307
Biotechnol. Prog. 1990, 6, 307-309
NOTES N-Laurylbiotinamide as Affinity Surfactant Robert W. Coughlin' and Jeffrey B. Baclaski Department of Chemical Engineering and Biotechnology Center, University of Connecticut, Storrs, Connecticut 06269
N-laurylbiotinamide (NLB), which retains strong affinity for the protein avidin, was synthesized from biotin and N-laurylamine via the biotin ester of N-hydroxysuccinimide and characterized by NMR. When the synthesized NLB was used as a cosurfactant with AOT to form a reverse micellar system in isooctane, it was found to extend the p H range over which avidin can be transferred from a continuous aqueous solution to the reverse-micellar phase. This behavior is similar to that already reported for a different affinity surfactant, n-octyl P-D-glucopyranoside.
Considerable information is now available regarding the solubilization of biomolecules in reversed micelles, ranging from the early work of Luisi and co-workers (I)to the more recent work of Hatton and co-workers (2), who have demonstrated how partitioning between an aqueous phase and an organic phase incorporating reversed micelles can be used to separate proteins on the basis of their isoelectric points and sizes. Generally, pH, ionic strength, and surfactant concentration are major factors that govern the distribution of proteins between reversed micelles in an organic solvent and an aqueous solution. The phenomenon is also strongly influenced by the types of salt present, the types of surfactant and organic solvent employed, and the nature of the proteins. A very important role is played by pH, which governs the charge borne by the protein. Incorporation of proteins into reversed micelles depends strongly on the electrostatic interaction between protein and charged head groups of the ionic surface-active agents usually employed to induce micelle formation. Ionic effects of salts arise from their Debye shielding of the interactions between proteins and surfactants and the shielding between charged surfactants, which can result in smaller micelles. Salts can also interact nonspecifically with proteins and surfactants and even displace proteins from the micellar water domains. The possibility of incorporating into micelles certain affinity molecules that strongly bind proteins was proposed in 1986 (3), and an example was first reported by Hatton and co-workers in 1987, with further details given in ref 4. In the latter work, small amounts of the affinity cosurfactant n-octyl P-Dglucopyranoside were employed with the anionic surfactant Aerosol OT and found to increase substantially the amount of the protein concanavalin A transferred from aqueous solution to reversed micelles; at the same time, there was no influence on the transfer of ribonuclease A, which does not have the strong affinity possessed by concanavalin A for the glycosyl protion of the cosurfactant. The increased transfer was observed at pH values near that of the isoelectric point and ascribed to affinity interaction between the concanavalin A and the glycosyl moiety of the cosurfactant. 8756-7938/90/3006-0307$02.50/0
In the present work, we have synthesized the affinity surfactant N-laurylbiotinamide (NLB) and demonstrated its significant enhancement of transfer of the protein avidin from aqueous solution into isooctane containing reversed micelles formed with the aid of Aerosol OT.
Experimental Procedures The reagents employed included the following: dioxane from Fluka Chemical; D-biotin, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide,N,N'-dimethylformamide, n-laurylamine (n-dodecylamine), dimethyl sulfoxide, and tetramethylsilane from Sigma Chemical; streptavidin from Chemical Dynamics; isooctane and Aerosol OT (AOT) from Fisher Scientific; 2-propanol, NaZHP04 (sodium phosphate, dibasic), NaHZP04 (sodium phosphate, monobasic), NaHC03 (sodium bicarbonate), and NazC03 (sodium carbonate) from Baker Chemical. Biotin (B) was activated by forming the ester between its carboxylic group and N-hydroxysuccinimide (NHS), with N,"-dicyclohexylcarbodiimide (DCC) as dehydrating agent. D-Biotin (976 mg) was dissolved at 80 "C in 12 mL of dimethylformamide (DMF). To this were added 472 mg of NHS and 824 mg of DCC. After the resulting mixture was stirred at room temperature for about 2 h, a white precipitate formed. The precipitate (presumably dicyclohexylurea) was filtered, and the supernatant containing the biotin ester intermediate was evaporated to dryness under vacuum at 60 "C. The resulting residue was dissolved in refluxing 2-propanol and recrystallized to give a product of melting point 210 "C, which is that reported by Becker et al. (5) for the expected biotin NHS ester. The yield was 60%. The biotin ester of NHS (341 mg) was added to 15 mL of dry dioxane at 70-80 "C, followed by the addition of 185 mg of n-laurylamine, after which all solids dissolved. The reaction mixture was maintained at 40 "C for 2 h, during which time a white precipitate formed. Dioxane was removed by vacuum evaporation at 50 "C and the waxy white solid product was washed in double-distilled water, filtered, and dried. Its melting point was 184-189 "C.
