Metabolism of the antiandrogenic drug (Flutamide) by human CYP1A2.

The antiandrogenic drug, flutamide, is widely used in the treatment of carcinoma of the prostate. The present study examines the metabolism of flutamide by human liver microsomes and purified recombinant human cytochrome P450s (CYP), expressed as fusion proteins. These studies show the principal role of CYP1A2 in the metabolism of flutamide to 2-hydroxyflutamide. A minor metabolite is formed during the metabolism of flutamide by CYP3A4 in the presence of an excess of added purified NADPH-P450 reductase. The metabolism of flutamide is inhibited by low concentrations of alpha-naphthoflavone and ketoconazole. Other substrates of CYP1A2, such as phenacetin, imipramine, caffeine, and estradiol, are also inhibitors of flutamide metabolism by CYP1A2. Of interest is the inhibition of flutamide metabolism by its metabolite, 2-hydroxyflutamide, and the inhibition of the 2- and 4- hydroxylation of estradiol by flutamide. CV1 cells do not metabolize flutamide to 2-hydroxyflutamide. In assays performed using this cell line transfected with the cDNA for the androgen receptor, flutamide is a pure antagonist, and 2-hydroxyflutamide, while a more potent androgen receptor (AR) antagonist, activates the AR at higher concentrations. Stable expression of CYPIA2 in these CV1 cells causes flutamide to exhibit agonistic properties at higher concentrations, a behavior not exhibited by cells stably transfected only with the expression vector encoding the AR. These findings raise the possibility that increased conversion of flutamide to 2-hydroxyflutamide or accumulation of 2-hydroxyflutamide in cells may contribute to the anomalous responses to flutamide that are observed in some advanced prostate cancers.

The function of recombinant cytochrome P450s in intact Escherichia coli cells: the 17 alpha-hydroxylation of progesterone and pregnenolone by P450c17.

Studies are reported showing that recombinant P450c17, coexpressed with rat NADPH-P450 reductase or expressed as a fusion protein containing the domain of the P450 linked to the domain of NADPH-P450 reductase, function effectively in intact Escherichia coli cells. Progesterone is rapidly hydroxylated by transformed E. coli cells at rates as rapid as 50 nmol of steroid hydroxylated/min/nmol of P450 at 37 degrees C. This rate measured in vivo equals or exceeds the best rates we have measured when reconstituting progesterone hydroxylase activity in vitro using purified recombinant bovine P450c17 and purified recombinant rat NADPH-P450 reductase. The limits imposed in vivo by the availability of reducing equivalents (NADPH) and molecular oxygen are identified by showing the nearly fivefold increase in hydroxylation activity when glucose is present and the tendency for the constitutive respiratory activity of E. coli to limit the availability of oxygen required for the P450-catalyzed reaction. The rate of progesterone metabolism is about 200 times faster by P450c17 coexpressed with NADPH-P450 reductase than when P450c17 functions with the constitutive electron transfer system of E. coli (flavodoxin and flavodoxin reductase). Expression of the fusion protein, termed rF450[mBov17A/mRatOR]L1, results in a rate of progesterone metabolism in vivo at 37 degrees C of about 15 nmol of steroid hydroxylated/min/nmol of P450. Pregnenolone is actively metabolized to dehydroepiandrosterone at rates similar to those seen when the P450 activity is reconstituted in vitro with cytochrome b5. Experiments are described showing that the limited solubility of progesterone in water imposes a limit on the extent of steroid hydroxylated. The practicality of this type of P450-containing system for the bioconversion of large amounts of a chemical for the manufacture of speciality chemicals is discussed.

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

The interaction of NADPH-P450 reductase with P450: an electrochemical study of the role of the flavin mononucleotide-binding domain.

