resin allows rapid flop rates, does not lcnk dithionite, and yields an ion-free. neutral effluent. Its dye is a useful indicator of remaining columnbinding capacity. Glass columns I 2.5 cm id. X 30 cm lwight) are packed at t-he hott.om with a 1 cm Ixyer of glnss pool. TIN columns ztre filled with water. and 8 ml of resin is added with stirring to assure uniform settling of t,he beads. A reservoir containing distilled water feeds into the tops of t’lie columns, and the airtight tops also 11xve an inlet for introducing hemoglobin or clithionitc. The column:: arc conipletcly filled with wntct and re*in so that, there are no gas bubbles. The effluent, is collected in an osggcn-free container. C’ltnrgin~~ of the col~mt~ with. clithionite: The calculated sodium dithionite requirement is 0.114 gm/liter of H,O in equilibrium with ambient air and 0.94 gin/liter of 2 mcq (in respect to heme groups) stock hemoglobin eolut.ion. ;Z twofold esccss of the day’s calculated dithionite requirement is int1,oduced onto the column, and 1 liter of water passed (20 ml/min) through the column to sweep the dithionite onto the resin and deoxygcnnte the entire coluiim; 500 ml of H,O is sufficient to apply t.he new dithionite charge on subsequent days providing the system remains closed to xir. OPERATION
Operation
OF
THE
COLUMS
of the Colrrmn
Normally two colu~zmsare employed, one for deosygenating water and one for drosygenating hemoglobin. Two liters of deosygenated water and 60 1111 of 2 meq Ihcnle b wo11psJstock lie1noglobin solution are neces-
302
WALLACH,
GAIL,
Ah-D
JANEWAY
sarv for our rapid flop experiments Dithionite (0.5 gin) is dissolved in 20 ml of water and this solution added to the hemoglobin column. ,\fter washing with I liter of water (20 ml/min) , 60 ml of stock hemoglol)in solution is int’roduced and followed by natcr i20 ml,/min) until all the deoxyliemoglobin is elutctl. The hemoglobin is collected in an oxygen-free container. The color of the effluent is deep purple (in contradistinction to the initial oxyhemoglobin solutionj, and the effluent ~0, as meusured on an Inst’rumentation Laboratories model 125A oxygen electrode system is les;sthan 1 1nm Hg. Dcoxygenated water is prepared similarI;\- on the other rolumn. For our purposes the capacit?- of the hemoglobin col~mm suffices for t8hree deoxygcnation, q and that of the water column for folir deoxygcnatione. RESULTS C’onductivity and pH of the deoxyyenated efluent: =In Arthur H. Thomas Co. model RCM 15Bl conductivity meter was used, and the conductivity of the effluent, was found to be 4.9 X lo-‘; Inho-cn-‘. The same instrument yielded I-alues of 2.6 X IO-‘: mho-en-l for water l~~cd over a Bar&cad model BD1 deionizing column and 1.44 X 10 ’ mhoCl11 ’ for a 0.01 31 KC1 solution (all readings at 20°C). The effluent thcrcfore has an ionic strengtlt of less t.hwn 5 X lo-“. The effluent pH W:W between 6.8 and 7.2 iInstrumentat,ion T,aboratory Inc. model 135.4 plI met8erwith catalog number 14040 glass electrode). These findings taken tog&her establish that the effluent contains neither significant quantities of ionic contaminant:: introduced in the stock osyltemoglobin solution no1 ditliionite or oxidation I)roduct. q of dithionite (,including sulfate, siilfite, and associated hydrogen ions). The protein: The main objection to the UPCof dithionite for deosygcnating hemoglobin has heen that it may denature human hemoglobin and produrc other hrllle piglllents (3). WC thcrcforr tried to detect abnonnnl pigments or alterations in the processed hemoglobin yljcctrophotoinctrically. ( 1) (~‘ompnriron of stock or!/hemoglobin with reosygentrted processed hemo~globin. A sample of deoxygenated human hemoglobin was reosy-
genat,cd by diluting with a large volume of 0.15 1V NaCl solution wltich had hccn ljreviously equilibrated with room air. The spectrum of the solution was th(bn collq~trcd with that of a similarly diluted solution of stock oxyht~moglohin. this control solution never haying been deoxygenated. The absorption spectra of the two samples as recorded on a Perkin-Elmer model 202 spectrophotometer were identical in t,he region 350-740 m/r. and had absorption maxima characteristic of oxyhemoglobin at 415, 543, and 577 mu.