0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6, No. 4
308
a
Table I. NMR SDectra. (200 MHz)
D-bioiln 8
D-Biotin NMR in DMSO 6.7 (bs, 1 H, NH), 6.5 (bs, 1 H, NH), 4.3 (m, 2 H, NCHCHN), 3.15 (bd, 1 H, CHS), 2.75 (m, 2 H, CHzS), 2.22 (t, 2 H, CHz), and 1.5 (bm, 6 H) D-Biotin NHS Ester NMR in DMSO 6.7 (bs, 1 H, NH), 4.3 (m, 2 H, NCHCHN), 3.15 (bd, 1 H, CHS), 2.81 (s,4 H, COCHzCHzCO), 2.75 (m, 2 H, CHzS), 2.22 (t, 2 H, CHz), and 1.5 (bm, 6 H)
NLB Product NMR in DMSO 6.7 (bs, 1 H, NH), 6.5 (bs, 1 H, NH), 4.3 (m, 2 H, NCHCHN), 3.15 (bd, 1 H, CHS), 2.75 (m, 2 H, CHzS), 2.22 (t, 2 H, CHz), and 1.5 (bm, 31 H) a Numerical values given are chemical shifts (ppm) measured with respect to the standard (trimethylsilane). Designations in parentheses indicate resonance shape, the number of protons represented by the area of the resonance peak, and the assignment of proton location, in that order. s = singlet, b = broad, d = doublet, t = triplet, and m = multiplet.
7
6
b
B
1
5
4 3 2 Chemical Shift Ippm]
1
0
Bioiinyl-N-hydmxysuccinimlde eder
D
4
' H I H IH
A\
"7 HN
7
-(CH2)4CO
6
/--
/
1
5
4
3
2
1
0
Chemicd Shift [ppm] N-lauryl biollnamide
C
I
8
7
6
. .
B
-
2
-
HX)
i
i
i
0
Chemical ShM [Ppml
Figure 1. NMR spectra of (a) biotin, (b) biotin N-hydroxysuccinimide ester, and (c) N-laurylbiotinamide. The various protons are designated by a capital letter to show the correspondence between the individual resonances and positions in the chemical structures.
NMR spectra were obtained in deuterated DMSO by using tetramethylsilane as a standard and a 200-MHz spectrometer, Bruker Company, Model IBMWP200SY. The resulting spectra for biotin NHS ester and the NLB product are given in Figure 1 and the characteristic chemical shifts summarized in Table I. The biotin spectra included several resonances not previously reported by Confalone et al. (6). These were presumably impurities, and the same lines appeared in the spectrum of the unpurified biotin NHS ester but disappeared upon recrystallization from 2-propanol. Partitioning of avidin was accomplished by contacting an aqueous avidin solution (5 mL) of desired pH, ionic strength, and protein concentration with an equal volume of isooctane containing AOT (100 mM) and, in some cases, the affinity cosurfactant NLB (3 mM). The starting aqueous solution contained 0.5 mg of avidin/mL. It was made up by mixing 2.5 mL of stock solution containing protein (1mg/mL) and NaCl [0.1 MI with 2.5 mL of a 0.1 M buffer solution. Sodium phosphate buffer was used for pH 6-8 and carbonate buffer for the pH range 9-10.5. The pH values reported as part of the results were measured after the phases had equilibrated. After the mixtures were agitated for 5 min in 50-mL beakers at room temperature in a water bath, they were transferred to test tubes, centrifuged for 15 min, and then equilibrated for 24 h a t 20 "C. After equilibration, the phases were separated and the pH and absorbance (280 nm) of the aqueous phase measured. Absorbance was measured in a double-beam Perkin-Elmer spectrometer, Model Lambda 3B, using as a standard a similar aqueous solution that did not contain protein and that had been similarly equilibrated with the organic phase. The fraction of avidin transferred to the organic phase was about 90% in the pH range of about 6.3 to about 9.5, regardless of whether the NLB affinity surfactant was present or not. A t higher pH, however, there was a significant decrease in the amounts transferred, as indicated in Figure 2. I t is evident that a t pH 10 no transfer of avidin from the aqueous phase was detected in the absence of affinity surfactant, whereas in the presence of 3 mM NLB about 85 $3 transfer was observed. Thus, the effect of the affinity surfactant appears to become significant only at pH's in the region of the isoelectric point, which is 10.5 for avidin. A similar enhancement of transfer in the presence of an affinity surfactant was obtained by Woll et al. ( 4 ) for the transfer of concanavalin A above a pH of about 6.5; a t lower pH, transfer was high, regardless of the presence or absence
309
Biotechnol. Prog., 1990, Vol. 6, NO. 4 1
.