The electrochemically reduced mediator cobalt sepulchrate requires the presence of a flavoprotein for the rapid transfer of electrons to cytochrome P450. This electrochemical method has been used here to show the interaction of NADPH-P450 reductase (either the detergent-solubilized form, d-OR, or the proteolytic-cleaved truncated form, t-OR), as well as Escherichia coli flavodoxin (FLD), with P450c17 by measuring the rate of 17 alpha-hydroxylation of progesterone. When NADPH is used as electron donor with a reconstituted system composed of d-OR and P450c17, the addition of t-OR, flavodoxin, or cytochrome c inhibited the rate of formation of 17 alpha-hydroxyprogesterone. These results suggest the presence of a common protein binding site on the surface of d-OR, t-OR, and flavodoxin which plays a role in the interaction of the flavoproteins with the P450. It is speculated that a domain composed of acidic amino acids, located near the flavin mononucleotide-binding region of the flavoproteins, may serve as this site. No inhibition by t-OR, flavodoxin, or cytochrome c is observed when comparable experiments are carried out using the artificial recombinant fusion protein rF450[mBov17A/mRatOR]L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase.

The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase.

The recombinant fusion protein rF450[mRat4Al/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega- oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a Ki of about 1.9 microM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b5, and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

Identification of the human liver cytochrome P450 enzymes involved in the metabolism of zileuton (ABT-077) and its N-dehydroxylated metabolite, Abbott-66193.

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) forms involved in the oxidative metabolism of [14C]zileuton (ABT-077) and its N-dehydroxylated metabolite, [14C]Abbott-66193, by human liver microsomes. The two compounds were metabolized by parallel pathways to form the corresponding ring-hydroxylated and diastereomer sulfoxide metabolites. Results suggested that whereas the metabolism of zileuton and Abbott-66193 were mediated by the same CYP forms, the CYP forms responsible for hydroxylation (CYP1A2 and CYP2C9/10) were distinct from those involved in sulfoxidation (CYP3A > CYP2C9/10). Sulfoxidation (zileuton, Km = 0.82 +/- 0.40 mM, Vmax = 39.1 +/- 21.8 pmol/min/mg; Abbott-66193, Km = 0.23 +/- 0.06 mM, Vmax = 507 +/- 215 pmol/min/mg; mean +/- SD, N=3) was highly correlated with the CYP3A-specific erythromycin N-demethylase activity (r=0794-0.856; p<0.01, N=11) in human microsomes and was inhibited (32-67%) by ketoconazole and troleandomycin. In addition, purified recombinant human CYP3A4/rat NADPH-P450 reductase fusion protein catalyzed only the sulfoxidation of zileuton and Abbott-66193; no hydroxylated metabolites were detected. On the other hand, hydroxylation of the two compounds (zileuton, Km = 0.34 +/- 0.25 mM, Vmax = 17.8 +/- 5.58 pmol/min/mg; Abbott-66193,Km = 0.39 +/- 0.14 mM, Vmax = 1061 +/- 220 pmol/min/mg) was significantly correlated with 7-ethoxyresorufin O-deethylase (CYP1A2; r=0.652-0.762; p<0.01, N=11) and tolbutamide methyl hydroxylase (CYP2C9/10; r=0.863-0.935; p<0.01, N=10) activity in human liver microsome, and was inhibited (26-51%) by well-known CYP1A2 inhibitors (furafylline and alpha-naphthoflavone). Furthermore, microsomes from human B-lymphoblastoid cells expressing CYP1A2 catalyzed only the hydroxylation of zileuton and Abbott-66193; sulfoxide were not formed. Abbott-66193 was a better substrate for CYP2C9/10, when compared with zileuton: 1) the effect of sulfaphenazole on hydroxylation in human liver microsomes was more pronounced for Abbott-66193 than zileuton (56% vs. 9% inhibition); and 2) the rate of Abbott-66193 hydroxylation by purified CYP2C9 was almost 30-fold greater than that of zilueton.

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.

In vitro metabolism of terfenadine by a purified recombinant fusion protein containing cytochrome P4503A4 and NADPH-P450 reductase. Comparison to human liver microsomes and precision-cut liver tissue slices.

