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Amperometric argentometric titration of thiol groups in imidazole buffer
SHORT
31-7
COMMUNICATIONS
3. KAY, E. R., Nature 202,390 (1964). 4. WANNEMACHER, R. W., JR., BANKS, W. L., JR., .~ND WUNNER, them. 11, 320 (1965). 5. BURTON, K., Biochem. J. 62,315 (1956). 6. GAEAZSI, M., AND DEME, I., .I. Invest. Dermatol. 27, 49 (1956).
W. H.,
D. F.
Anal.
LOGSDON,
Bio-
JR.
USAF, Radiobiology Branch USAF SchooE of Aerospace Medicine Aerospace Medical Division (AFSC) Brooks Air Force Base, Texas Received July 27, 1966
Amperometric
Argentometric in lmidazole
Titration
of
Thiol
Groups
Buffer
The amperometric titration of thiol groups in proteins and protein constituents with silver ions has been frequently used. Benesch and Benesch (1) and Kolthoff and Stricks (2) carried out such titrations in buffers of ammonia (pK 9.2) at pH around 9. Benesch, Lardy, and Benesch (3) recommended the application of a buffer of tris(hydroxymethyl)aminomethane (pK 8.2) at pH 7.4. In these media theoretical values were obtained for the low molecular weight compounds glutathione (1, 3, 4-6)) tert-dodecyl mercaptan (3, 5)) and 2-mercaptoethanol (6). However, high values of ll@-150% were obtained for cysteine (5-g), cysteine ethyl ester (5)) cysteamine (9), and thioglycolic acid (5, 6). Kolthoff and Eisenstldter (6) observed that addition of 0.1 M sulfite may reduce or even abolish such errors. However, the presence of sulfite is undesirable in many cases, since sulfite is able to produce additional thiol groups from disulfide bonds. The present author needed a titration medium of a lower pH range than afforded by the buffers mentioned above. Since the pK value of imidaaole is 7.1 and since the formation constants for silver-imidazole complexes are approximately the same as those for silver-ammonia complexes (lo), the applicability of imidazole buffers at pH 6-7 was tested. The results (Table 1) show that this medium is a suitable one. Moreover, not only glutathione but also cysteine gave theoretical values. On the other hand, cysteine at pH 8-9 and cysteine ester and thioglycolic acid
6.0 7.0 8.0 9.0 7.7 101 99 100 100 98
rt 4 * f f
1% (5Y 2% (20) 1% (3) 1% (3) 1%0
Glutathione
99 99 146 152 155
f f + + *
3% (10) 2% (10) 4% (4) 2% (5) 3%‘,c
L-Cyst&e hydrochloride
eater
143 + 4%=
138 f 2% (3) 136 f 2% (3)
~-Cyst&e ethyl hydmchbmde
TABLE 1 SH VALUES AB PERCENTAGE OF THEORETICAL VALUES*
136 f 3%”
119 f 2% (3) 126 f 4% (7)
Thioglycolic acid
0 The experimental methods were those described earlier (5). For protein analysis an apparatus was used with a vibrating platinum electrode (50 vibrations/set) which required only 3 ml of protein solution. Model compounds gave identical values in this apparatus and in the conventional one. All buffers were adjusted to the desired pH with nitric acid and contained 10 mM KCl, 1 mM ethylenediaminetetraacetic acid, and 0.01% of gelatin. Titrations were conducted at room temperature (N2O”C), at - 150 mv versus saturated calomel electrode, where current-voltage curves, recorded at pH 7 and pH 9, had shown up a well-defined plateau region. A current of nitrogen was found to be unnecessary at pH 6-7. b Numbers in parentheses indicate the number of titrations carried out. c From ref. (5).
