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Interferences by sulfhydryl, disulfide reagents and potassium ions on protein determination by Lowry's method
ANALYTSC4L
BIOCHEMISTRY
Interferences
36,
!i%T-%!
(1970)
by Sulfhydryl,
Potassium
Disulfide
Ions on Protein by Lowry’s
Reagents
and
Determination
Method
CARAMEN G. VALLEJO AKDROSARIO LA4GUNAS Institute Consejo
de Enxinao2ogia, Centra de lnvestigaciones Biol6gicas, Superior de Investigaciones Cientificas, M&id, Spain
Received December
31, 1969
Lowry’s method (1) for protein determination is a routine procedure in most biochemistry laboratories. A number of interfering substances have been described and include ethylenediaminetetraacetate (Z), penicillin and derivatives (3), different buffers and other chemicals (4, 5), sucrose (6-9) I and oxidized lipids (IO), as well as those mentioned by the authors of the method (1). We have found that sulfhydryl and disulfide reagents and pot,assium ions can also produce importa.nt interferences. Sulfhydryl reagents, especially dithioerythritol and 2-mercaptoethanol are extensively used as protectors of t’he sulfhydryl groups of proteins. Gradients containing potassium salts are frequently used in the development of column chromatography. This paper presents a study of the interference by these widely employed substances. MATERIALS
SND
METHODS
All chemicals were of analytical grade. Dithioerythritol, 2-mercaptoethanol, oxidized glutathione, and bovine serum albumin were obtained from Sigma Chemical Co., St. Louis, MO. Reduced glutathione was obt,ained from Calbiochem, Loewengraben 14, 6002 Luzern, Switzerland. L-Cyst)eine monochloride (hydrate) was from Pfanstiehl Laboratories, Inc., Waukegan, Illinois, and Ii-cystine from Nutritional Biochemicals Corporation, Cleveland, Ohio. The usual Lowry procedure was followed in the present experiments. Freshly prepared reagents were employed and known amounts of bovine serum albumin were used as a standard. Measurements of optical density were made with a Klett-Summerson photoelect8riccalorimeter using the number 66 fiber. Substancestested for interference with the method were dissolved in water except L-cystine (in 0.5 N sodium hydroxide) because 207
SuljhydryI and tdisulfide reugmfs interfwmws. DithioeIythritol, 2mercaptoethanol, reduced glutat,hione, cysteinci, oxidized glutathione, and cystine were assayed ard all of them pruvcd to intctrferc:, producing a blue color whose visible akl;orption q&rum was like that caused by proteins (Fig. 1). The values of the blanks wit’11 these interfering subetnnrcs iit
I LOO
500
600 WAVELENGTH
700
000
(nm)
FIG. 1. Visible absorption xpect,ra of color productbd by protein, sulfhydryl. and disulfide reagents in Lowry’s met.hod. 100 ,~g bovine s:Prum albumin, 0.05 mM dithioerythritol, or 0.3 mM oxidized glutathione was used in a final voIume of 2.5 ml. Optical densities were recorded with a Gary model 15 spectrophotometer, and I cm cells were employed.
different concentrat.ions a,re shown in Table 1. The greatest interference was found with dithiaerythritol, which at 0.2 mM showed a color too intense to’ be tolerable. From 0.7 mM 2-mercaptoethanol onward, the blank values sharply increased, and at. 1.0 mM the color of the blank was so intense that no reliable results could be expected. The other four tested reagents behaved in a similar way with respect to thp concentration at which the interference was import,ant. In each case ;tt, 0.7 rnM the color development due to these reagents was so strong that’ ,their ~NWW~ did not permit t.hu estimation of proteins by the Lowry method. Above vcrt:ziu ranges, the optical densities are not proportional ta concentration and
INTERFERENCES
IN
LOWRY’S
209
METHOD
TABLE I E&ct of Sulfhydryl and Disulfide Reagents on Blank of Lowry’s Method The blanks contained the reagents required by the method and the interfering substances at the indicated concentrations. Values represent the means of three experiments and are expressed in optical densities measured a indicated in “Methods.” mM
Dithioerythritol
2-Mercaptoethanol
Reduced glutathione
Cysteme
-
0.08
0.06
0.06
0.07
0.02 0.04 0.2 0.4 0.7 1.0 2.0
0.20 0.35 1.59
0.05 0.05 0.05 0.05
0.0s 0.10
0.08 0.09
0.26 0.46 1.00 >2.00
0.16 0.26 0.98
>2.00
0.07 1.24 >2.00
>2.00
Oxidized gIut&hione 0.06 0.12 0.17 0.45 0.73 1.00 1,50
Cystine 0.07 0.10 0.13 0.30 0.47 1.30 >2.00
>2.00
larger increases than those expected are observed. The response of the method to increasing amounts of protein was linear when moderate concentrations, the interference can be avoided by running blanks containing centrations of these reagents were present (Fig. 2). Therefore, at low con-
PROTEIN Iygl
FIG. 2. Effects of different sulfhydryl and disulfide reagents on determination of increasing amounts of protein: (II) without additions; (13) with 0.04 mM dithioerythritol; (A) with 0.2 mM reduced glutathione; with 0.2 mM oxidized ghrtathione.
