Biochimica et Biophysica Acts, 386 0975) 470---478
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37012 SYNTHESIS OF I M M O B I L I Z E D FLAVIN DERIVATIVES A N D T H E I R USE IN P U R I F I C A T I O N OF C H I C K E N E G G - W H I T E O V O F L A V O P R O T E I N
GUNTER BLANKENHORN*, DAVID T. OSUGA, HONSON S. LEE and ROBERT E. FEENEY Department of Food Science and Technology, University of California, Davis, Calif. 95616 ( U.S..4.J
(Received August 21st, 1974) (Revised manuscript received January 9th, 1975)
SUMMARY Affinity adsorbents for flavoproteins were prepared by the covalent attachment of polyacrylamide and agarose to flavin derivatives linked through position N(3) of the ftavin nucleus. 3-Carboxymethyl-FMN covalently linked to aminoalkyl substituted agarose was successfully used for the separation and purification of the apo form of the ovoflavoprotein from chicken egg white. High yields and high purities were achieved by two different isolation procedures employing the affinity adsorbent.
INTRODUCTION Affinity chromatography of proteins has been recently reviewed and demonstrated to be a valuable technique [1, 2]. The interaction of the flavin cofactor with the apoprotein moiety of many flavoenzymes and flavoproteins is reversible [3-11]. Thus, cofactor-affinity chromatography should be valuable for purifying these proteins. This was recognized as early as 1964, when Arsenis and McCormick [12] synthesized isoalloxazine derivatives of cellulose compounds to purify flavokinase from rat liver. FMN-cellulose compounds, with the cofactor linked to cellulose by ester bonds of its hydroxylic side chain, were effective in the purification of glycolate apooxidase from spinach [13]. Waters et al. [14] have recently reported the purification of bacterial luciferase on Sepharose 6Bohexanoate with F M N covalently attached. Studies on cofactor specificity for binding and for activity of various flavoenzymes revealed that the N(10)-ribitylphosphate side chain was essential for binding the cofactor, as well as for activity of most enzymes. Substitution in position N(3), however, did not lead to extensive losses of binding in most instances [3, 7, 9, 10]. Rhodes et al. [11] isolated a flavoprotein from chicken egg white which bound riboflavin (Ks ---- 7.9.10 s M -1) stronger than it did either F M N (Ks = 7.3. l05 M -1) or FAD (Ks ~ 7.104 M-~), the flavin cofactors present in most flavoenzymes. Becvar [15] showed that a large variety of flavin analogs was also bound by the riboflavinbinding protein from chicken egg white. The flavin-apoprotein interaction for this * Present address: Universit/it Konstanz, G.F.R.
471 protein was reversible, and the apoprotein was stable over a wide range of pH (3.09.0). Therefore, this protein should be well suited for studying purification by affinity chromatography on resins substituted with flavin analogs. In this paper we report the synthesis of polyacrylamide and agarose substituted by flavin derivatives linked through position N(3) of the flavin nucleus. These derivatives were used to purify chicken egg-white flavoprotein. EXPERIMENTAL PROCEDURE
Materials. The flavin derivatives used in this study were synthesized by published methods [16]. We thank Gisela Scola-Nagelschneider, University of Konstanz, FB Biologic, for the preparation of 3-carboxymethyl-FMN. Polyacrylamides (BioGel P-100 and P-150) were purchased from BioRad Laboratories, agarose (Sepharose 4B) and Sephadex G-25 from Pharmacia, Inc., DEAE-eellulose (DE 32) from Whatman Products, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate from Aldrich Chemicals, and CNBr from K and K Laboratories. White Leghorn chicken eggs were obtained from the Poultry Department of the University. Other chemicals and solvents used in this investigation were reagent grade commercial products. General analytical procedures. Purity of the flavin derivatives was checked by thin-layer chromatography on 6060 silica gel plates (Eastman Kodak). These were