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A precise method for the determination of whole blood and plasma sulfhydryl groups
ANALYTICAL BIOCHEMISTRY 93, 98-102 (1979)
A Precise Method for the Determination of Whole Blood and Plasma Sulfhydryl Groups GEORGE E L L M A N AND HIRO LYSKO
Brain-Behavior Research Center, University of California, Langley Porter Neuropsychiatric Institute, Sonoma State Hospital, Eldridge, California 95431 Received July 12, 1978 A colorimetric method for the determination of total sulfhydryl content of whole blood or plasma is presented. For whole blood, a mixture of fresh blood and 5,5'-dithiobis(2nitrobenzoic acid) in diluted buffer is separated on a gel column into the yellow anion and the hemoglobin fraction. The plasma-DTNB mixture was read directly, and a correction for turbidity and/or color made after addition of N-ethylmaleimide. Results of a sample of human bloods are presented. The effects of several detergents yield high values of sulfhydryl in whole blood, which we believe to be artifactual. Studies of the stability of stored blood samples indicate that whole blood, but not plasma, can be kept refrigerated for several days without significant loss of sulfhydryl reaction.
Intracellular sulfhydryl levels generally are higher in cells actively performing some anabolic activity, e.g., protein synthesis, lipogenesis, or glycogenesis (1). This may reflect the high content of rough endoplasmic reticulum or other cellular components involved in synthetic activities. For example, it is likely that the islet cells of the pancreas, which synthesize insulin, have a higher sulfhydryl level than other cells in the pancreas (1). Absence or lowered amounts would therefore reflect a more catabolic or possibly stationary metabolic state. Sulfhydryl levels therefore represent a measure of the activity potential of cells for maintenance, recovery from damage, or more generally, their viability, i.e., their ability to react to stressors (2,3). As reactive groups, sulfhydryls represent potentially significant sites for reaction with a variety of environmental agents, e.g., metal ions, oxidizing agents, and hydrocarbon-like (hydrophobic) regions accessible to them. They are known to be involved at cell surfaces, as receptors, or as portions of proteins that determine the three-dimensional structure of cell receptors (4). 0003-2697/79/030098-05502.00/0 Copyright© 1979by AcademicPress, Inc. All rioht~ af r~nradlletifln
in snv fn~
ra~ervecl
98
For these reasons, we desired to assess the concentration in a readily available cell from living humans, the erythrocyte. Blood cells represent important and available tissues for assessment of various biological functions. The procedures we have developed are relatively simple and should be adaptable to a variety of clinical problems. Prior methods seemed cumbersome and therefore subject to considerable variation. The procedures devised yield a standard deviation which is 2-3% of the mean value.
MATERIALS AND METHODS
Preparation Chemicals. Sodium DTNBl:Dithionitro benzoic acid (1.98 g) was added to 50 ml of n-propanol with stirring at room temperature. To the resulting suspension 10.0 ml of 1.0 M (8.4%) sodium bicarbonate was 1Abbreviations used: DTNB, 5,5'-dithiobis(2nitrobenzoic acid); NEM, N-ethylmaleimide; NTB, nitrothiobenzoate; RBC, red blood cells; SH, sulfhydryl; SDS, sodium doceyl sulfate; HTAB, hexadecyltrimethylammonium bromide.