The metabolism of terfenadine was studied with a cDNA-expressed/purified recombinant fusion protein containing human liver microsomal cytochrome P4503A4 (CYP3A4) linked to rat NADPH-P450 reductase (rF450[mHum3A4/mRatOR]L1) and was compared with that observed in the presence of human liver microsomes and precision-cut human liver tissue slices. In all three cases, [3H]terfenadine was metabolized to at least three major metabolites. LC/MS (electrospray) analysis confirmed that these metabolites were alpha, alpha-diphenyl-4-piperidinomethanol (M5), t-butyl hydroxy terfenadine (M4), and t-butyl carboxy terfenadine (M3), although the level of M5 detected in the presence of fusion protein was greater than that found with microsomes or tissue slices. Two additional metabolites, M1 (microsomes and tissue slices) and M2 (fusion protein), were also detected, but remain uncharacterized. Consumption of parent drug (microsomes: KM = 9.58 +/- 2.79 microM, Vmax = 801 +/- 78.3 pmol/min/nmol CYP; fusion protein: KM = 14.1 +/- 1.13 microM, Vmax = 1670 +/- 170 pmol/min/nmol CYP) and t-butyl hydroxylation to M4 (microsomes: KM = 12.9 +/-3.74 microM, Vmax = 643 +/- 62.5 pmol/min/nmol CYP, ; fusion protein: KM = 30.0 +/- 2.55 microM, Vmax = 1050 +/- 141 pmol/min/nmol CYP) obeyed Michaelis-Menten kinetics over the terfenadine concentration range of 1-200 microM. Ketoconazole, a well-documented CYP3A inhibitor, effectively inhibited terfenadine metabolism in all three models. The conversion of M4 to M3, studied with human liver microsomes and fusion protein, was NADPH-dependent and inhibited by ketoconazole. It is concluded that cDNA-expressed CYP3A4, in the form of a NADPH-P450 reductase-linked fusion protein, may also serve as a model for studying the metabolism of terfenadine in vitro and many other drugs.

The effects of cytochrome b5, NADPH-P450 reductase, and lipid on the rate of 6 beta-hydroxylation of testosterone as catalyzed by a human P450 3A4 fusion protein.

The recombinant fusion protein containing the heme domain of human P450 3A4 and the flavin domains of rat NADPH-cytochrome P450 (P450) reductase (rF450[mHum3A4/mRatOR]L1) requires both phospholipid and detergent as well as cytochrome b5 (b5) for the NADPH-dependent catalysis of the 6 beta-hydroxylation of testosterone. NADPH oxidation results in the formation of hydrogen peroxide in the presence or absence of phospholipid and detergent. NADPH oxidation and hydrogen peroxide formation are inhibited by the addition of b5 and stimulated greater than 3-fold by the addition of testosterone. Marked differences in the ability of various phospholipids to support the P450-dependent 6 beta-hydroxylation of testosterone by the fusion protein were seen. Addition of a 4-fold excess of purified NADPH-P450 reductase, in the presence of phospholipid, detergent, and b5, stimulates the rate of testosterone 6 beta-hydroxylation approximately 10-fold, providing turnover rates as high as 80 min-1 for P450 3A4. Approximately 30% of the rate of hydrogen peroxide formation is not sensitive to inhibition by the P450 inhibitor ketoconazole, suggesting hydrogen peroxide (or superoxide anion) formation directly from the reduced flavin domains of the fusion protein. It is proposed that the stimulation of NADPH oxidation observed following the addition of testosterone to the fusion protein may serve as a useful means of monitoring the interaction of other substrates with this P450 and thereby permit the rapid screening of chemicals to evaluate their potential metabolism by a human P450.

The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function.

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b5. The cDNAs of b5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b5 proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b5. Purified b5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b5 for the rapid transfer of electrons from NADPH.

Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase.

