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A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters
ANALYTICAL
BIOCHEMISTRY
126, 433-435 (1982)
A Spectrophotometric Assay for Soluble and Immobilized A/-Hydroxysuccinimide Esters TALIA MIRON
AND MEIR
WILCHEK
Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel Received June 12, 1982 A quantitative spectrophotometric assay for N-hydroxysuccinimide and its esters based on the formation of a chromophore upon titration with mild base has been developed. The maximum absorbance for the chromophore is at 260 nm with an extinction coefficient of 9700 M-’ cm-‘. The method can be used for measuring the active esters on carriers and their interaction with amines during peptide synthesis.
cinimide can be detected on thin-layer chromatography plates with short-wave uv lamps, indicating that this compound has an absorption spectra in the uv region. Subsequently, we determined the uv spectrum of N-hydroxysuccinimide under various pH conditions and found no absorption spectra under acidic condition; under basic conditions, however, an absorption peak appeared at 260 nm. Based on these observations we have developed a method for the rapid determination of N-hydroxysuccinimide esters. (See Scheme 1.)
N-hydroxysuccinimide (NHS)’ esters are active esters that are widely used in peptide synthesis (l), the modification of amino groups of proteins and cells (2,3), and in affinity chromatography (4,5). Current methods for the assay of hydroxysuccinimide esters are based on nitrogen analysis or titration with strong bases in organic solvents (6) and require milligram quantities of the esters. Therefore, a more sensitive method is needed. Recently, we observed that hydroxysuc0 +!!!!e!L
R-t-ON
Qo-,”
R-&-N,,,
5 0
5 0 Xmox=260 6 =9700 SCHEME
MATERIALS
AND METHODS
Agarose-c-amino caproic acid, succinylpolyacrylic hydrazide agarose, (Sue-PAHA), and hydroxysuccinimide and its esters were from Miles-Yeda, Rehovot, Israel. Sepharose 4B and Sepharose-C l-4B were from Pharmacia, Uppsala, Sweden. Affigel 10, an Nhydroxysuccinimide-containing gel was from Bio-Rad, Richmond, California. N-hydroxy’ Abbreviations used: NHS, N-hydroxysuccinimide; Sue-PAHA, succinylpolyacrylic hydrazide agarose. 433
1.
succinimide chloroformate was synthesized as described (7). The N-hydroxysuccinimide derivatives of agarose-c-amino caproic acid and the Sue-PAHA were prepared by the carbodiimide method (4). Sepharose-N-hydroxysuccinimide carbonate was prepared as described (5). Standard assay condition. The general procedure for the calorimetric assay of soluble N-hydroxysuccinimide ester is as follows. 0003-2697/82/160433-03$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form resewed.
434
MIRON AND WILCHEK
For a determination of immobilized Nhydroxysuccinimide esters, 2-3 mg of dry powder or about 50 mg wet gel is incubated in the presence of 0.1 N NH40H in 2 ml for 2 to 5 min at room temperature. The carrier is removed by filtration, washed with 0.1 N NH40H, and the filtrate collected. After appropriate dilutions, the absorbance is read at 260 nm.
06
RESULTS AND DISCUSSION
Xnm
FIG. 1. Ultraviolet absorption spectra of NHS. NHS 1 X low4 M was titrated with increasing amounts of 0.1 M NH40H up to one equivalent.
A 0. l-ml sample of the ester in methanol is added to 0.9 ml of 0.1 N NH40H to yield a final concentration of about 100-PM of the ester. After the solution has stood for 2 min and not more than 20 min, at room temperature, the absorbance of the solution is read at 260 nm. Concentrations are calculated based on t 9700 M-’ cm-‘. TABLE
The increase in the absorption spectrum at 260 nm of N-hdroxysuccinimide upon its ionization with weak bases such as NH40H or bicarbonate is shown in Fig. 1. When NaOH is used for titration, care is necessary in avoiding excess NaOH because the N-hydroxysuccinimide is hydrolyzed rapidly under such conditions and uv absorption is lost. The increase in absorption was linear with ionization until an absorption maximum of 9700 M-’ cm-‘, at one equivalent of base, had been added. No further increase was observed with additional NH,OH and the color remained stable for several hours. When N-hydroxysuccinimide esters of low molecular weight were treated with 0.1 M NH,OH, the ester reacted quickly (see Scheme l), and the absorption at 260 nm could be measured immediately. The results of the spectrophotometric titration of several NHS esters with NH40H are presented in 1
SPEC~ROPHOTOMETRICDETERMINATIONOFN-HYDROXYSUCCINIMIDEESTERS Molecular weight Active estef BromoAc-NHS t-Boc-Bz-AspNHS t-Boc-Leu-NHS t-BooN-f-Z-Lys-NHS Z-D-Ala-NHS Z-DPhe-NHS
Stock solution6 (w/ml)
Calculated
Found
Factor of Purity
0.22 0.44 0.34 0.50 0.34 0.42
236 419 327 417 320 395
250 410 354 435 342 392
0.95 1.02 0.93 1.10 0.93 1.01
a Z, carbobenzoxy; NHS, N-hydroxysuccinimide esters; Bz, benzyl; t-Boc, tert.-butyloxycarbonyl. ‘After lOO-fold dilution. Average of three determinations.