WHOLE BLOODAND PLASMASULFHYDRYLS added. The mixture was placed in an ice bath. After 30 min of stirring, the mixture was filtered, and the residue air dried. Column. Bio-Gel P4 was mixed with 5 vol of solution A (see below). Columns (glass, 1 × 50 cm) were poured into a packed height of 20-22 cm. Solutions. (A) Disodium EDTA, 1 mM, (372 mg) was dissolved in 1 liter of 5 mM phosphate buffer, pH 7.4. (B) NEM; Nethylmaleimide, saturated aqueous solution. (C) DTNB; sodium DTNB was dissolved in solution A, 1 mg/ml. Procedures Whole Blood (heparinized or EDTA). Two milliliters of the DTNB solution (solution C) were mixed with 0.2 ml of whole blood. The mixture was allowed to stand for at least 15 min, during which time hemolysis occurred and the sulfhydryl groups reacted with DTNB. One milliliter of this solution was placed on top of the column of Bio-Gel P4, followed by solution A after the sample had entered the column. The separation of the yellow anion from the hemoglobin proceeded readily on a column as described. The hemoglobin was separated by about 2 ml from the yellow anionic nitrothiobenzoate (NTB). For multiple assays, many columns can be run at one time. The yellow fraction was collected separately and its volume was measured (Ve). The absorbance at 410 nm was measured against a blank consisting of the DTNB solution diluted 1:15 with solution A. Sulfhydryl concentration of the original whole blood sample was calculated as follows: SW-
AAW 13,600
Ve
x --
Vs
2.2
x - - x
0.2
103
= 0.8088 (Ve) AAW (raM/liter), where: AAW is the measured absorbance difference (sample-blalik), 13600 is the molar extinction coefficient of the yellow anion (NTB), Ve is the volume of the column
99
eluate containing the yellow anion (ml) and Vs is the volume of sample applied to the column (1 ml). Plasma. Plasma sulfhydryl concentrations were determined as follows: two milliliters of the DTNB solution was mixed with 0.2 ml of plasma and allowed to stand for 15 min. Absorbance was measured against water at 410 nm. Fifteen minutes reaction gave a color which was stable for at least 45 min. Twenty microliters of Nethylmaleimide (solution B) was added to the cuvette. After 10 min the absorption was measured again; this blank accounts for the absorbance of the DTNB in the solution and any color or turbidity in the plasma. The difference in absorbance (AAP) was calculated. The s~lfhydryl concentration of the plasma sample was calculated as follows: SP-
AAP 2.2 - x--× 13,600 0.2
103
= 0.8088 AA (mM/liter). Red cell. The sulfhydryl content of the red cells was calculated from the hematocrit and the whole blood and plasma sulfhydryl levels as follows: SR=
SW-SP(1-H) H
where SR is the concentration of sulfhydryl in the red cells (mM/liter) and H is the hematocrit. RESULTS AND DISCUSSION
Quadruplicate assays of three whole blood samples done as described gave means _+ standard deviations of 6.10 _+ 0.15, 6.28 + 0.12, and 7.02 + 0.20 mM/liter blood, respectively. Quadruplicate assays of three plasma samples gave means _+ standard deviations of 0.431 _+ 0.003, 0.364 _+ 0.002, and 0.395 +_ 0.004 mM/liter plasma, respectively. The red cell sulfhydryl concentrations calculated from the above values
100
ELLMAN AND LYSKO
were 13.03 ___ 0.33, 14.12 _+ 0.28, and 14.20 _ 0.42, mM/liter RBC, respectively.
Effects of Sulfhydryl Modifying Reagents on Measured Sulfhydryl Values Detergents. It was thought desirable that detergents which are k n o w n to unfold proteins be utilized to obtain values for various types of reactive sulfhydryl groups. F o r these purposes, cationic, anionic, and nonionic detergents were examined. The results of these studies are shown in Table 1. It is evident that the use of a nonionic detergent gives values v e r y similar to those found not using any detergent, whereas cationic and anionic detergents gave higher values. We o b s e r v e d that when the higher values were seen there was also evidence of conversion of the hemoglobin to methemoglobin, i.e., oxidation of the h e m e iron. As was o b s e r v e d previously (3), this oxidation was a c c o m p a n i e d by an equivalent reduction of disulfides, hence the elevated values o b s e r v e d with SDS and H T A B are probably artifacts, and do not represent sulfhydryl groups present in blood. Other compounds. Ethanol and acetone gave b r o w n colors, i.e., methemoglobin.
Alcohols and acetone had been used as solvents earlier (5) in blood SH m e a s u r e m e n t s , and it was thought desirable to replicate these observations. Trichloracetic acid is used in procedures for making a protein-free filtrate; sulfhydryl values of the filtrate were 2 mM/liter of red cells, usually ascribed to glutathione (6).
Effects of Storage Table 2 shows the effect of storage at 4°C on the sulfhydryl content o f whole blood and of red cell-free plasma. It is evident that whole blood retained its sulfhydryl content reasonably well o v e r a 5-day period, whereas p l a s m a lost sulfhydryl at the rate of about 5%/day. Since plasma accounts for about 5% of the whole blood sulfhydryl, a loss of e v e n 25% of the p l a s m a S H in 5 days would a m o u n t to a loss of only 1.3% in the whole blood levels; hence, it would be difficult to assess.
Time of Reaction with DTNB Whole blood was allowed to react for various periods of time with D T N B in the m a n n e r described; no changes in absorbance were o b s e r v e d after the mixture was
TABLE 1 EFFECTS OF DETERGENTSON MEASUREDSULFHYDRYLLEVELSOF WHOLEBLOODa Reagent Anionic 1% Sodium Dodecyl Sulfate Cationic 0.1%, 1%, Hexadecyltrimethyl-ammonium bromide (HTAB) Nonionic 1% Triton
Results Maximum absorbance (410 nm) at 10 min which rapidly diminishes; solution becomes brown (methemoglobin).Maximumvalue corresponds to 15 m~Jliter. Results as with SDS (above).