0.1 M, 50% Carbonate Buffer, 1 OOmM AOT 0 7
O*gl
I
w 0.8 n
\ \ \\
0.1
f
0.0 4 9.0
\
\ 9.5
pH
w
10.0
action involves a part of the biotin molecule far removed from its carboxylic side chain.
Acknowledgment
i
I
We thank the National Science Foundation for support under Grants CBT-8717768 and INT-8903509. Additional support came from the Research Foundation and the Biotechnology Center of the University of Connecticut. Dennis McCann of the School of Pharmacy helped in obtaining and interpreting NMR spectra.
Literature Cited i
10.5
Figure 2. Results of equilibrium experiments of the transfer of avidin from aqueous, continuous phase to reversed micelles in isooctane. 0 , 3.0 mM N-laurylbiotinamide; 0,no N-laurylbiotinamide.
of the affinity agent. Thus, where the charge on the protein is opposite to that on the polar head group of the major surfactant, the affinity attraction does not appear to enhance transfer significantly beyond that attributed to the electrostatic effect a t pH values. The effect does appear to be significant a t pH’s where the protein is without charge, however. At pH’s where the protein takes on a charge identical with that of the polar head group of the major surfactant (pH > 10.5 in the present case), the effect of the affinity cosurfactant is also expected to be small, as in the case of the much lower pH’s. In the present case, the hydrophobic moiety of the affinity cosurfactant was bonded to biotin via the carboxylic group at the end of the biotin side chain. This is the same site at which biotin is attached to enzymes for which it serves as a prosthetic group (7). For example, in pyruvate decarboxylase (8),the role of biotin is to form a carboxy intermediate by bonding COz at the ring nitrogen opposite the ring carbon to which the side chain of biotin is attached. The inhibition of this enzymatic reaction by avidin suggests that the avidin-biotin affinity inter-
(1)Luisi, P. L.; Henninger, F.; Joppich, M.; Dossena, A.; Casnati, G. Biochem. Biophys. Res. Commun. 1977, 74(4), 1384; Luisi, P. L.; Bonner, F. L.; Pellegrini, A.; Wiget, P.; Wolf, R. Helu. Chim. Acta 1979, 62(3), 740. (2) Woll, J. M.; Dillon, A. S.; Rahaman, R. S.; Hatton, T. A. In Protein Purification: Micro to Macro;Alan R. Liss Inc.: New York, 1987; pp 117-130; Goklin, K. E.; Hatton, T. A. Biotechnol. Prog. 1985, 1, 1;Abbott, N. L.; Hatton, T. A. Chem. Eng. Prog. 1988, 84, 31-41. (3) Coughlin, R. W. Engineering Approaches to Continuous Affinity Purification. Proposal submitted to National Science Foundation, 1986. (4) Woll, J. M.; Hatton, T. A.; Yarmush, M. L. Biotechnol. Prog. 1989,5(2), 57-63. (5) Becker, J. M.; Wilchek, M.; Katchalski, E. Proc. Natl. Acad. Sci. U.S.A. 1971,68, 2604. ( 6 )Confalone,P. N.; Pizzolato, G.; Uskokovic, M. R. J. Org. Chem. 1977,42, 1630. (7) Wood, H. G.; Barden, R. E. Annu. Reu. Biochem. 1977, 46, 385-414. ( 8 ) Scrutton, M. C.; Young, M. R. In The Enzymes, 3rd Ed.; Boyer, P. D., Ed.; Academic Press: Orlando, FL, 1972; Vol. 6, pp 1-35. Accepted May 30, 1990.
Registry No. NLB, 128631-44-7; NHS, 6066-82-6; B, 58-855; Aerosol OT, 577-11-7; biotin NHS ester, 128631-45-8; laurylamine, 124-22-1.