An examination of t.hc contribution of the different ingwdicnts of Lowry’s assay mixture (Table 2) showed that the Folk reagent is essential for the appearanceof blue color \vitNhall the above compounds. In the case of t’he sulfhydvl compounds, sotlirm hydroxide, soc~iumesrbonate, or copper sulfate is aleo needed.The wagcuts required for tlw appearance of the interference with the rlkulfidc? compounds arc not idcrltical in the two casesstudied: oxidized glutathione wquires sodium hydroxide or sodium carbonate, whereas cpstine nr-reckonr of t,hesereagents and copper sulfate. Tartrate showecI no cffcct. eit.ht>rOYIsulfhydryl or disulfide compounds. The fact that’ sulfhytiryl anti rliwlfitfc corqountl~~ - give a ~~0101~ with sirnilar absorption spectrum to that1~~roduccclhy prot.cins suggeststhat’ in both cases a reduction of the I~ho~])homolybtiic-p~~o~~~~~otungstic reagent takes hy increases in place, The large increasw of optical cicnsitic9 ~~i~orluced the concentrations of each of thaw subst.ancesabo~~ctcertain concentration ranges (Table I j may 1~: ~~~:lat~cd t,o tht~ir oxitlation-~cductioll potentials. The rise in concent,ratiou of these substarws would concomitantly increase their actual potentials! rwching a .~‘alw :at which thy would tw able to reduce the f?hosp~lolnolil)cli~l-~hospho~~ll~~~ti(~ rctagent.
Components
of L~wt,y’s Method Responsible for Interfererrves by t,he Sltlfhydryl and Disulfide Reagents The different component,ti of Low&s met.hod were added itr the sequence shown in the table at the final concentration mml in the routine proaedure. Aft,er addition of each component or group of compoilents, ihe reacthlg mixtures were incubated for 10 min except for the Polin reagent, 30 min. Xl1 possible combitta~t,ions of t,he different components were test,ed. Combinations producing MI effect are omilted. Optical densities of t,he blanks without interfering suljstaaces were < (1.117. Interfering substances (1.0 mMi Sulf hydry 1 reagent’s Oxidized glutathioue Cystine
Sodium hydroxide + -_ 4~ -. -t -
+ t _+
-+ -+ +
f + -5 + -i-I -t
,2.00 >2.00
1.60->2.00 >2.00 0 33 >2.00 >2 .oo
INTERFERENCES
IN
LOWRY’S
211
METHOD
TABLE 3 of Interference Produced by Potassium Ions in Lowry’s Method Bovine serum albumin was assayed at the indicated concent,rat’ions with and wit’hout potassium chloride, The reacting mixtures were centrifuged at ca. 12,000g for 5 min after color development and the sediments discarded. The optical densit;ies were measured in the clear supernatants as indicat,ed in “Methods.” Removal by Cent,rifugation
Protein, pg -
2;, 50 7.’ 100
No addition 0.04 0.2.; 0.40 0.59 0.72
40 mM K+ added 0.04 0.26 0.42 0.61 0.74
Potassium ions Gaterference. A white precipitate was formed during protein estimation by Lowry’s method when potassium phosphate or potassium chloride was present. Since sodium phosphate or sodium chloride
was without effect, potassium ions are most likely to be responsible for this interference. The precipitate was observed from 12 mM onward but lower concentrations were allowed without significant trouble. It was found, however, that this interference by potassium ions can be overcome by centrifuging the reaction mixtures after coIor development. By this procedure, no difference in color was observed with respect to a parallel determination without potassium (Table 3). The components of the reaction mixture responsible for this precipit,ation were found to be sodium carbonate together with the Folin reagent. SUMMARY
Sulfhydryl, disulfide reagents, and also pot’a’ssiumions interfere with the protein determination by Lowry’s met’hod.The concentrat,ionsof these substances which can be used without serious interference are given. Sulfhydryl and disulfide reagents yield a color with the same absorption spectrum as that caused by proteins. At moderate concentrations this interference can be overcome by running appropriate blanks. Potaasium ions produce a white precipitate that can be removed without change of color intensity
by centrifuging
the reaction mixtures after color development. ACKNOWLEDGMENTS
The authors wish to thank Dr. A. Sols and Dr. W. M. Ingledew for critical reading and discussion of the manuscript. The able technical assistance of Miss Anabel de Diego is gratefully acknowledged. One of us (C. G. V,) was in receipt of a fellowship from the Spanish Ministry of Education and Science.