Maximum at about 10 min, color is stable for at least 10 min; color is as for a dilute hemolyzed blood (hemoglobin). Maximum values correspond to 6 raM/liter.
a Whole blood was treated as described in the first procedure except that detergents in the final concentration indicated were included in the mixture.
101
W H O L E BLOOD AND PLASMA S U L F H Y D R Y L S TABLE2 EFFECTOFREF~GE~TEDSTORAGEONSULFHYDRYLLEVELSOF WHOLEBLOODANDPLASMA Whole blood (mM/liter)
Plasma (mM/liter)
Subject
Day 1
Day 5
Day 5/Day 1
Day 1
Day 5
Day 5/Day 1
1 2 3
6.55 5.89 6.12
6.34 5.86 6.20
0.97 0.99 1.01
0.64 0.63 0.63
0.50 0.47 0.49
0.79 0.74 0.77
incubated for more than 2 min. The increases in absorbance up to 2 min. appeared to be correlated with the extent of hemolysis. Thus, the rate-determining step for the reaction of red cell sulfhydryl and DTNB is likely to be the hemolysis of the cell. It is likely that the major portion of the sulfhydryl that reacts in whole blood samples is principally the material inside the red blood cells. Further support for this argument is shown in Table 3 which lists the whole blood, plasma, and red cell sulfhydryl levels in a number of subjects. Since the red cell sulfhydryl levels are about 22-fold those of the plasma, it is not surpris-
ing that the measured total blood levels are due principally to the availability of RBC sulfhydryl groups to the reagent, i.e., they occur only after hemolysis of the red cells. The mean whole blood sulfhydryl levels of men and women were significantly different (P < 0.01). This is, however, accounted for by the well-known difference in hematocrit, as the red cell and plasma sulfhydryl levels did not differ. If one were interested in red cell sulfhydryl only, the assay could be done on packed red cells, without the necessity for plasma determinations. As the procedures given are
TABLE3 SULFHYDRYL CONTENT OF BLOOD Male
Subject
Whole blood (mM/liter)
1 2 3 4 5 6 7 8 9 10
7.62 6.16 6.17 6.77 6.30 6.78 6.53 6.07 6.13 6.50
~" SD
6.51 0.47
Female Plasma (mM/liter)
RBC a (m~dliter)
Subject
Whole blood (mM/liter)
Plasma (mM/liter)
RBC a (mM/liter)
0.59 0.54 0.49 0.64 0.58 0.64 0.57 0.63 0.59 0.62
14.10 12.51 14.02 13.15 13.02 12.44 13.00 13.00 12.91 13.68
1 2 3 4 5 6 7 8 9 10
5.43 6.06 6.13 5.90 5.47 5.51 5.46 5.72 5.70 6.33
0.57 0.61 0.57 0.62 0.60 0.51 0.54 0.59 0.65 0.62
12.72 13.30 13.81 13.82 12.47 11.87 12.84 12.26 13.28 12.77
0.59 0.05
13.19 0.58
5.78 0.32
0.59 0.04
12.92 0.64
a Calculated as described under Materials and Methods.
102
ELLMAN AND LYSKO
quite general, they could be applied, e.g., to r e d cells s e p a r a t e d on a d e n s i t y g r a d i e n t , i . e . , as a f u n c t i o n o f t h e i r " a g e . " S i m i l a r l y , s t u d i e s o f w h i t e cells a n d p l a t e l e t s a r e possible.
ACKNOWLEDGMENT We appreciate the financial help provided by the Biomedical Research Support Grant awarded to this Institute.
REFERENCES 1. Ellman, G. L., and Gala, G. L. (1969) Exp. Brain Res. 9, 261-8. 2. Black, S. (1963)Annu. Rev. Biochem. 32, 556-82. 3. Meister, A., and Tate, S. S. (1976) Annu. Rev. Biochem. 45, 559-604. 4. Steveninck, J. V., Weed, R. I., and Rothstein, A. (1965) J. Gen. Physiol. 48, 617-29. 5. Ellman, G. L. (1959)Arch. Biochem. Biophys. 82, 70-77. 6. Beutler, E., Duron, O., and Kelley, B. M. (1963) J. Lab. Clin. Med. 61, 882-887.