ASSAY FOR N-HYDROXYSUCCINIMIDE TABLE 2 SPECTROPHOTOMETRIC DETERMINATION OF IMMOBILIZED N-HYDROXYSUCCINIMIDE ESTERS NHS column Sepharose-amino caproic-NHS SW-PAHA-NHS Sepharose-C 1-4B-Nhydroxysuccinimide carbonate All&gel 10”
rmol NHS/ wet gel 2-9 20 35 32
a From Bio-Rad.
Table 1. In a series of determinations with sample sizes of 10 to 100 nmol, accuracy was within -t5%. Aromatic amino acids interfere slightly with this determination. Commercial agarose and polyacrylamide carriers containing N-hydroxysuccinimide esters for binding to amino-containing ligands and proteins were assayed for NHS with the technique described. In the past, the amount of hydroxysuccinimide (active ester) had been determined indirectly by the amount of ligand that was coupled. For example, Cuatrecasas and Par&h (4) estimated the efficacy of agarose N-hydroxysuccinimide
ESTERS
435
esters by the amount of [C14]alanine coupled. With such an analysis, the ligand is needlessly sacrificed in order to determine the extent of activation by the ester. Our procedure avoids this step and quantitates the coupling capacity directly. Table 2 shows the spectrophotometric determination of the NHS content of several immobilized esters using the outlined reaction conditions. With this method, a determination of the upper limit coupling capacity of NHS esters is possible. REFERENCES 1. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1963) J. Amer. Chem. Sot. 85, 3039. 2. Bauminger, S., and Wilchek, M. (1980) in Methods in Enzymology (Van Vunakis, H., and Langone, J. J., eds.), Vol. 70, pp. 15 l- 159, Academic Press, New York. 3. Bayer, E. A., and Wilchek, M. (1980) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 26, pp. l-45. 4. Cuatrecasas, P., and Parikh, I. (1972) Biochemistry 12,2291-2299. 5. Wilchek, M., and Miron, T. (1982) Biochem. Int. 4,629-635. 6. Wilchek, M., Fridkin, M., and Patchomik, A. (1970) Anal. Chem. 42, 275-277. 7. Gross, H., and Bilk, L. (1967) Angew. Chem. 79, 532-533.
BIOCHEMISTRY
126, 433-435 (1982)
A Spectrophotometric Assay for Soluble and Immobilized A/-Hydroxysuccinimide Esters TALIA MIRON
AND MEIR
WILCHEK
Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel Received June 12, 1982 A quantitative spectrophotometric assay for N-hydroxysuccinimide and its esters based on the formation of a chromophore upon titration with mild base has been developed. The maximum absorbance for the chromophore is at 260 nm with an extinction coefficient of 9700 M-’ cm-‘. The method can be used for measuring the active esters on carriers and their interaction with amines during peptide synthesis.
cinimide can be detected on thin-layer chromatography plates with short-wave uv lamps, indicating that this compound has an absorption spectra in the uv region. Subsequently, we determined the uv spectrum of N-hydroxysuccinimide under various pH conditions and found no absorption spectra under acidic condition; under basic conditions, however, an absorption peak appeared at 260 nm. Based on these observations we have developed a method for the rapid determination of N-hydroxysuccinimide esters. (See Scheme 1.)
N-hydroxysuccinimide (NHS)’ esters are active esters that are widely used in peptide synthesis (l), the modification of amino groups of proteins and cells (2,3), and in affinity chromatography (4,5). Current methods for the assay of hydroxysuccinimide esters are based on nitrogen analysis or titration with strong bases in organic solvents (6) and require milligram quantities of the esters. Therefore, a more sensitive method is needed. Recently, we observed that hydroxysuc0 +!!!!e!L
R-t-ON
Qo-,”
R-&-N,,,
5 0
5 0 Xmox=260 6 =9700 SCHEME
MATERIALS
AND METHODS
Agarose-c-amino caproic acid, succinylpolyacrylic hydrazide agarose, (Sue-PAHA), and hydroxysuccinimide and its esters were from Miles-Yeda, Rehovot, Israel. Sepharose 4B and Sepharose-C l-4B were from Pharmacia, Uppsala, Sweden. Affigel 10, an Nhydroxysuccinimide-containing gel was from Bio-Rad, Richmond, California. N-hydroxy’ Abbreviations used: NHS, N-hydroxysuccinimide; Sue-PAHA, succinylpolyacrylic hydrazide agarose. 433
1.
succinimide chloroformate was synthesized as described (7). The N-hydroxysuccinimide derivatives of agarose-c-amino caproic acid and the Sue-PAHA were prepared by the carbodiimide method (4). Sepharose-N-hydroxysuccinimide carbonate was prepared as described (5). Standard assay condition. The general procedure for the calorimetric assay of soluble N-hydroxysuccinimide ester is as follows. 0003-2697/82/160433-03$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form resewed.