lIFPJ~‘Jl?l‘N~‘~F~ 2, J Iii 1. rlowllY, 0. II., J1oHEIIRoUGH, K. ,l., J“.\liI~, =1. L.. :\?+I) T~.NI>u,I.. I(, .r.. .I. Hl.ol. ~‘!hr:m. 193, 265 !1!)51). 2. SEURATlI. A. li .? E’zpel-icnfla 22, 290 ( 1966). 3. SlLVERkI.4N, D. J.: hznl. Biochcm. 27, 189 (lc969) I 4. GWLERT, -1/1, ti‘., VOX HIPPEL, P. H.. ~CXIACHJI.~N, H. li.. ;AND MORALES. M. I;., J.
d??&.
chc??~~
,%JC.
81,
1384
(1!%9).
5. PETERS, M. A., AND For-m, J, R., dnnb. Hiochem, 30, 290 (1969) _ 6. BACH~LARD, H. s., &rxhtm. J. 104, ‘E36 (1967). 7. ~%XUEL, H.. AND %XIWX,, R., dna!. hkhem. 20, 86 (1967). 8. GERHARDT, B.. AND BEICVERS, Ii., Amr~. Ri~chc:m. 24, 337 C1968.).
9. HINTON, Ii. H.? RUKGF, M. L. E., ASII HAIIT~\~AS. G. C‘., Anab. Biochem. (1969) _ 10, EICHBERG, J .. AND MOKRASCH, IA. C.: AnoE. Bioche~m. 30, 386 (1969). 11. FIYZER, B., Rull. Sot. Chim.. Biol. 46, 27 (1964).
29, 248
BIOCHEMISTRY
Interferences
36,
!i%T-%!
(1970)
by Sulfhydryl,
Potassium
Disulfide
Ions on Protein by Lowry’s
Reagents
and
Determination
Method
CARAMEN G. VALLEJO AKDROSARIO LA4GUNAS Institute Consejo
de Enxinao2ogia, Centra de lnvestigaciones Biol6gicas, Superior de Investigaciones Cientificas, M&id, Spain
Received December
31, 1969
Lowry’s method (1) for protein determination is a routine procedure in most biochemistry laboratories. A number of interfering substances have been described and include ethylenediaminetetraacetate (Z), penicillin and derivatives (3), different buffers and other chemicals (4, 5), sucrose (6-9) I and oxidized lipids (IO), as well as those mentioned by the authors of the method (1). We have found that sulfhydryl and disulfide reagents and pot,assium ions can also produce importa.nt interferences. Sulfhydryl reagents, especially dithioerythritol and 2-mercaptoethanol are extensively used as protectors of t’he sulfhydryl groups of proteins. Gradients containing potassium salts are frequently used in the development of column chromatography. This paper presents a study of the interference by these widely employed substances. MATERIALS
SND
METHODS
All chemicals were of analytical grade. Dithioerythritol, 2-mercaptoethanol, oxidized glutathione, and bovine serum albumin were obtained from Sigma Chemical Co., St. Louis, MO. Reduced glutathione was obt,ained from Calbiochem, Loewengraben 14, 6002 Luzern, Switzerland. L-Cyst)eine monochloride (hydrate) was from Pfanstiehl Laboratories, Inc., Waukegan, Illinois, and Ii-cystine from Nutritional Biochemicals Corporation, Cleveland, Ohio. The usual Lowry procedure was followed in the present experiments. Freshly prepared reagents were employed and known amounts of bovine serum albumin were used as a standard. Measurements of optical density were made with a Klett-Summerson photoelect8riccalorimeter using the number 66 fiber. Substancestested for interference with the method were dissolved in water except L-cystine (in 0.5 N sodium hydroxide) because 207
SuljhydryI and tdisulfide reugmfs interfwmws. DithioeIythritol, 2mercaptoethanol, reduced glutat,hione, cysteinci, oxidized glutathione, and cystine were assayed ard all of them pruvcd to intctrferc:, producing a blue color whose visible akl;orption q&rum was like that caused by proteins (Fig. 1). The values of the blanks wit’11 these interfering subetnnrcs iit
I LOO
500
600 WAVELENGTH
700
000
(nm)
FIG. 1. Visible absorption xpect,ra of color productbd by protein, sulfhydryl. and disulfide reagents in Lowry’s met.hod. 100 ,~g bovine s:Prum albumin, 0.05 mM dithioerythritol, or 0.3 mM oxidized glutathione was used in a final voIume of 2.5 ml. Optical densities were recorded with a Gary model 15 spectrophotometer, and I cm cells were employed.