A Precise Method for the Determination of Whole Blood and Plasma Sulfhydryl Groups GEORGE E L L M A N AND HIRO LYSKO
Brain-Behavior Research Center, University of California, Langley Porter Neuropsychiatric Institute, Sonoma State Hospital, Eldridge, California 95431 Received July 12, 1978 A colorimetric method for the determination of total sulfhydryl content of whole blood or plasma is presented. For whole blood, a mixture of fresh blood and 5,5'-dithiobis(2nitrobenzoic acid) in diluted buffer is separated on a gel column into the yellow anion and the hemoglobin fraction. The plasma-DTNB mixture was read directly, and a correction for turbidity and/or color made after addition of N-ethylmaleimide. Results of a sample of human bloods are presented. The effects of several detergents yield high values of sulfhydryl in whole blood, which we believe to be artifactual. Studies of the stability of stored blood samples indicate that whole blood, but not plasma, can be kept refrigerated for several days without significant loss of sulfhydryl reaction.
Intracellular sulfhydryl levels generally are higher in cells actively performing some anabolic activity, e.g., protein synthesis, lipogenesis, or glycogenesis (1). This may reflect the high content of rough endoplasmic reticulum or other cellular components involved in synthetic activities. For example, it is likely that the islet cells of the pancreas, which synthesize insulin, have a higher sulfhydryl level than other cells in the pancreas (1). Absence or lowered amounts would therefore reflect a more catabolic or possibly stationary metabolic state. Sulfhydryl levels therefore represent a measure of the activity potential of cells for maintenance, recovery from damage, or more generally, their viability, i.e., their ability to react to stressors (2,3). As reactive groups, sulfhydryls represent potentially significant sites for reaction with a variety of environmental agents, e.g., metal ions, oxidizing agents, and hydrocarbon-like (hydrophobic) regions accessible to them. They are known to be involved at cell surfaces, as receptors, or as portions of proteins that determine the three-dimensional structure of cell receptors (4). 0003-2697/79/030098-05502.00/0 Copyright© 1979by AcademicPress, Inc. All rioht~ af r~nradlletifln
in snv fn~
ra~ervecl
98
For these reasons, we desired to assess the concentration in a readily available cell from living humans, the erythrocyte. Blood cells represent important and available tissues for assessment of various biological functions. The procedures we have developed are relatively simple and should be adaptable to a variety of clinical problems. Prior methods seemed cumbersome and therefore subject to considerable variation. The procedures devised yield a standard deviation which is 2-3% of the mean value.
MATERIALS AND METHODS
Preparation Chemicals. Sodium DTNBl:Dithionitro benzoic acid (1.98 g) was added to 50 ml of n-propanol with stirring at room temperature. To the resulting suspension 10.0 ml of 1.0 M (8.4%) sodium bicarbonate was 1Abbreviations used: DTNB, 5,5'-dithiobis(2nitrobenzoic acid); NEM, N-ethylmaleimide; NTB, nitrothiobenzoate; RBC, red blood cells; SH, sulfhydryl; SDS, sodium doceyl sulfate; HTAB, hexadecyltrimethylammonium bromide.