HERIOGLOBIX
303
DEOXYGENATIOX
(2) Spectrum of the deo.ryhernogZobi~~ solution. l>eosygenated hemoglobin solution w:~s collected in an air-free flow cell, and the absorbance in tdw Poret region was determined on a Beckman nK q~ectropl~otometcr. This >olution was found to haw the spectral characteristic!: of cleosyhemoglobin as reported by Bcnesch et cd. (,-I). Tllere ww no “shouldet~” in the 408-412 m+ region, indicating t,hat methemoglobin was not present, in quantities exceeding 5% of the total heme pigment. This conclusion is confirmed by the fact that, upon reoxygenwtion of t#he hemoglobin the alwrption maximum in the Soret region remained at 415 m,~. Thr following table contains our obeerred and literature values for h,,,;,,. valuebtar XI,>%,. Osyhen~oglobin (control) Deosyhemoglobin Reoxygc~nated h~rnoglol~in
Olwrwd.
415 430 415
,,I+
13erNwh.
I,,$4
415 430
13t Biochemiccrl tests of the protein. The deoxyhemoglobin prepared 1~~ this method was used to demonstrate the Bohr effect and to study osygcnation kinetic+, and it seemed to be functioning properly. To demonstrate the Bohr effect, the difference in H+ binding by deoxyhcmoglobin and oxyhemoglobin was determined using the llwthod of hntonini et trl. (5). Our results agree with those of hntonini et rrl.. yic~ltling :I pH change of --0.20 pH unit on oxygenation of a 1.0 gm s deoxyhemoglobin solution containing 0.3 M N:iCl at 20°C. Preliminary StuclieSof oxygenation kinetics using t.he continuous flow method of Hartridge :ind Boughton (1 ) yield I-nlues greater than 2 X I@;W’ wc ’ for the o~chr-all :woc.i:~tioii coii.~t;~iit (k’j at. 20°C anal p1-T7.0. ;\n estiniatc of k’ lw(v1 on the disso&t.ion const,ant, (k) as tletcrmined by Dal&l ant1 ( )‘Brivn (6) aml the equilil)rium constant ah clctcl,niinccl by Lystclr ( 7 1 is 5 X 10, W’ st’c I. The fact that 0111‘ob~trvcd k’ is consi&nt with the theoretical estimate sugg(‘t: that our deos~henloglol)ill preparation is not clwat,urcd or nltcwd and that, the efllucnt cloc>bnot wntain ~lol~gll tlitllionitc~ to :tltcLr osygcwltion kinctiw. DISCUSSIOK
Thih bystem ha:: many advantages. It is easy to set up and to use, and i* lwtll l,apid and reliable. The duration and extent of contact of the hcnwglobin n-it11 the dithionite is minimal, since most. of the dithionite ir bouml inside the resin beads and is in a narrow band on the column. The :~ole of dit,hionite is to provide an oxygen-poor aqueous phase in n-hicll the oxyhemoglobin dissociates. Furthermore, because the concentration of hemoglobin on the column is high (ea. 0.5 mM). and because no oxygen is available in the column for peroxide formation, there is little ridl< of forming abnormal hence pigments (3). The effluent is also free of
304
WALLACH:
dithionite. oxidation l)rotlucts duced with the oxyheiiioglobin
GAIL,
Ah-D
JAKEWAP
of dithionite, and ionic contaminants stock solution.
intro-
SUMMARY
The mixed-bed resin column charged with dithionite is shown to be an efficient deoxygenator. Deoxyhcmoglobin prepnrcd on this column from stock oxyhemoglobin in solution has normal spectral characteristics, and upon reoxygenation the oxyhemoglobin spectrum is identical with that of the original stock oxyhemoglobin solution. The deoxyhelnoglobin solution has a ~0, of less t,han 1 mu Hg and an ionic strength of less than .5 X lo-“. and exhibits a Bohr effect and deoxygenation kinetirs consistent with litcrature values Dcoxygenated water is also prepared by this nlcthod. Dit.hionite, oxidation products of dithionite, and ionic contaluinante in t,hc elluent remain bound to the resin. REFERENCES 1. H.IRTRIDGE, H., AND R~UGHTON, F. J. W., Proc. Roy. fkx. London, Series -\ 104, 376 (1923). 2. Joua~, R., J. Chim. Phys., 56, 327 (1959). 3. DALZIEL, K., .~ND O’BRIEN, J. R. P., Biochmn. J. 49, slvii (1951). 4. BENESCH, R., BEXESCH, R., AND M.ICDUFF, G., &?.ence 144, 69 (1964). 5. ANTONINI, E., WYMAN, J., BRUNORI, M., BUCCI, E., FROXTICF,LLI, C., ,~XD RawFANELLI, A., J. Biol. Chem. 238, 2950 (1963). 6. DALZIEL, K., .~ND O’BRIEN, J. R. P., Biochem. J. 78, 236 (1961). 7. LYSTER, R., Ph.D. Thesis, Cambridge, England, 1955, which is referrtvl IO 1)~ Antonini, E., Rossi-Fanelli, A., and Caputo, A., Adam. Prolein. ChrYnc. 19, 167 (1964).