different concentrat.ions a,re shown in Table 1. The greatest interference was found with dithiaerythritol, which at 0.2 mM showed a color too intense to’ be tolerable. From 0.7 mM 2-mercaptoethanol onward, the blank values sharply increased, and at. 1.0 mM the color of the blank was so intense that no reliable results could be expected. The other four tested reagents behaved in a similar way with respect to thp concentration at which the interference was import,ant. In each case ;tt, 0.7 rnM the color development due to these reagents was so strong that’ ,their ~NWW~ did not permit t.hu estimation of proteins by the Lowry method. Above vcrt:ziu ranges, the optical densities are not proportional ta concentration and
INTERFERENCES
IN
LOWRY’S
209
METHOD
TABLE I E&ct of Sulfhydryl and Disulfide Reagents on Blank of Lowry’s Method The blanks contained the reagents required by the method and the interfering substances at the indicated concentrations. Values represent the means of three experiments and are expressed in optical densities measured a indicated in “Methods.” mM
Dithioerythritol
2-Mercaptoethanol
Reduced glutathione
Cysteme
-
0.08
0.06
0.06
0.07
0.02 0.04 0.2 0.4 0.7 1.0 2.0
0.20 0.35 1.59
0.05 0.05 0.05 0.05
0.0s 0.10
0.08 0.09
0.26 0.46 1.00 >2.00
0.16 0.26 0.98
>2.00
0.07 1.24 >2.00
>2.00
Oxidized gIut&hione 0.06 0.12 0.17 0.45 0.73 1.00 1,50
Cystine 0.07 0.10 0.13 0.30 0.47 1.30 >2.00
>2.00
larger increases than those expected are observed. The response of the method to increasing amounts of protein was linear when moderate concentrations, the interference can be avoided by running blanks containing centrations of these reagents were present (Fig. 2). Therefore, at low con-
PROTEIN Iygl
FIG. 2. Effects of different sulfhydryl and disulfide reagents on determination of increasing amounts of protein: (II) without additions; (13) with 0.04 mM dithioerythritol; (A) with 0.2 mM reduced glutathione; with 0.2 mM oxidized ghrtathione.
An examination of t.hc contribution of the different ingwdicnts of Lowry’s assay mixture (Table 2) showed that the Folk reagent is essential for the appearanceof blue color \vitNhall the above compounds. In the case of t’he sulfhydvl compounds, sotlirm hydroxide, soc~iumesrbonate, or copper sulfate is aleo needed.The wagcuts required for tlw appearance of the interference with the rlkulfidc? compounds arc not idcrltical in the two casesstudied: oxidized glutathione wquires sodium hydroxide or sodium carbonate, whereas cpstine nr-reckonr of t,hesereagents and copper sulfate. Tartrate showecI no cffcct. eit.ht>rOYIsulfhydryl or disulfide compounds. The fact that’ sulfhytiryl anti rliwlfitfc corqountl~~ - give a ~~0101~ with sirnilar absorption spectrum to that1~~roduccclhy prot.cins suggeststhat’ in both cases a reduction of the I~ho~])homolybtiic-p~~o~~~~~otungstic reagent takes hy increases in place, The large increasw of optical cicnsitic9 ~~i~orluced the concentrations of each of thaw subst.ancesabo~~ctcertain concentration ranges (Table I j may 1~: ~~~:lat~cd t,o tht~ir oxitlation-~cductioll potentials. The rise in concent,ratiou of these substarws would concomitantly increase their actual potentials! rwching a .~‘alw :at which thy would tw able to reduce the f?hosp~lolnolil)cli~l-~hospho~~ll~~~ti(~ rctagent.