developed in the solvent mixture butanol/ethanol/water (7:2:1, by vol.), and spots were made visible by ultraviolet light (;tmax = 254 nm). Starch gel electrophoretic analyses were done as previously described [17], using discontinuous buffer systems at pH 8.6; disc gel electrophoretie analyses were at a running pH of 7.2 in an imidazole buffer system [18] to check the purity of the protein preparation. Absorbance measurements were made with a Beckman Model 25 spectrophotometer. Fluorescencequenching titrations to determine amounts of apoflavoprotein were made with a Turner Model 111 fluorimeter. Apoprotein (0.2-0.4 mg or egg white to give an equivalent amount) was diluted in 3 ml of 0.01 M phosphate buffer, pH 7.0, and titrated with 2.10 -5 M riboflavin solution in the same buffer. The titration curve was extrapolated to zero fluorescence to determine the amount of riboflavin bound [19]. Flavoprotein was determined by measuring the amount of protein-bound riboflavin spectrophotometrically at 444 nm against a blank of proteins (or egg white) bleached with sodium dithionite (approx. 20 mg per 10 ml egg white). Synthesis o f lumiflavin-polyacrylamide compounds. The aminoethyl form of polyacrylamide was synthesized according to Inman and Dintzis [20], with 2 mmol of aminoethyl groups being incorporated per g of dry resin. 5 g of this product were treated with 200 ml of an aqueous solution containing 300 mg of 3-carboxymethyllumiflavin (5- 10-3 M) and a 2-fold excess (2 mmol) of water-soluble carbodiimide. Acetylation of unreacted amino groups on lumiflavin-polyacrylamide. 5 g of lumiflavin-polyacrylamidewere suspended in 150 ml of water, and the pH was adjusted to 7.5. Under stirring, 10.2 g (0.1 mol corresponding to an excess of approx. 100-fold) of acetic anhydride were added dropwise. The pH was maintained at 7.5 by adding 0.05 M NaOH as needed, and the temperature was maintained at 20-25 °C by adding crushed ice. The reaction was complete after 5 min. The acetylated resin was then washed thoroughly with water on a Biichner funnel and stored at 4 °C.
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Synthesis of FMN-agarose. Agarose was activated by reaction with 50 mg of CNBr per ml of agarose. 1,8-Diaminooctane was then coupled to the activated agarose as described above. All further procedures were done in the dark. 3-CarboxymethylF M N (350 mg) was dissolved in 150 ml of an aqueous suspension containing the aminoalkyl-agarose to achieve a final concentration of 5- 10 -a M in flavin. A 10-fold excess of water-soluble carbodiimide was added in two portions as described above for lumiflavin-agarose compounds. The final product was washed with 2000 ml of 0.01 M phosphate buffer, pH 7.0, then with 1000 ml of water, and stored at 4 °C. Determination of specific binding capacity of flavin resins for apoflavoprotein. 10 ml of flavin resin were washed with 100 ml water and filtered on a Biichner funnel. The resin was then suspended in 25 ml of 0.5 M NaC1, pH 7.0, and 0.1-ml portions of 0.01 M apoflavoprotein from egg white were added. The resin was allowed to settle after each addition, and 0.1-ml samples of the supernatant were removed and added to 2.5 ml of a 1.5.10 -6 M solution of riboflavin in water. The relative fluorescence of the riboflavin solution was recorded as a function of apoflavoprotein additions. Determinations of non-specific binding capacity of flavin resins. Chicken ovomucoid (300 mg) was dissolved in 10 ml of 0.1 M ammonium acetate buffer, pH 5.0. This was applied to a column containing the flavin resin (approx. 70 ml). The column was washed thoroughly with starting buffer until A280n m in the eluate was less than 0.05. The non-specifically bound ovomucoid was then eluted with 0.1 M ammonium acetate, pH 5.0, containing 0.5 M NaC1. This protein fraction was dialyzed against several changes of water, lyophilized, and then weighed to determine the amount of chicken ovomucoid non-specifically bound by the flavin resin.
