Characterization of a Stable Form of Carboxypeptidase G2 (Glucarpidase), a Potential Biobetter Variant, From Acinetobacter sp. 263903-1.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s-1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

Soluble expression, purification and functional characterisation of carboxypeptidase G2 and its individual domains.

Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.

Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli.

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (approximately 53-kDa and approximately 47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K(m) value for PABA-GLU of 60 +/- 0.08 microM and a specific activity of 63,300 +/- 600 nmol min(-1) mg(-1). Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

Functional overexpression and purification of a codon optimized synthetic glucarpidase (carboxypeptidase G2) in Escherichia coli.

Glucarpidase (former name: carboxypeptidase G2, or CPG2) is a bacterial enzyme that is widely used in detoxification of the cytotoxic drug, methotrexate, and in Antibody Directed Enzyme Prodrug Therapy for cancer treatment. The glucarpidase gene of Pseudomonas sp. strain RS-16 was previously cloned in E coli, but expresses at a level that is approximately 100-fold lower than in the native strain. In this study, a synthetic gene coding for glucarpidase was codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work indicated that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step by Ni(2+) charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg(2+) as well as Mn(2+) ions inhibit the activity of the recombinant enzyme.

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate.

Phosphorus-containing pseudopeptides, racemic at the C-terminal alpha-carbon, are potent mechanism-based inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(gamma-l-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R(f) pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH(4)Pte) heterocycle is most potent (K(is) = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R(f) species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., (l-Glu-gamma-l-Glu). Unexpectedly, the low R(f) (presumed l-Glu-gamma-d-Glu) species are only slightly less potent (<30-fold) than their diastereomers. Further study of this phenomenon comparing l-Glu-gamma-l-Glu and l-Glu-gamma-d-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial d-Glu precursors by l-Glu may give misleading information if l-Glu-gamma-l-Glu substrates have low K(m) values.

Characterization of poly-gamma-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-gamma-glutamate.

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a gamma-linkage, called poly-gamma-glutamate (gamma-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for gamma-PGA (designated gamma-PGA hydrolase, PghP) from bacteriophage PhiNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed gamma-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-gamma-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn(2+) or Mn(2+) ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M(r) of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel gamma-glutamyl hydrolase. Whereas phage PhiNIT1 proliferated in B. subtilis cells encapsulated with gamma-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce gamma-PGA. Analogous to polysaccharide capsules, gamma-PGA appears to serve as a physical barrier to phage absorption. Phages break down the gamma-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.

gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.

Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.

Purification and molecular analysis of an extracellular gamma-glutamyl hydrolase present in young tissues of the soybean plant.

A polypeptide present in intercellular wash fluids of young leaves of Glycine max has been purified to electrophoretic homogeneity. The protein has been identified as gamma-glutamyl hydrolase (GGH) based on the shared homology with a recently cloned cDNA from rat. The enzyme is present within the extracellular space of young leaves and a portion is bound to the cell wall. Northern and Western analysis confirm that this polypeptide is expressed only in young (1-15 d old) leaf, stem and root tissue and is therefore expressed under a strict developmental program. The primary sequence of gamma-glutamyl hydrolase shares amino acid identity with a cDNA clone from rat and two partially sequenced cDNAs from Arabidopsis. Although the complete in vivo function of gamma-glutamyl hydrolase in plants is unclear, it is known that the protein plays a critical role in folate metabolism and therefore likely in meeting the physiological demands of growing plant tissues.

The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells.

gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells.

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16.

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.

Functional specificity of jejunal brush-border pteroylpolyglutamate hydrolase in pig.