Components
of L~wt,y’s Method Responsible for Interfererrves by t,he Sltlfhydryl and Disulfide Reagents The different component,ti of Low&s met.hod were added itr the sequence shown in the table at the final concentration mml in the routine proaedure. Aft,er addition of each component or group of compoilents, ihe reacthlg mixtures were incubated for 10 min except for the Polin reagent, 30 min. Xl1 possible combitta~t,ions of t,he different components were test,ed. Combinations producing MI effect are omilted. Optical densities of t,he blanks without interfering suljstaaces were < (1.117. Interfering substances (1.0 mMi Sulf hydry 1 reagent’s Oxidized glutathioue Cystine
Sodium hydroxide + -_ 4~ -. -t -
+ t _+
-+ -+ +
f + -5 + -i-I -t
,2.00 >2.00
1.60->2.00 >2.00 0 33 >2.00 >2 .oo
INTERFERENCES
IN
LOWRY’S
211
METHOD
TABLE 3 of Interference Produced by Potassium Ions in Lowry’s Method Bovine serum albumin was assayed at the indicated concent,rat’ions with and wit’hout potassium chloride, The reacting mixtures were centrifuged at ca. 12,000g for 5 min after color development and the sediments discarded. The optical densit;ies were measured in the clear supernatants as indicat,ed in “Methods.” Removal by Cent,rifugation
Protein, pg -
2;, 50 7.’ 100
No addition 0.04 0.2.; 0.40 0.59 0.72
40 mM K+ added 0.04 0.26 0.42 0.61 0.74
Potassium ions Gaterference. A white precipitate was formed during protein estimation by Lowry’s method when potassium phosphate or potassium chloride was present. Since sodium phosphate or sodium chloride
was without effect, potassium ions are most likely to be responsible for this interference. The precipitate was observed from 12 mM onward but lower concentrations were allowed without significant trouble. It was found, however, that this interference by potassium ions can be overcome by centrifuging the reaction mixtures after coIor development. By this procedure, no difference in color was observed with respect to a parallel determination without potassium (Table 3). The components of the reaction mixture responsible for this precipit,ation were found to be sodium carbonate together with the Folin reagent. SUMMARY
Sulfhydryl, disulfide reagents, and also pot’a’ssiumions interfere with the protein determination by Lowry’s met’hod.The concentrat,ionsof these substances which can be used without serious interference are given. Sulfhydryl and disulfide reagents yield a color with the same absorption spectrum as that caused by proteins. At moderate concentrations this interference can be overcome by running appropriate blanks. Potaasium ions produce a white precipitate that can be removed without change of color intensity
by centrifuging
the reaction mixtures after color development. ACKNOWLEDGMENTS
The authors wish to thank Dr. A. Sols and Dr. W. M. Ingledew for critical reading and discussion of the manuscript. The able technical assistance of Miss Anabel de Diego is gratefully acknowledged. One of us (C. G. V,) was in receipt of a fellowship from the Spanish Ministry of Education and Science.
lIFPJ~‘Jl?l‘N~‘~F~ 2, J Iii 1. rlowllY, 0. II., J1oHEIIRoUGH, K. ,l., J“.\liI~, =1. L.. :\?+I) T~.NI>u,I.. I(, .r.. .I. Hl.ol. ~‘!hr:m. 193, 265 !1!)51). 2. SEURATlI. A. li .? E’zpel-icnfla 22, 290 ( 1966). 3. SlLVERkI.4N, D. J.: hznl. Biochcm. 27, 189 (lc969) I 4. GWLERT, -1/1, ti‘., VOX HIPPEL, P. H.. ~CXIACHJI.~N, H. li.. ;AND MORALES. M. I;., J.
d??&.
chc??~~
,%JC.
81,
1384
(1!%9).
5. PETERS, M. A., AND For-m, J, R., dnnb. Hiochem, 30, 290 (1969) _ 6. BACH~LARD, H. s., &rxhtm. J. 104, ‘E36 (1967). 7. ~%XUEL, H.. AND %XIWX,, R., dna!. hkhem. 20, 86 (1967). 8. GERHARDT, B.. AND BEICVERS, Ii., Amr~. Ri~chc:m. 24, 337 C1968.).
9. HINTON, Ii. H.? RUKGF, M. L. E., ASII HAIIT~\~AS. G. C‘., Anab. Biochem. (1969) _ 10, EICHBERG, J .. AND MOKRASCH, IA. C.: AnoE. Bioche~m. 30, 386 (1969). 11. FIYZER, B., Rull. Sot. Chim.. Biol. 46, 27 (1964).
29, 248