Preparation of crude apoflavoprotein from chicken egg white using DEAEcellulose adsorption and gel exclusion. One volume (approx. 1000 ml) of homogenized chicken egg white was diluted with one volume of deionized water. The crude suspension was adjusted to pH 4.3 with 3.0 M acetic acid, and then centrifuged at 10 000 rev./min for 15 min at 4 °C. The mucilaginous precipitate was discarded. The pale yellow supernatant was diluted with one and one-half volumes of water to lower the ionic strength, and the pH was readjusted to 4.3. About 200 ml of DEAE-cellulose previously equilibrated with 0.1 M ammonium acetate buffer, pH 4.3, was mixed in, and the mixture was stirred for 30 min in the cold room. The mixture was then suction filtered, and the filtrate discarded. The DEAE-cellulose was packed into a glass column (3.2 × 20 cm) and washed with 2000 ml of the 0.1 M ammonium acetate buffer, pH 4.3. The protein was eluted with 0.1 M ammonium acetate buffer, pH 3.6, containing 0.5 M NaC1. Fractions with A2s0 ,m above 0.1 were pooled, and enough solid (NH4)2SO4 was added to give an 85 700 saturated solution. After being stirred for 30 min and centrifuged, the supernatant was discarded. The yellow precipitate was dissolved in a small volume of 0.1 M ammonium acetate buffer, pH 3.15, and the'pH adjusted to 3.15 with 6.0 M acetic acid. This solution was put through a Sephadex G-25 column (4.5 × 45 cm) in the pH 3.15 buffer and fractions were collected. Protein-bound riboflavin was dissociated and separated from the flavoprotein under these conditions. The contents of the tubes of the first protein peak eluted from the column were pooled and adjusted to pH 5.0, 0.25 M in NaC1, and diluted with two volumes of 0.1 M ammonium acetate buffer, also pH 5.0 and containing 0.25 M NaC1.
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Preparation of crude apoflavoprotein from chicken egg white by dialysis against KBr. One volume (approx. 300 ml) of homogenized chicken egg white was diluted with a half volume of 0.1 M ammonium acetate buffer, pH 3.5. The crude egg-white suspension was adjusted to pH 3.5-3.6 with 3.0 M acetic acid and then centrifuged. The pale yellow supernatant was dialyzed for 48 h in the cold room against two changes of five volumes each of the ammonium acetate buffer, pH 3.5, containing 1.0 M KBr. Protein-bound riboflavin was dissociated into the dialyzing buffer under these conditions. The sample was centrifuged and the supernatant was dialyzed against water until the conductance of the dialyzing water was below 100-Q-1. This required approx. 48 h with frequent changes of water. The dialyzed sample was then made to 0.1 M in acetate, 0.25 M in NaC1, and adjusted to pH 5.0. Affinity adsorption and elution from FMN-agarose. The FMN-agarose affinity adsorbent was equilibrated with 0.1 M ammonium acetate buffer, pH 5.0, containing 0.25 M NaC1. Depending upon the estimate of the amount of apoflavoprotein in the sample, 25-40 ml of the equilibrated affinity adsorbent were added to the protein solutions prepared (in 0.1 M ammonium acetate, pH 5.0, and containing 0.25 M NaC1) as described above. This and all subsequent steps involving the FMN-agarose affinity adsorbent were done in the dark. The mixture was stirred for 30 min then suction filtered, and the adsorbent was packed into glass columns (1.8 x 9 cm or 1.8 × 12 cm) and washed with 500 ml of the pH 5.0 ammonium acetate buffer. The adsorbed apoflavoprotein was eluted with 0.1 M ammonium acetate buffer, pH 3.2, containing 0.5 M NaCI. Fractions with A280nm above 0.1 were pooled, dialyzed against water, and lyophilized. RESULTS AND DISCUSSION
Synthesis and properties of polyacrylamides substituted with 3-carboxymethyllumiflavin Most flavoenzymes have very specific steric requirements for the reversible binding of their cofactors. Usually modification at the N(10)-ribitylphosphate side chain decreases binding, whereas substitution at position N(3) does not greatly affect binding. We consequently selected 3-carboxymethyllumiflavin as a cofactor analog to be linked through its carboxy group to aminoalkyl derivatives of polyacrylamide, using a water-soluble carbodiimide as the coupling reagent. Unreacted amino groups had ion-exchange capacity, since they were largely protonated at pH values less than 9. When crude preparations of apoftavoprotein were applied to flavin affinity columns, these amino groups were responsible for nonspecific binding, mainly of ovomucoid. This problem could be prevented by their acetylation with either acetic anhydride or N-acetyl imidazole in aqueous suspension at pH 7.5 (Figs 1A and 1B; Fig. 2A). Polyacrylamide resins allow a high degree of substitution by low molecular weight ligands but they have some disadvantages. When highly concentrated protein solutions or salt gradients were applied to a polyacrylamide column, the beads shrank to less than half their original volume, and flow rate decreased drastically.