Biotechnol. Prog. 1990, 6, 307-309
NOTES N-Laurylbiotinamide as Affinity Surfactant Robert W. Coughlin' and Jeffrey B. Baclaski Department of Chemical Engineering and Biotechnology Center, University of Connecticut, Storrs, Connecticut 06269
N-laurylbiotinamide (NLB), which retains strong affinity for the protein avidin, was synthesized from biotin and N-laurylamine via the biotin ester of N-hydroxysuccinimide and characterized by NMR. When the synthesized NLB was used as a cosurfactant with AOT to form a reverse micellar system in isooctane, it was found to extend the p H range over which avidin can be transferred from a continuous aqueous solution to the reverse-micellar phase. This behavior is similar to that already reported for a different affinity surfactant, n-octyl P-D-glucopyranoside.
Considerable information is now available regarding the solubilization of biomolecules in reversed micelles, ranging from the early work of Luisi and co-workers (I)to the more recent work of Hatton and co-workers (2), who have demonstrated how partitioning between an aqueous phase and an organic phase incorporating reversed micelles can be used to separate proteins on the basis of their isoelectric points and sizes. Generally, pH, ionic strength, and surfactant concentration are major factors that govern the distribution of proteins between reversed micelles in an organic solvent and an aqueous solution. The phenomenon is also strongly influenced by the types of salt present, the types of surfactant and organic solvent employed, and the nature of the proteins. A very important role is played by pH, which governs the charge borne by the protein. Incorporation of proteins into reversed micelles depends strongly on the electrostatic interaction between protein and charged head groups of the ionic surface-active agents usually employed to induce micelle formation. Ionic effects of salts arise from their Debye shielding of the interactions between proteins and surfactants and the shielding between charged surfactants, which can result in smaller micelles. Salts can also interact nonspecifically with proteins and surfactants and even displace proteins from the micellar water domains. The possibility of incorporating into micelles certain affinity molecules that strongly bind proteins was proposed in 1986 (3), and an example was first reported by Hatton and co-workers in 1987, with further details given in ref 4. In the latter work, small amounts of the affinity cosurfactant n-octyl P-Dglucopyranoside were employed with the anionic surfactant Aerosol OT and found to increase substantially the amount of the protein concanavalin A transferred from aqueous solution to reversed micelles; at the same time, there was no influence on the transfer of ribonuclease A, which does not have the strong affinity possessed by concanavalin A for the glycosyl protion of the cosurfactant. The increased transfer was observed at pH values near that of the isoelectric point and ascribed to affinity interaction between the concanavalin A and the glycosyl moiety of the cosurfactant. 8756-7938/90/3006-0307$02.50/0
In the present work, we have synthesized the affinity surfactant N-laurylbiotinamide (NLB) and demonstrated its significant enhancement of transfer of the protein avidin from aqueous solution into isooctane containing reversed micelles formed with the aid of Aerosol OT.
Experimental Procedures The reagents employed included the following: dioxane from Fluka Chemical; D-biotin, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide,N,N'-dimethylformamide, n-laurylamine (n-dodecylamine), dimethyl sulfoxide, and tetramethylsilane from Sigma Chemical; streptavidin from Chemical Dynamics; isooctane and Aerosol OT (AOT) from Fisher Scientific; 2-propanol, NaZHP04 (sodium phosphate, dibasic), NaHZP04 (sodium phosphate, monobasic), NaHC03 (sodium bicarbonate), and NazC03 (sodium carbonate) from Baker Chemical. Biotin (B) was activated by forming the ester between its carboxylic group and N-hydroxysuccinimide (NHS), with N,"-dicyclohexylcarbodiimide (DCC) as dehydrating agent. D-Biotin (976 mg) was dissolved at 80 "C in 12 mL of dimethylformamide (DMF). To this were added 472 mg of NHS and 824 mg of DCC. After the resulting mixture was stirred at room temperature for about 2 h, a white precipitate formed. The precipitate (presumably dicyclohexylurea) was filtered, and the supernatant containing the biotin ester intermediate was evaporated to dryness under vacuum at 60 "C. The resulting residue was dissolved in refluxing 2-propanol and recrystallized to give a product of melting point 210 "C, which is that reported by Becker et al. (5) for the expected biotin NHS ester. The yield was 60%. The biotin ester of NHS (341 mg) was added to 15 mL of dry dioxane at 70-80 "C, followed by the addition of 185 mg of n-laurylamine, after which all solids dissolved. The reaction mixture was maintained at 40 "C for 2 h, during which time a white precipitate formed. Dioxane was removed by vacuum evaporation at 50 "C and the waxy white solid product was washed in double-distilled water, filtered, and dried. Its melting point was 184-189 "C.