A fusion protein containing the heme domain of bovine cytochrome P450 17A and the flavin domains of rat NADPH-cytochrome P450 reductase has been genetically engineered by linking the modified cDNAs for each gene with the codons for serine and threonine. Transformation of Escherichia coli (DH5 alpha) and growth under defined conditions permits expression of 600-700 nmol of membrane-bound fusion protein per liter of growth medium (approximately 4% of cellular protein). A method has been developed for the solubilization, isolation, and purification to homogeneity of this protein. In the presence of NADPH the purified fusion protein catalyzes the 17 alpha-hydroxylation of progesterone and pregnenolone as well as the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone. The 17,20-lyase activity is enhanced sixfold by the addition of purified rat liver cytochrome b5. Further, dehydroepiandrosterone is slowly metabolized to a number of additional more polar metabolites while 17 alpha-hydroxy-progesterone is slowly converted to dihydroxy-progesterone metabolites as well as a small amount of androstenedione in a reaction not influenced by cytochrome b5. Use of 5 alpha-pregnan steroids as substrates show the importance of the 3 beta-hydroxyl group for cytochrome b5 stimulated 17,20-lyase activity. Studies investigating the factors affecting electron transport between the flavin and heme domains suggest that the protein exists as a tight complex functioning as a self-contained biocatalytic unit.

Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.

Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids; such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5 for the 6 beta-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.

High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.

This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Escherichia coli as enzymatically active fusion proteins containing the flavoprotein domain of rat NADPH-cytochrome P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by engineering the cDNAs for the steroid-metabolizing bovine adrenal P450 17A with the cDNA for rat liver NADPH-P450 reductase with the introduction of a Ser-Thr linker to give a protein we have named rF450[mBov17A/mRatOR]L1. Similarly, the cDNA for the omega-hydroxylase of rat liver (P450 4A1) was linked with the cDNA for rat liver NADPH-P450 reductase to give rF450[mRat4A1/mRatOR]L1. A procedure involving disruption of transformed E. coli by sonication, isolation of membranes by differential centrifugation, solubilization with detergent, and affinity chromatography provided significant amounts of purified fusion proteins of approximately 118 kDa. The purified fusion proteins had turnover numbers for the metabolism of steroids (rF450[mBov17A/mRatOR]L1) or fatty acids (rF450[mRat4A1/mRatOR]L1) ranging from 10/min to 30/min in the absence of added phospholipid. Addition of purified rat liver cytochrome b5 stimulated the 17,20-lyase reaction for the conversion of 17-hydroxypregnenolone to dehydroepiandrosterone, and addition of purified rat NADPH-cytochrome P450 reductase enhanced the formation of omega--1 metabolites from lauric and arachidonic acids. NADPH oxidation was tightly coupled to substrate hydroxylation with the purified fusion proteins.

A lectin from the thigh muscle of Rana tigerina.

Lectin activity has been detected in the thigh muscle extracts of Rana tigerina, which was found to agglutinate both trypsinized and untrypsinized rabbit erythrocytes. The lectin has been purified to homogeneity by MEPBS (0.01 M phosphate-buffered saline (pH 7.2) with 4 mM beta-mercaptoethanol) buffer extraction of the tissue and affinity chromatography on acid-treated Sepharose 6B. The molecular weight (Mr) of the purified lectin was determined by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-75, which gave values of 15,500 +/- 1000 and 32,000 +/- 1000, respectively, suggesting that the lectin is a dimer. Amino acid composition data of the lectin has revealed that it contains a high proportion of glycine and alanine, and low amounts of sulphur-containing amino acids. Hapten-inhibition study of this lectin has shown that it is galactose-specific. Hemagglutination activity of the lectin can also be inhibited by beta-galactoside containing oligosaccharides.

Effect of raw winged bean [Psophocarpus tetragonolobus (L.) DC] tuber lectin on gastrointestinal tract of growing rats.

The fraction containing high hemagglutinating activity was prepared from raw winged bean tubers and orally administered to growing rats. The food intake and body weights of these rats decreased as the level of lectin increased and significant lectin activity was detected in the faeces extracted from these rats which is anti-genically similar to the native lectin preparation. Microscopic examination has revealed morphological changes in the intestinal epithelial cells. The binding action of the lectin to the mucosal epithelia of the gastrointestinal tract is indicative of the deleterious effects caused by the winged bean tuber lectin.

Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations.

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl alpha-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59.10(5) M-1 at 25 degrees C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent with alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.