WHOLE BLOODAND PLASMASULFHYDRYLS added. The mixture was placed in an ice bath. After 30 min of stirring, the mixture was filtered, and the residue air dried. Column. Bio-Gel P4 was mixed with 5 vol of solution A (see below). Columns (glass, 1 × 50 cm) were poured into a packed height of 20-22 cm. Solutions. (A) Disodium EDTA, 1 mM, (372 mg) was dissolved in 1 liter of 5 mM phosphate buffer, pH 7.4. (B) NEM; Nethylmaleimide, saturated aqueous solution. (C) DTNB; sodium DTNB was dissolved in solution A, 1 mg/ml. Procedures Whole Blood (heparinized or EDTA). Two milliliters of the DTNB solution (solution C) were mixed with 0.2 ml of whole blood. The mixture was allowed to stand for at least 15 min, during which time hemolysis occurred and the sulfhydryl groups reacted with DTNB. One milliliter of this solution was placed on top of the column of Bio-Gel P4, followed by solution A after the sample had entered the column. The separation of the yellow anion from the hemoglobin proceeded readily on a column as described. The hemoglobin was separated by about 2 ml from the yellow anionic nitrothiobenzoate (NTB). For multiple assays, many columns can be run at one time. The yellow fraction was collected separately and its volume was measured (Ve). The absorbance at 410 nm was measured against a blank consisting of the DTNB solution diluted 1:15 with solution A. Sulfhydryl concentration of the original whole blood sample was calculated as follows: SW-
AAW 13,600
Ve
x --
Vs
2.2
x - - x
0.2
103
= 0.8088 (Ve) AAW (raM/liter), where: AAW is the measured absorbance difference (sample-blalik), 13600 is the molar extinction coefficient of the yellow anion (NTB), Ve is the volume of the column
99
eluate containing the yellow anion (ml) and Vs is the volume of sample applied to the column (1 ml). Plasma. Plasma sulfhydryl concentrations were determined as follows: two milliliters of the DTNB solution was mixed with 0.2 ml of plasma and allowed to stand for 15 min. Absorbance was measured against water at 410 nm. Fifteen minutes reaction gave a color which was stable for at least 45 min. Twenty microliters of Nethylmaleimide (solution B) was added to the cuvette. After 10 min the absorption was measured again; this blank accounts for the absorbance of the DTNB in the solution and any color or turbidity in the plasma. The difference in absorbance (AAP) was calculated. The s~lfhydryl concentration of the plasma sample was calculated as follows: SP-
AAP 2.2 - x--× 13,600 0.2
103
= 0.8088 AA (mM/liter). Red cell. The sulfhydryl content of the red cells was calculated from the hematocrit and the whole blood and plasma sulfhydryl levels as follows: SR=
SW-SP(1-H) H
where SR is the concentration of sulfhydryl in the red cells (mM/liter) and H is the hematocrit. RESULTS AND DISCUSSION
Quadruplicate assays of three whole blood samples done as described gave means _+ standard deviations of 6.10 _+ 0.15, 6.28 + 0.12, and 7.02 + 0.20 mM/liter blood, respectively. Quadruplicate assays of three plasma samples gave means _+ standard deviations of 0.431 _+ 0.003, 0.364 _+ 0.002, and 0.395 +_ 0.004 mM/liter plasma, respectively. The red cell sulfhydryl concentrations calculated from the above values
100
ELLMAN AND LYSKO
were 13.03 ___ 0.33, 14.12 _+ 0.28, and 14.20 _ 0.42, mM/liter RBC, respectively.
Effects of Sulfhydryl Modifying Reagents on Measured Sulfhydryl Values Detergents. It was thought desirable that detergents which are k n o w n to unfold proteins be utilized to obtain values for various types of reactive sulfhydryl groups. F o r these purposes, cationic, anionic, and nonionic detergents were examined. The results of these studies are shown in Table 1. It is evident that the use of a nonionic detergent gives values v e r y similar to those found not using any detergent, whereas cationic and anionic detergents gave higher values. We o b s e r v e d that when the higher values were seen there was also evidence of conversion of the hemoglobin to methemoglobin, i.e., oxidation of the h e m e iron. As was o b s e r v e d previously (3), this oxidation was a c c o m p a n i e d by an equivalent reduction of disulfides, hence the elevated values o b s e r v e d with SDS and H T A B are probably artifacts, and do not represent sulfhydryl groups present in blood. Other compounds. Ethanol and acetone gave b r o w n colors, i.e., methemoglobin.
Alcohols and acetone had been used as solvents earlier (5) in blood SH m e a s u r e m e n t s , and it was thought desirable to replicate these observations. Trichloracetic acid is used in procedures for making a protein-free filtrate; sulfhydryl values of the filtrate were 2 mM/liter of red cells, usually ascribed to glutathione (6).
Effects of Storage Table 2 shows the effect of storage at 4°C on the sulfhydryl content o f whole blood and of red cell-free plasma. It is evident that whole blood retained its sulfhydryl content reasonably well o v e r a 5-day period, whereas p l a s m a lost sulfhydryl at the rate of about 5%/day. Since plasma accounts for about 5% of the whole blood sulfhydryl, a loss of e v e n 25% of the p l a s m a S H in 5 days would a m o u n t to a loss of only 1.3% in the whole blood levels; hence, it would be difficult to assess.
Time of Reaction with DTNB Whole blood was allowed to react for various periods of time with D T N B in the m a n n e r described; no changes in absorbance were o b s e r v e d after the mixture was
TABLE 1 EFFECTS OF DETERGENTSON MEASUREDSULFHYDRYLLEVELSOF WHOLEBLOODa Reagent Anionic 1% Sodium Dodecyl Sulfate Cationic 0.1%, 1%, Hexadecyltrimethyl-ammonium bromide (HTAB) Nonionic 1% Triton
Results Maximum absorbance (410 nm) at 10 min which rapidly diminishes; solution becomes brown (methemoglobin).Maximumvalue corresponds to 15 m~Jliter. Results as with SDS (above).