Synthesis and properties of agarose substituted with flavin analogs Although agarose could not be substituted by low molecular weight ligands to
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© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37012 SYNTHESIS OF I M M O B I L I Z E D FLAVIN DERIVATIVES A N D T H E I R USE IN P U R I F I C A T I O N OF C H I C K E N E G G - W H I T E O V O F L A V O P R O T E I N
GUNTER BLANKENHORN*, DAVID T. OSUGA, HONSON S. LEE and ROBERT E. FEENEY Department of Food Science and Technology, University of California, Davis, Calif. 95616 ( U.S..4.J
(Received August 21st, 1974) (Revised manuscript received January 9th, 1975)
SUMMARY Affinity adsorbents for flavoproteins were prepared by the covalent attachment of polyacrylamide and agarose to flavin derivatives linked through position N(3) of the ftavin nucleus. 3-Carboxymethyl-FMN covalently linked to aminoalkyl substituted agarose was successfully used for the separation and purification of the apo form of the ovoflavoprotein from chicken egg white. High yields and high purities were achieved by two different isolation procedures employing the affinity adsorbent.
INTRODUCTION Affinity chromatography of proteins has been recently reviewed and demonstrated to be a valuable technique [1, 2]. The interaction of the flavin cofactor with the apoprotein moiety of many flavoenzymes and flavoproteins is reversible [3-11]. Thus, cofactor-affinity chromatography should be valuable for purifying these proteins. This was recognized as early as 1964, when Arsenis and McCormick [12] synthesized isoalloxazine derivatives of cellulose compounds to purify flavokinase from rat liver. FMN-cellulose compounds, with the cofactor linked to cellulose by ester bonds of its hydroxylic side chain, were effective in the purification of glycolate apooxidase from spinach [13]. Waters et al. [14] have recently reported the purification of bacterial luciferase on Sepharose 6Bohexanoate with F M N covalently attached. Studies on cofactor specificity for binding and for activity of various flavoenzymes revealed that the N(10)-ribitylphosphate side chain was essential for binding the cofactor, as well as for activity of most enzymes. Substitution in position N(3), however, did not lead to extensive losses of binding in most instances [3, 7, 9, 10]. Rhodes et al. [11] isolated a flavoprotein from chicken egg white which bound riboflavin (Ks ---- 7.9.10 s M -1) stronger than it did either F M N (Ks = 7.3. l05 M -1) or FAD (Ks ~ 7.104 M-~), the flavin cofactors present in most flavoenzymes. Becvar [15] showed that a large variety of flavin analogs was also bound by the riboflavinbinding protein from chicken egg white. The flavin-apoprotein interaction for this * Present address: Universit/it Konstanz, G.F.R.
471 protein was reversible, and the apoprotein was stable over a wide range of pH (3.09.0). Therefore, this protein should be well suited for studying purification by affinity chromatography on resins substituted with flavin analogs. In this paper we report the synthesis of polyacrylamide and agarose substituted by flavin derivatives linked through position N(3) of the flavin nucleus. These derivatives were used to purify chicken egg-white flavoprotein. EXPERIMENTAL PROCEDURE
Materials. The flavin derivatives used in this study were synthesized by published methods [16]. We thank Gisela Scola-Nagelschneider, University of Konstanz, FB Biologic, for the preparation of 3-carboxymethyl-FMN. Polyacrylamides (BioGel P-100 and P-150) were purchased from BioRad Laboratories, agarose (Sepharose 4B) and Sephadex G-25 from Pharmacia, Inc., DEAE-eellulose (DE 32) from Whatman Products, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate from Aldrich Chemicals, and CNBr from K and K Laboratories. White Leghorn chicken eggs were obtained from the Poultry Department of the University. Other chemicals and solvents used in this investigation were reagent grade commercial products. General analytical procedures. Purity of the flavin derivatives was checked by