0.13Mtris
0.15 M imidazole 0.05 M imidazole 0.05 M imidazole + 0.02 M borate
PH
EXPERIMENTAL
$ g $ 2 m
22 i%
8 3
R
E 00
SHORT
319
COMMUNICATIONS
at pH 6-7 again gave high values. There was some indication of electrode poisoning in these cases. In order to find out whether imidaaole can prevent undesirable interactions with other protein groups, a sample of nonactivated papain was titrated. In accordance with the literature (ll), the SH content was zero. Furthermore, a sample of bovine serum albumin (Armour Pharmaceutical Company Ltd.) was found to have the same thiol content when titrated in imidazole buffer of pH 7.0 (0.65 + 0.01 group per molecule) as in tris buffer, pH 7.7 (0.64 rt 0.03 group). This value equals the one reported by Benesch, Lardy, and Benesch in tris buffer (3). Apparently there are no undesirable interactions of silver ions with at least these two proteins. It may be concluded that imidazole buffer is a promising one for amperometric titration of protein thiol groups, of use especially when titration within the pH range of 6-7 is required. In view of the theoretical values obtained for L-cysteine, results in imidazole buffer of pH 6-7 may be even more reliable than those in the buffer systems hitherto used. ACKNOWLEDGMENT The author van Troost.
is pleased to acknowledge
the able technical assistance of Miss J. S.
REFERENCES 1. BENESCR, R., AND BENESCH, R. E., Arch. Biochem. 19,35 (1948). 2. KOLTHOFF, I. M., AND STRICKS, W., J. Am. Chem. Sot. 72, 1952 (1950). 3. BENESCH, R. E., LARDY, H. A., AND BENESCH, R., J. Biol. Chem. 216,663 4. INGRAM, v. M., &o&em. J. 59, 653 (1955). 5. SLUYTERMAN, L. A. AE., Biochim. Biophys. Acta 25,402 (1957). 6. KOLTHOFF, I. M., AND EISENSTXDTER, J., Anal. Chim. Acta 24,83 (1961). 7. STAIB, W., AND TVRBA, F., B&hem. 2.327,473 (1956). 8. BURTON, H., Biochim. Biophys. Acta 29, 193 (1958). 9. B~RRESEN, H. C., Anal. Chem. 35, 1096 (1963). 10. GOLD, D. H., AND GREGOR, H. P., J. Phys. Chem. 64, 1461 (1960). 11. FINKLE, B. J., AND SMITH, E. L., J. Biol. Chem. 230, 669 (1958).
L. A. AE. Philips
Research
Laboratories Gloeilampenfabrieken Netherlands May 20, 1966
N. V. Philips’ Eindhoven, Received
(1955).
SLUYTERMAN
31-7
COMMUNICATIONS
3. KAY, E. R., Nature 202,390 (1964). 4. WANNEMACHER, R. W., JR., BANKS, W. L., JR., .~ND WUNNER, them. 11, 320 (1965). 5. BURTON, K., Biochem. J. 62,315 (1956). 6. GAEAZSI, M., AND DEME, I., .I. Invest. Dermatol. 27, 49 (1956).
W. H.,
D. F.
Anal.
LOGSDON,
Bio-
JR.
USAF, Radiobiology Branch USAF SchooE of Aerospace Medicine Aerospace Medical Division (AFSC) Brooks Air Force Base, Texas Received July 27, 1966
Amperometric
Argentometric in lmidazole
Titration
of
Thiol
Groups
Buffer
The amperometric titration of thiol groups in proteins and protein constituents with silver ions has been frequently used. Benesch and Benesch (1) and Kolthoff and Stricks (2) carried out such titrations in buffers of ammonia (pK 9.2) at pH around 9. Benesch, Lardy, and Benesch (3) recommended the application of a buffer of tris(hydroxymethyl)aminomethane (pK 8.2) at pH 7.4. In these media theoretical values were obtained for the low molecular weight compounds glutathione (1, 3, 4-6)) tert-dodecyl mercaptan (3, 5)) and 2-mercaptoethanol (6). However, high values of ll@-150% were obtained for cysteine (5-g), cysteine ethyl ester (5)) cysteamine (9), and thioglycolic acid (5, 6). Kolthoff and Eisenstldter (6) observed that addition of 0.1 M sulfite may reduce or even abolish such errors. However, the presence of sulfite is undesirable in many cases, since sulfite is able to produce additional thiol groups from disulfide bonds. The present author needed a titration medium of a lower pH range than afforded by the buffers mentioned above. Since the pK value of imidaaole is 7.1 and since the formation constants for silver-imidazole complexes are approximately the same as those for silver-ammonia complexes (lo), the applicability of imidazole buffers at pH 6-7 was tested. The results (Table 1) show that this medium is a suitable one. Moreover, not only glutathione but also cysteine gave theoretical values. On the other hand, cysteine at pH 8-9 and cysteine ester and thioglycolic acid
