- Email: [email protected]
Enzymatic-potentiometric determination of oxalic acid
Bioebcfroc~enzisfry and Bi0ensrgefic.s Short
2,
348-350 (1975)
communication
Enzymatic-Pofentiometric Determination of Oxalic Acid ~~S~GJ_YAO,
SIDNEYK.WOLFSON,
Department of Neurological Surgery, and Surgical Research Laboratories. U.S.A. 1Manuscript received
July
'~1st
University Montefiore
JR. ~~~JoYcEM.
TOKARSKY
of Pittsburgh School of Medicine Hospital, Pittsburgh, Pa 15213.
1975
-4 method utilizing oxalate decarboxylase and potentiometry is described for the determination of oxalic acid. The oxalic acid was enzymatically decomposed to form formic acid and carbon dioxide. The released CO, was measured potentiometrically using a CO, electrode. A nemstian relationship between the voltage output of the CO, sensor and range, 0.3-1-0 mg the concentration of oxalic acid, in the-phy&IogicaI per IOO cm3, has been observed_
Introduction OxaIate concentration in body fluids has been estimated by the method of permanganate-titration of an insoluble salt, ls2 by gasometric permanganate-oxidation 3s4 in the VAN SLY=--NLELL apparatus 5 and by enzymatic-manometric method using the WARBURG technique_ 6~7 Those methods employing precipitation and subsequent titration involve a number of sequential steps employing several vessels and separations, all potential sources of error_ The manometric technique is rather inconvenient and requires temperature and pressure adjustments. This note presents a new approach to the determination of oxalate- The method involves the enzymatic decomposition of oxalate by oxaiate decarboxylase and the potentiometric measurement of the CO, released from the decarboxylation- From the voltage output of the CO, electrode, the concentration of the oxalate is determined. The method, using an existing CO, electrode for oxalate determination, is simple and accurate. Moreover, the entire analysis is carried out in a single reaction vessel.
ExperimentaIandresults The determination of oxalate was carried out in a system modified from one which we described previously for the determination of urea 8 and glutamic acid O. A SEvERIxGHAus-type CO, electrode connected to
Enzymatic-Potentiometric
Determination
of- Oz&!ic -.-
Acid
349 _
an IL MODEL‘-123~s~ ultramicro $ (COi) Analyzer w& employed for: the measurement of partial pressure of CO, i& $ (CO~;in.-units. of Torr:ormmHg, generated- during the decarboxylation. -: .The reactionlwa.sllcarrie~ out in a glass tube conttining 5 cxiP of 1.0 M citr&e ;buff&,’ pE 3.0, ! Tw& activity units of oxalate decarboxyltie (partially -puriGed:.froni -.&&+z veZt&@e, SIGMA CHEMICAL COMPAXY, grade II) I was dispetied+in :-the buffer by ,magnetic stirring. The CO, electrode -..was then. immersed. hi the -enzyme-buffer solution_ The electrode was suspended- from abovethe glass reaction tube by means of a rubber stopper. me syst+n wassealed and maintained at 37 0C with .mild stirring.. Anhydrous _oxali~_ acid, dissolved in citrate buffer (pH 3.0), was injected in to t_he’&&io& vessel through a needle inserted through the rubber stopper pecarboxylation began immediately after.the substrate was introduced into the reactor. -The CO2 Treleased.during the decarboxylaiion’&fmsed~ _through the gas permeable membrane .at the .tip of the CO, .electrode in @ropertion to the par&l pressure difference of- CO, across -the-membrane; ’ ..Tlie pH (glass) : electrode, arranged behind the gas ; permeable; ~&+d’~ the eiectrolyte . placed 1behind’3he .--gS @er[H+] changes of the HCOs --Cl2 meable membrane_ It required about. six: minutes- for-rthe: dec%boxylaG tion and product diffusion to come to equilibrium~and,~conseque&ly~ the CO, electrode response to steady state_ I. -- ._r ::- ;-:. -7: _“-‘- =Since the decarboxylation of I mole of oxakte yieldk~r. -mole-_of CO, 10~11,the voltage output of the CO, electrode should be linear to- the the logarithm of the partial-pressure of CO, (log #CO,) Snd,‘conskqu~ntly, 10~ [oxalatel. Our results, as Shown in- IGg_ I, -indeed protide Y--confi-rL mation of this nernstian relationship, at least, in the physiological range (0.3-1.0 mg oxalic acid per . I00 ems sample) _
!