0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6, No. 4
308
a
Table I. NMR SDectra. (200 MHz)
D-bioiln 8
D-Biotin NMR in DMSO 6.7 (bs, 1 H, NH), 6.5 (bs, 1 H, NH), 4.3 (m, 2 H, NCHCHN), 3.15 (bd, 1 H, CHS), 2.75 (m, 2 H, CHzS), 2.22 (t, 2 H, CHz), and 1.5 (bm, 6 H) D-Biotin NHS Ester NMR in DMSO 6.7 (bs, 1 H, NH), 4.3 (m, 2 H, NCHCHN), 3.15 (bd, 1 H, CHS), 2.81 (s,4 H, COCHzCHzCO), 2.75 (m, 2 H, CHzS), 2.22 (t, 2 H, CHz), and 1.5 (bm, 6 H)
NLB Product NMR in DMSO 6.7 (bs, 1 H, NH), 6.5 (bs, 1 H, NH), 4.3 (m, 2 H, NCHCHN), 3.15 (bd, 1 H, CHS), 2.75 (m, 2 H, CHzS), 2.22 (t, 2 H, CHz), and 1.5 (bm, 31 H) a Numerical values given are chemical shifts (ppm) measured with respect to the standard (trimethylsilane). Designations in parentheses indicate resonance shape, the number of protons represented by the area of the resonance peak, and the assignment of proton location, in that order. s = singlet, b = broad, d = doublet, t = triplet, and m = multiplet.
7
6
b
B
1
5
4 3 2 Chemical Shift Ippm]
1
0
Bioiinyl-N-hydmxysuccinimlde eder
D
4
' H I H IH
A\
"7 HN
7
-(CH2)4CO
6
/--
/
1
5
4
3
2
1
0
Chemicd Shift [ppm] N-lauryl biollnamide
C
I
8
7
6
. .
B
-
2
-
HX)
i
i
i
0
Chemical ShM [Ppml
Figure 1. NMR spectra of (a) biotin, (b) biotin N-hydroxysuccinimide ester, and (c) N-laurylbiotinamide. The various protons are designated by a capital letter to show the correspondence between the individual resonances and positions in the chemical structures.
NMR spectra were obtained in deuterated DMSO by using tetramethylsilane as a standard and a 200-MHz spectrometer, Bruker Company, Model IBMWP200SY. The resulting spectra for biotin NHS ester and the NLB product are given in Figure 1 and the characteristic chemical shifts summarized in Table I. The biotin spectra included several resonances not previously reported by Confalone et al. (6). These were presumably impurities, and the same lines appeared in the spectrum of the unpurified biotin NHS ester but disappeared upon recrystallization from 2-propanol. Partitioning of avidin was accomplished by contacting an aqueous avidin solution (5 mL) of desired pH, ionic strength, and protein concentration with an equal volume of isooctane containing AOT (100 mM) and, in some cases, the affinity cosurfactant NLB (3 mM). The starting aqueous solution contained 0.5 mg of avidin/mL. It was made up by mixing 2.5 mL of stock solution containing protein (1mg/mL) and NaCl [0.1 MI with 2.5 mL of a 0.1 M buffer solution. Sodium phosphate buffer was used for pH 6-8 and carbonate buffer for the pH range 9-10.5. The pH values reported as part of the results were measured after the phases had equilibrated. After the mixtures were agitated for 5 min in 50-mL beakers at room temperature in a water bath, they were transferred to test tubes, centrifuged for 15 min, and then equilibrated for 24 h a t 20 "C. After equilibration, the phases were separated and the pH and absorbance (280 nm) of the aqueous phase measured. Absorbance was measured in a double-beam Perkin-Elmer spectrometer, Model Lambda 3B, using as a standard a similar aqueous solution that did not contain protein and that had been similarly equilibrated with the organic phase. The fraction of avidin transferred to the organic phase was about 90% in the pH range of about 6.3 to about 9.5, regardless of whether the NLB affinity surfactant was present or not. A t higher pH, however, there was a significant decrease in the amounts transferred, as indicated in Figure 2. I t is evident that a t pH 10 no transfer of avidin from the aqueous phase was detected in the absence of affinity surfactant, whereas in the presence of 3 mM NLB about 85 $3 transfer was observed. Thus, the effect of the affinity surfactant appears to become significant only at pH's in the region of the isoelectric point, which is 10.5 for avidin. A similar enhancement of transfer in the presence of an affinity surfactant was obtained by Woll et al. ( 4 ) for the transfer of concanavalin A above a pH of about 6.5; a t lower pH, transfer was high, regardless of the presence or absence
309
Biotechnol. Prog., 1990, Vol. 6, NO. 4 1
.