434
MIRON AND WILCHEK
For a determination of immobilized Nhydroxysuccinimide esters, 2-3 mg of dry powder or about 50 mg wet gel is incubated in the presence of 0.1 N NH40H in 2 ml for 2 to 5 min at room temperature. The carrier is removed by filtration, washed with 0.1 N NH40H, and the filtrate collected. After appropriate dilutions, the absorbance is read at 260 nm.
06
RESULTS AND DISCUSSION
Xnm
FIG. 1. Ultraviolet absorption spectra of NHS. NHS 1 X low4 M was titrated with increasing amounts of 0.1 M NH40H up to one equivalent.
A 0. l-ml sample of the ester in methanol is added to 0.9 ml of 0.1 N NH40H to yield a final concentration of about 100-PM of the ester. After the solution has stood for 2 min and not more than 20 min, at room temperature, the absorbance of the solution is read at 260 nm. Concentrations are calculated based on t 9700 M-’ cm-‘. TABLE
The increase in the absorption spectrum at 260 nm of N-hdroxysuccinimide upon its ionization with weak bases such as NH40H or bicarbonate is shown in Fig. 1. When NaOH is used for titration, care is necessary in avoiding excess NaOH because the N-hydroxysuccinimide is hydrolyzed rapidly under such conditions and uv absorption is lost. The increase in absorption was linear with ionization until an absorption maximum of 9700 M-’ cm-‘, at one equivalent of base, had been added. No further increase was observed with additional NH,OH and the color remained stable for several hours. When N-hydroxysuccinimide esters of low molecular weight were treated with 0.1 M NH,OH, the ester reacted quickly (see Scheme l), and the absorption at 260 nm could be measured immediately. The results of the spectrophotometric titration of several NHS esters with NH40H are presented in 1
SPEC~ROPHOTOMETRICDETERMINATIONOFN-HYDROXYSUCCINIMIDEESTERS Molecular weight Active estef BromoAc-NHS t-Boc-Bz-AspNHS t-Boc-Leu-NHS t-BooN-f-Z-Lys-NHS Z-D-Ala-NHS Z-DPhe-NHS
Stock solution6 (w/ml)
Calculated
Found
Factor of Purity
0.22 0.44 0.34 0.50 0.34 0.42
236 419 327 417 320 395
250 410 354 435 342 392
0.95 1.02 0.93 1.10 0.93 1.01
a Z, carbobenzoxy; NHS, N-hydroxysuccinimide esters; Bz, benzyl; t-Boc, tert.-butyloxycarbonyl. ‘After lOO-fold dilution. Average of three determinations.
ASSAY FOR N-HYDROXYSUCCINIMIDE TABLE 2 SPECTROPHOTOMETRIC DETERMINATION OF IMMOBILIZED N-HYDROXYSUCCINIMIDE ESTERS NHS column Sepharose-amino caproic-NHS SW-PAHA-NHS Sepharose-C 1-4B-Nhydroxysuccinimide carbonate All&gel 10”
rmol NHS/ wet gel 2-9 20 35 32
a From Bio-Rad.
Table 1. In a series of determinations with sample sizes of 10 to 100 nmol, accuracy was within -t5%. Aromatic amino acids interfere slightly with this determination. Commercial agarose and polyacrylamide carriers containing N-hydroxysuccinimide esters for binding to amino-containing ligands and proteins were assayed for NHS with the technique described. In the past, the amount of hydroxysuccinimide (active ester) had been determined indirectly by the amount of ligand that was coupled. For example, Cuatrecasas and Par&h (4) estimated the efficacy of agarose N-hydroxysuccinimide
ESTERS
435
esters by the amount of [C14]alanine coupled. With such an analysis, the ligand is needlessly sacrificed in order to determine the extent of activation by the ester. Our procedure avoids this step and quantitates the coupling capacity directly. Table 2 shows the spectrophotometric determination of the NHS content of several immobilized esters using the outlined reaction conditions. With this method, a determination of the upper limit coupling capacity of NHS esters is possible. REFERENCES 1. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1963) J. Amer. Chem. Sot. 85, 3039. 2. Bauminger, S., and Wilchek, M. (1980) in Methods in Enzymology (Van Vunakis, H., and Langone, J. J., eds.), Vol. 70, pp. 15 l- 159, Academic Press, New York. 3. Bayer, E. A., and Wilchek, M. (1980) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 26, pp. l-45. 4. Cuatrecasas, P., and Parikh, I. (1972) Biochemistry 12,2291-2299. 5. Wilchek, M., and Miron, T. (1982) Biochem. Int. 4,629-635. 6. Wilchek, M., Fridkin, M., and Patchomik, A. (1970) Anal. Chem. 42, 275-277. 7. Gross, H., and Bilk, L. (1967) Angew. Chem. 79, 532-533.