thin-layer chromatography on 6060 silica gel plates (Eastman Kodak). These were developed in the solvent mixture butanol/ethanol/water (7:2:1, by vol.), and spots were made visible by ultraviolet light (;tmax = 254 nm). Starch gel electrophoretic analyses were done as previously described [17], using discontinuous buffer systems at pH 8.6; disc gel electrophoretie analyses were at a running pH of 7.2 in an imidazole buffer system [18] to check the purity of the protein preparation. Absorbance measurements were made with a Beckman Model 25 spectrophotometer. Fluorescencequenching titrations to determine amounts of apoflavoprotein were made with a Turner Model 111 fluorimeter. Apoprotein (0.2-0.4 mg or egg white to give an equivalent amount) was diluted in 3 ml of 0.01 M phosphate buffer, pH 7.0, and titrated with 2.10 -5 M riboflavin solution in the same buffer. The titration curve was extrapolated to zero fluorescence to determine the amount of riboflavin bound [19]. Flavoprotein was determined by measuring the amount of protein-bound riboflavin spectrophotometrically at 444 nm against a blank of proteins (or egg white) bleached with sodium dithionite (approx. 20 mg per 10 ml egg white). Synthesis o f lumiflavin-polyacrylamide compounds. The aminoethyl form of polyacrylamide was synthesized according to Inman and Dintzis [20], with 2 mmol of aminoethyl groups being incorporated per g of dry resin. 5 g of this product were treated with 200 ml of an aqueous solution containing 300 mg of 3-carboxymethyllumiflavin (5- 10-3 M) and a 2-fold excess (2 mmol) of water-soluble carbodiimide. Acetylation of unreacted amino groups on lumiflavin-polyacrylamide. 5 g of lumiflavin-polyacrylamidewere suspended in 150 ml of water, and the pH was adjusted to 7.5. Under stirring, 10.2 g (0.1 mol corresponding to an excess of approx. 100-fold) of acetic anhydride were added dropwise. The pH was maintained at 7.5 by adding 0.05 M NaOH as needed, and the temperature was maintained at 20-25 °C by adding crushed ice. The reaction was complete after 5 min. The acetylated resin was then washed thoroughly with water on a Biichner funnel and stored at 4 °C.
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Synthesis of FMN-agarose. Agarose was activated by reaction with 50 mg of CNBr per ml of agarose. 1,8-Diaminooctane was then coupled to the activated agarose as described above. All further procedures were done in the dark. 3-CarboxymethylF M N (350 mg) was dissolved in 150 ml of an aqueous suspension containing the aminoalkyl-agarose to achieve a final concentration of 5- 10 -a M in flavin. A 10-fold excess of water-soluble carbodiimide was added in two portions as described above for lumiflavin-agarose compounds. The final product was washed with 2000 ml of 0.01 M phosphate buffer, pH 7.0, then with 1000 ml of water, and stored at 4 °C. Determination of specific binding capacity of flavin resins for apoflavoprotein. 10 ml of flavin resin were washed with 100 ml water and filtered on a Biichner funnel. The resin was then suspended in 25 ml of 0.5 M NaC1, pH 7.0, and 0.1-ml portions of 0.01 M apoflavoprotein from egg white were added. The resin was allowed to settle after each addition, and 0.1-ml samples of the supernatant were removed and added to 2.5 ml of a 1.5.10 -6 M solution of riboflavin in water. The relative fluorescence of the riboflavin solution was recorded as a function of apoflavoprotein additions. Determinations of non-specific binding capacity of flavin resins. Chicken ovomucoid (300 mg) was dissolved in 10 ml of 0.1 M ammonium acetate buffer, pH 5.0. This was applied to a column containing the flavin resin (approx. 70 ml). The column was washed thoroughly with starting buffer until A280n m in the eluate was less than 0.05. The non-specifically bound ovomucoid was then eluted with 0.1 M ammonium acetate, pH 5.0, containing 0.5 M NaC1. This protein fraction was dialyzed against several changes of water, lyophilized, and then weighed to determine the amount of chicken ovomucoid non-specifically bound by the flavin resin.