0.1 M, 50% Carbonate Buffer, 1 OOmM AOT 0 7
O*gl
I
w 0.8 n
\ \ \\
0.1
f
0.0 4 9.0
\
\ 9.5
pH
w
10.0
action involves a part of the biotin molecule far removed from its carboxylic side chain.
Acknowledgment
i
I
We thank the National Science Foundation for support under Grants CBT-8717768 and INT-8903509. Additional support came from the Research Foundation and the Biotechnology Center of the University of Connecticut. Dennis McCann of the School of Pharmacy helped in obtaining and interpreting NMR spectra.
Literature Cited i
10.5
Figure 2. Results of equilibrium experiments of the transfer of avidin from aqueous, continuous phase to reversed micelles in isooctane. 0 , 3.0 mM N-laurylbiotinamide; 0,no N-laurylbiotinamide.
of the affinity agent. Thus, where the charge on the protein is opposite to that on the polar head group of the major surfactant, the affinity attraction does not appear to enhance transfer significantly beyond that attributed to the electrostatic effect a t pH values. The effect does appear to be significant a t pH’s where the protein is without charge, however. At pH’s where the protein takes on a charge identical with that of the polar head group of the major surfactant (pH > 10.5 in the present case), the effect of the affinity cosurfactant is also expected to be small, as in the case of the much lower pH’s. In the present case, the hydrophobic moiety of the affinity cosurfactant was bonded to biotin via the carboxylic group at the end of the biotin side chain. This is the same site at which biotin is attached to enzymes for which it serves as a prosthetic group (7). For example, in pyruvate decarboxylase (8),the role of biotin is to form a carboxy intermediate by bonding COz at the ring nitrogen opposite the ring carbon to which the side chain of biotin is attached. The inhibition of this enzymatic reaction by avidin suggests that the avidin-biotin affinity inter-
(1)Luisi, P. L.; Henninger, F.; Joppich, M.; Dossena, A.; Casnati, G. Biochem. Biophys. Res. Commun. 1977, 74(4), 1384; Luisi, P. L.; Bonner, F. L.; Pellegrini, A.; Wiget, P.; Wolf, R. Helu. Chim. Acta 1979, 62(3), 740. (2) Woll, J. M.; Dillon, A. S.; Rahaman, R. S.; Hatton, T. A. In Protein Purification: Micro to Macro;Alan R. Liss Inc.: New York, 1987; pp 117-130; Goklin, K. E.; Hatton, T. A. Biotechnol. Prog. 1985, 1, 1;Abbott, N. L.; Hatton, T. A. Chem. Eng. Prog. 1988, 84, 31-41. (3) Coughlin, R. W. Engineering Approaches to Continuous Affinity Purification. Proposal submitted to National Science Foundation, 1986. (4) Woll, J. M.; Hatton, T. A.; Yarmush, M. L. Biotechnol. Prog. 1989,5(2), 57-63. (5) Becker, J. M.; Wilchek, M.; Katchalski, E. Proc. Natl. Acad. Sci. U.S.A. 1971,68, 2604. ( 6 )Confalone,P. N.; Pizzolato, G.; Uskokovic, M. R. J. Org. Chem. 1977,42, 1630. (7) Wood, H. G.; Barden, R. E. Annu. Reu. Biochem. 1977, 46, 385-414. ( 8 ) Scrutton, M. C.; Young, M. R. In The Enzymes, 3rd Ed.; Boyer, P. D., Ed.; Academic Press: Orlando, FL, 1972; Vol. 6, pp 1-35. Accepted May 30, 1990.
Registry No. NLB, 128631-44-7; NHS, 6066-82-6; B, 58-855; Aerosol OT, 577-11-7; biotin NHS ester, 128631-45-8; laurylamine, 124-22-1.