6.0 7.0 8.0 9.0 7.7 101 99 100 100 98
rt 4 * f f
1% (5Y 2% (20) 1% (3) 1% (3) 1%0
Glutathione
99 99 146 152 155
f f + + *
3% (10) 2% (10) 4% (4) 2% (5) 3%‘,c
L-Cyst&e hydrochloride
eater
143 + 4%=
138 f 2% (3) 136 f 2% (3)
~-Cyst&e ethyl hydmchbmde
TABLE 1 SH VALUES AB PERCENTAGE OF THEORETICAL VALUES*
136 f 3%”
119 f 2% (3) 126 f 4% (7)
Thioglycolic acid
0 The experimental methods were those described earlier (5). For protein analysis an apparatus was used with a vibrating platinum electrode (50 vibrations/set) which required only 3 ml of protein solution. Model compounds gave identical values in this apparatus and in the conventional one. All buffers were adjusted to the desired pH with nitric acid and contained 10 mM KCl, 1 mM ethylenediaminetetraacetic acid, and 0.01% of gelatin. Titrations were conducted at room temperature (N2O”C), at - 150 mv versus saturated calomel electrode, where current-voltage curves, recorded at pH 7 and pH 9, had shown up a well-defined plateau region. A current of nitrogen was found to be unnecessary at pH 6-7. b Numbers in parentheses indicate the number of titrations carried out. c From ref. (5).
0.13Mtris
0.15 M imidazole 0.05 M imidazole 0.05 M imidazole + 0.02 M borate
PH
EXPERIMENTAL
$ g $ 2 m
22 i%
8 3
R
E 00
SHORT
319
COMMUNICATIONS
at pH 6-7 again gave high values. There was some indication of electrode poisoning in these cases. In order to find out whether imidaaole can prevent undesirable interactions with other protein groups, a sample of nonactivated papain was titrated. In accordance with the literature (ll), the SH content was zero. Furthermore, a sample of bovine serum albumin (Armour Pharmaceutical Company Ltd.) was found to have the same thiol content when titrated in imidazole buffer of pH 7.0 (0.65 + 0.01 group per molecule) as in tris buffer, pH 7.7 (0.64 rt 0.03 group). This value equals the one reported by Benesch, Lardy, and Benesch in tris buffer (3). Apparently there are no undesirable interactions of silver ions with at least these two proteins. It may be concluded that imidazole buffer is a promising one for amperometric titration of protein thiol groups, of use especially when titration within the pH range of 6-7 is required. In view of the theoretical values obtained for L-cysteine, results in imidazole buffer of pH 6-7 may be even more reliable than those in the buffer systems hitherto used. ACKNOWLEDGMENT The author van Troost.
is pleased to acknowledge
the able technical assistance of Miss J. S.
REFERENCES 1. BENESCR, R., AND BENESCH, R. E., Arch. Biochem. 19,35 (1948). 2. KOLTHOFF, I. M., AND STRICKS, W., J. Am. Chem. Sot. 72, 1952 (1950). 3. BENESCH, R. E., LARDY, H. A., AND BENESCH, R., J. Biol. Chem. 216,663 4. INGRAM, v. M., &o&em. J. 59, 653 (1955). 5. SLUYTERMAN, L. A. AE., Biochim. Biophys. Acta 25,402 (1957). 6. KOLTHOFF, I. M., AND EISENSTXDTER, J., Anal. Chim. Acta 24,83 (1961). 7. STAIB, W., AND TVRBA, F., B&hem. 2.327,473 (1956). 8. BURTON, H., Biochim. Biophys. Acta 29, 193 (1958). 9. B~RRESEN, H. C., Anal. Chem. 35, 1096 (1963). 10. GOLD, D. H., AND GREGOR, H. P., J. Phys. Chem. 64, 1461 (1960). 11. FINKLE, B. J., AND SMITH, E. L., J. Biol. Chem. 230, 669 (1958).
L. A. AE. Philips
Research
Laboratories Gloeilampenfabrieken Netherlands May 20, 1966
N. V. Philips’ Eindhoven, Received
(1955).
SLUYTERMAN