0 0.1
^
c(mg/loucm) 9
I
Q2
0.4
,
I
I
0-S QB IO
2.0
1.
Relationship between concentration of oxalic acid and voltage output of a CO, electrode.
conclnslon
The determination of oxalate by the. present enzymatic-potent& metric method is simple, specific and accurate. The- nernsti&n relation between voltage output and the oxalate concentration @reseated--here Therefore, it is possible to calibrate the provides concrete evidence.
Yao, Wolfson, Jr_ and Tokarsky
350
$ &OS) meter directly in terms of oxalate concentration_ This analytical techruque is general. It could be applied to the determination of many decarboxylable compounds as long as their respective decarboxylases are existing and available_ It is convenient since it utilizes existing CO, electrodes and avoids difficulti_es confronting the manometric, spectrophotometric and titration techniques which include problems in temperature -and pressure compensation, optical clarity and precipitation.
References W. MERZ and S. MAUGERI, Ho+$e-Seylev’s
2. Physiol.
Chem.
201,
31 (rgbx)
S_ Suzri~r, Jup_ J_ Med. Sci. (and Series, Biochem.) 2, ZQI (1934) H-H_ BARBER and E. J. GALLIMORE, Biochem- J- 34, 144 (1940) J_F_B_ BARRETT. Biochem. I_ 37, 254 (1943) D.D. VAN SLYKE and J. SENDROY. J_ BioZ. Gem_ 84, 217 (Igzg) J-C. CRAWHALL and R-W-EWATTS, C&z_ Sci. 20, 357 (1961) G.G. MAYER, D. MARKOW and F. KARP, CZin. Chem. 9, 334 (1~63) S.J. YAO and J.M. TOKARSKY, Unpublished observations (1972) B.K. AHN. S-K_ WOLFSON, JR_ and S-J. YAO, BioeZectrochem_ Seaerg_ 10 11
14" (1975) H_ SHI~~AZONO and 0. HAYAISHI, J_ BioZ. Chem. 227, E. E~LIAXI and P_ BEKJZS. Arch. Biochem. Biophys.
ISI (1957) 105, 488 (1964)
2,
2,
348-350 (1975)
communication
Enzymatic-Pofentiometric Determination of Oxalic Acid ~~S~GJ_YAO,
SIDNEYK.WOLFSON,
Department of Neurological Surgery, and Surgical Research Laboratories. U.S.A. 1Manuscript received
July
'~1st
University Montefiore
JR. ~~~JoYcEM.
TOKARSKY
of Pittsburgh School of Medicine Hospital, Pittsburgh, Pa 15213.
1975
-4 method utilizing oxalate decarboxylase and potentiometry is described for the determination of oxalic acid. The oxalic acid was enzymatically decomposed to form formic acid and carbon dioxide. The released CO, was measured potentiometrically using a CO, electrode. A nemstian relationship between the voltage output of the CO, sensor and range, 0.3-1-0 mg the concentration of oxalic acid, in the-phy&IogicaI per IOO cm3, has been observed_
Introduction OxaIate concentration in body fluids has been estimated by the method of permanganate-titration of an insoluble salt, ls2 by gasometric permanganate-oxidation 3s4 in the VAN SLY=--NLELL apparatus 5 and by enzymatic-manometric method using the WARBURG technique_ 6~7 Those methods employing precipitation and subsequent titration involve a number of sequential steps employing several vessels and separations, all potential sources of error_ The manometric technique is rather inconvenient and requires temperature and pressure adjustments. This note presents a new approach to the determination of oxalate- The method involves the enzymatic decomposition of oxalate by oxaiate decarboxylase and the potentiometric measurement of the CO, released from the decarboxylation- From the voltage output of the CO, electrode, the concentration of the oxalate is determined. The method, using an existing CO, electrode for oxalate determination, is simple and accurate. Moreover, the entire analysis is carried out in a single reaction vessel.