Preparation of crude apoflavoprotein from chicken egg white using DEAEcellulose adsorption and gel exclusion. One volume (approx. 1000 ml) of homogenized chicken egg white was diluted with one volume of deionized water. The crude suspension was adjusted to pH 4.3 with 3.0 M acetic acid, and then centrifuged at 10 000 rev./min for 15 min at 4 °C. The mucilaginous precipitate was discarded. The pale yellow supernatant was diluted with one and one-half volumes of water to lower the ionic strength, and the pH was readjusted to 4.3. About 200 ml of DEAE-cellulose previously equilibrated with 0.1 M ammonium acetate buffer, pH 4.3, was mixed in, and the mixture was stirred for 30 min in the cold room. The mixture was then suction filtered, and the filtrate discarded. The DEAE-cellulose was packed into a glass column (3.2 × 20 cm) and washed with 2000 ml of the 0.1 M ammonium acetate buffer, pH 4.3. The protein was eluted with 0.1 M ammonium acetate buffer, pH 3.6, containing 0.5 M NaC1. Fractions with A2s0 ,m above 0.1 were pooled, and enough solid (NH4)2SO4 was added to give an 85 700 saturated solution. After being stirred for 30 min and centrifuged, the supernatant was discarded. The yellow precipitate was dissolved in a small volume of 0.1 M ammonium acetate buffer, pH 3.15, and the'pH adjusted to 3.15 with 6.0 M acetic acid. This solution was put through a Sephadex G-25 column (4.5 × 45 cm) in the pH 3.15 buffer and fractions were collected. Protein-bound riboflavin was dissociated and separated from the flavoprotein under these conditions. The contents of the tubes of the first protein peak eluted from the column were pooled and adjusted to pH 5.0, 0.25 M in NaC1, and diluted with two volumes of 0.1 M ammonium acetate buffer, also pH 5.0 and containing 0.25 M NaC1.
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Preparation of crude apoflavoprotein from chicken egg white by dialysis against KBr. One volume (approx. 300 ml) of homogenized chicken egg white was diluted with a half volume of 0.1 M ammonium acetate buffer, pH 3.5. The crude egg-white suspension was adjusted to pH 3.5-3.6 with 3.0 M acetic acid and then centrifuged. The pale yellow supernatant was dialyzed for 48 h in the cold room against two changes of five volumes each of the ammonium acetate buffer, pH 3.5, containing 1.0 M KBr. Protein-bound riboflavin was dissociated into the dialyzing buffer under these conditions. The sample was centrifuged and the supernatant was dialyzed against water until the conductance of the dialyzing water was below 100-Q-1. This required approx. 48 h with frequent changes of water. The dialyzed sample was then made to 0.1 M in acetate, 0.25 M in NaC1, and adjusted to pH 5.0. Affinity adsorption and elution from FMN-agarose. The FMN-agarose affinity adsorbent was equilibrated with 0.1 M ammonium acetate buffer, pH 5.0, containing 0.25 M NaC1. Depending upon the estimate of the amount of apoflavoprotein in the sample, 25-40 ml of the equilibrated affinity adsorbent were added to the protein solutions prepared (in 0.1 M ammonium acetate, pH 5.0, and containing 0.25 M NaC1) as described above. This and all subsequent steps involving the FMN-agarose affinity adsorbent were done in the dark. The mixture was stirred for 30 min then suction filtered, and the adsorbent was packed into glass columns (1.8 x 9 cm or 1.8 × 12 cm) and washed with 500 ml of the pH 5.0 ammonium acetate buffer. The adsorbed apoflavoprotein was eluted with 0.1 M ammonium acetate buffer, pH 3.2, containing 0.5 M NaCI. Fractions with A280nm above 0.1 were pooled, dialyzed against water, and lyophilized. RESULTS AND DISCUSSION
Synthesis and properties of polyacrylamides substituted with 3-carboxymethyllumiflavin Most flavoenzymes have very specific steric requirements for the reversible binding of their cofactors. Usually modification at the N(10)-ribitylphosphate side chain decreases binding, whereas substitution at position N(3) does not greatly affect binding. We consequently selected 3-carboxymethyllumiflavin as a cofactor analog to be linked through its carboxy group to aminoalkyl derivatives of polyacrylamide, using a water-soluble carbodiimide as the coupling reagent. Unreacted amino groups had ion-exchange capacity, since they were largely protonated at pH values less than 9. When crude preparations of apoftavoprotein were applied to flavin affinity columns, these amino groups were responsible for nonspecific binding, mainly of ovomucoid. This problem could be prevented by their acetylation with either acetic anhydride or N-acetyl imidazole in aqueous suspension at pH 7.5 (Figs 1A and 1B; Fig. 2A). Polyacrylamide resins allow a high degree of substitution by low molecular weight ligands but they have some disadvantages. When highly concentrated protein solutions or salt gradients were applied to a polyacrylamide column, the beads shrank to less than half their original volume, and flow rate decreased drastically.
Synthesis and properties of agarose substituted with flavin analogs Although agarose could not be substituted by low molecular weight ligands to
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