Maximum at about 10 min, color is stable for at least 10 min; color is as for a dilute hemolyzed blood (hemoglobin). Maximum values correspond to 6 raM/liter.
a Whole blood was treated as described in the first procedure except that detergents in the final concentration indicated were included in the mixture.
101
W H O L E BLOOD AND PLASMA S U L F H Y D R Y L S TABLE2 EFFECTOFREF~GE~TEDSTORAGEONSULFHYDRYLLEVELSOF WHOLEBLOODANDPLASMA Whole blood (mM/liter)
Plasma (mM/liter)
Subject
Day 1
Day 5
Day 5/Day 1
Day 1
Day 5
Day 5/Day 1
1 2 3
6.55 5.89 6.12
6.34 5.86 6.20
0.97 0.99 1.01
0.64 0.63 0.63
0.50 0.47 0.49
0.79 0.74 0.77
incubated for more than 2 min. The increases in absorbance up to 2 min. appeared to be correlated with the extent of hemolysis. Thus, the rate-determining step for the reaction of red cell sulfhydryl and DTNB is likely to be the hemolysis of the cell. It is likely that the major portion of the sulfhydryl that reacts in whole blood samples is principally the material inside the red blood cells. Further support for this argument is shown in Table 3 which lists the whole blood, plasma, and red cell sulfhydryl levels in a number of subjects. Since the red cell sulfhydryl levels are about 22-fold those of the plasma, it is not surpris-
ing that the measured total blood levels are due principally to the availability of RBC sulfhydryl groups to the reagent, i.e., they occur only after hemolysis of the red cells. The mean whole blood sulfhydryl levels of men and women were significantly different (P < 0.01). This is, however, accounted for by the well-known difference in hematocrit, as the red cell and plasma sulfhydryl levels did not differ. If one were interested in red cell sulfhydryl only, the assay could be done on packed red cells, without the necessity for plasma determinations. As the procedures given are
TABLE3 SULFHYDRYL CONTENT OF BLOOD Male
Subject
Whole blood (mM/liter)
1 2 3 4 5 6 7 8 9 10
7.62 6.16 6.17 6.77 6.30 6.78 6.53 6.07 6.13 6.50
~" SD
6.51 0.47
Female Plasma (mM/liter)
RBC a (m~dliter)
Subject
Whole blood (mM/liter)
Plasma (mM/liter)
RBC a (mM/liter)
0.59 0.54 0.49 0.64 0.58 0.64 0.57 0.63 0.59 0.62
14.10 12.51 14.02 13.15 13.02 12.44 13.00 13.00 12.91 13.68
1 2 3 4 5 6 7 8 9 10
5.43 6.06 6.13 5.90 5.47 5.51 5.46 5.72 5.70 6.33
0.57 0.61 0.57 0.62 0.60 0.51 0.54 0.59 0.65 0.62
12.72 13.30 13.81 13.82 12.47 11.87 12.84 12.26 13.28 12.77
0.59 0.05
13.19 0.58
5.78 0.32
0.59 0.04
12.92 0.64
a Calculated as described under Materials and Methods.
102
ELLMAN AND LYSKO
quite general, they could be applied, e.g., to r e d cells s e p a r a t e d on a d e n s i t y g r a d i e n t , i . e . , as a f u n c t i o n o f t h e i r " a g e . " S i m i l a r l y , s t u d i e s o f w h i t e cells a n d p l a t e l e t s a r e possible.
ACKNOWLEDGMENT We appreciate the financial help provided by the Biomedical Research Support Grant awarded to this Institute.
REFERENCES 1. Ellman, G. L., and Gala, G. L. (1969) Exp. Brain Res. 9, 261-8. 2. Black, S. (1963)Annu. Rev. Biochem. 32, 556-82. 3. Meister, A., and Tate, S. S. (1976) Annu. Rev. Biochem. 45, 559-604. 4. Steveninck, J. V., Weed, R. I., and Rothstein, A. (1965) J. Gen. Physiol. 48, 617-29. 5. Ellman, G. L. (1959)Arch. Biochem. Biophys. 82, 70-77. 6. Beutler, E., Duron, O., and Kelley, B. M. (1963) J. Lab. Clin. Med. 61, 882-887.