ExperimentaIandresults The determination of oxalate was carried out in a system modified from one which we described previously for the determination of urea 8 and glutamic acid O. A SEvERIxGHAus-type CO, electrode connected to
Enzymatic-Potentiometric
Determination
of- Oz&!ic -.-
Acid
349 _
an IL MODEL‘-123~s~ ultramicro $ (COi) Analyzer w& employed for: the measurement of partial pressure of CO, i& $ (CO~;in.-units. of Torr:ormmHg, generated- during the decarboxylation. -: .The reactionlwa.sllcarrie~ out in a glass tube conttining 5 cxiP of 1.0 M citr&e ;buff&,’ pE 3.0, ! Tw& activity units of oxalate decarboxyltie (partially -puriGed:.froni -.&&+z veZt&@e, SIGMA CHEMICAL COMPAXY, grade II) I was dispetied+in :-the buffer by ,magnetic stirring. The CO, electrode -..was then. immersed. hi the -enzyme-buffer solution_ The electrode was suspended- from abovethe glass reaction tube by means of a rubber stopper. me syst+n wassealed and maintained at 37 0C with .mild stirring.. Anhydrous _oxali~_ acid, dissolved in citrate buffer (pH 3.0), was injected in to t_he’&&io& vessel through a needle inserted through the rubber stopper pecarboxylation began immediately after.the substrate was introduced into the reactor. -The CO2 Treleased.during the decarboxylaiion’&fmsed~ _through the gas permeable membrane .at the .tip of the CO, .electrode in @ropertion to the par&l pressure difference of- CO, across -the-membrane; ’ ..Tlie pH (glass) : electrode, arranged behind the gas ; permeable; ~&+d’~ the eiectrolyte . placed 1behind’3he .--gS @er[H+] changes of the HCOs --Cl2 meable membrane_ It required about. six: minutes- for-rthe: dec%boxylaG tion and product diffusion to come to equilibrium~and,~conseque&ly~ the CO, electrode response to steady state_ I. -- ._r ::- ;-:. -7: _“-‘- =Since the decarboxylation of I mole of oxakte yieldk~r. -mole-_of CO, 10~11,the voltage output of the CO, electrode should be linear to- the the logarithm of the partial-pressure of CO, (log #CO,) Snd,‘conskqu~ntly, 10~ [oxalatel. Our results, as Shown in- IGg_ I, -indeed protide Y--confi-rL mation of this nernstian relationship, at least, in the physiological range (0.3-1.0 mg oxalic acid per . I00 ems sample) _
!
0 0.1
^
c(mg/loucm) 9
I
Q2
0.4
,
I
I
0-S QB IO
2.0
1.
Relationship between concentration of oxalic acid and voltage output of a CO, electrode.
conclnslon
The determination of oxalate by the. present enzymatic-potent& metric method is simple, specific and accurate. The- nernsti&n relation between voltage output and the oxalate concentration @reseated--here Therefore, it is possible to calibrate the provides concrete evidence.
Yao, Wolfson, Jr_ and Tokarsky
350
$ &OS) meter directly in terms of oxalate concentration_ This analytical techruque is general. It could be applied to the determination of many decarboxylable compounds as long as their respective decarboxylases are existing and available_ It is convenient since it utilizes existing CO, electrodes and avoids difficulti_es confronting the manometric, spectrophotometric and titration techniques which include problems in temperature -and pressure compensation, optical clarity and precipitation.
References W. MERZ and S. MAUGERI, Ho+$e-Seylev’s
2. Physiol.
Chem.
201,
31 (rgbx)
S_ Suzri~r, Jup_ J_ Med. Sci. (and Series, Biochem.) 2, ZQI (1934) H-H_ BARBER and E. J. GALLIMORE, Biochem- J- 34, 144 (1940) J_F_B_ BARRETT. Biochem. I_ 37, 254 (1943) D.D. VAN SLYKE and J. SENDROY. J_ BioZ. Gem_ 84, 217 (Igzg) J-C. CRAWHALL and R-W-EWATTS, C&z_ Sci. 20, 357 (1961) G.G. MAYER, D. MARKOW and F. KARP, CZin. Chem. 9, 334 (1~63) S.J. YAO and J.M. TOKARSKY, Unpublished observations (1972) B.K. AHN. S-K_ WOLFSON, JR_ and S-J. YAO, BioeZectrochem_ Seaerg_ 10 11
14" (1975) H_ SHI~~AZONO and 0. HAYAISHI, J_ BioZ. Chem. 227, E. E~LIAXI and P_ BEKJZS. Arch. Biochem. Biophys.
ISI (1957) 105, 488 (1964)
2,