To determine the functional specificity of intestinal brush-border pteroylpolyglutamate hydrolase (PPH), we compared the regional location of in vivo hydrolysis of pteroyltriglutamate (PteGlu3) with the location of activity and immunoreactivity of the enzyme in the pig. After in vivo incubations, PteGlu3 hydrolytic products were recovered from intestinal segments in the jejunum but not from the ileum. Brush-border PPH activity in fractionated mucosa was 10-fold greater in the jejunum than in the ileum, whereas the activity of intracellular PPH was increased in the distal ileum. Antibodies to purified brush-border PPH identified a major protein band at 120 kDa and a minor protein band at 195 kDa in solubilized jejunal brush border. Immunohistochemistry identified the enzyme only on the brush-border surface of the jejunum, whereas an immunoblot of solubilized brush-border membranes identified brush-border PPH in the jejunum but not in the ileum. The parallel of the regional location of in vivo hydrolysis of PteGlu3 with the location of brush-border PPH activity and immunoreactivity demonstrates the functional specificity of this enzyme in folate digestion.

The measurement of gamma-glutamyl hydrolase (conjugase) activity in rat brain.

An assay using the artificial substrate, 2,4-diamino-10-methyl-pteroylglutamyl-gamma-glutamate (MTX-G1), was developed to measure gamma-glutamyl hydrolase (conjugase), which hydrolyzes folylpolyglutamates. This assay allows us to: 1) measure conjugase for the first time in rat brain and 2) measure conjugase in a reliable, sensitive and inexpensive manner. The MTX-binding assay results were compared to samples analyzed by HPLC and found to vary by only 13%. The artificial substrate, MTX-G1, had a lower rate of hydrolysis than pteroylglutamyl-gamma-glutamate (Pte-G2), 70.7 +/- 0.64 and 92.6 +/- 0.22 nmoles/hr/mg protein respectively. Conjugase was semi-purified 24 fold in H2O and found to have a pH optimum of 5.0.

Comparison of pteroylpolyglutamate hydrolase (folate conjugase) from porcine and human intestinal brush border membrane.

1. A comparative study of pteroylpolygluatamte hydrolase (folate conjugase) of brush border membrane vesicles from human and porcine intestine was conducted. 2. The enrichment of conjugase activity during membrane isolation was 5-fold greater for the human than the pig. 3. Porcine and human conjugases exhibited similar Km values and could completely hydrolyze pteroyltriglutamate (PteGlu3) to PteGlu1 via an exohydrolytic process. 4. Pteroic acid, PteGlu1 and anionic polysaccharides did not inhibit human or porcine conjugase. 5. Apparent mol. wts for detergent-enzyme complexes were 237,000 (pig) and greater than 500,000 (human). 6. These results indicate similar kinetic properties and mode of action but differences in physical behavior between the intestinal brush border folate conjugases of human and pig.

Intracellular pteroylpolyglutamate hydrolase from human jejunal mucosa. Isolation and characterization.

Human jejunal intracellular pteroylpolyglutamate hydrolase was purified 30-fold from intestinal mucosa. The apparent molecular weight of the enzyme was 75,000 by Sephadex G-200 gel filtration, and the isoelectric point was at pH 8.0. The enzyme was maximally active at pH 4.5 and was unstable at increasing temperatures. Intracellular pteroylpolyglutamate hydrolase cleaved both terminal and internal gamma-glutamate linkages. In contrast, brush-border pteroylpolyglutamate hydrolase catalyzed the hydrolysis of only terminal gamma-glutamate linkages. The intracellular enzyme showed greatest affinity for the complete folic acid molecule with longer glutamate chains. Subcellular fractionation studies showed the intracellular enzyme was localized in lysosomes. These data show that the properties of human jejunal intracellular pteroylpolyglutamate hydrolase are distinct from those of the brush-border enzyme but are similar to the properties of intracellular pteroylpolyglutamate hydrolase described in other tissues.

Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization.

Pteroylpolyglutamate hydrolase was solubilized with Triton X-100 from human jejunal mucosal brush borders and purified approximately 5,000-fold using organomercurial affinity chromatography, DEAE-cellulose chromatography, and gel filtration. The apparent molecular weight of the purified enzyme in the Triton micelle was estimated as 700,000 using Bio-Gel A-1.5m gel filtration. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis followed by Coomassie stain demonstrated two polypeptide bands at 145,000 and 115,000 daltons. The purified enzyme had an isoelectric point of 7.2, was maximally active at pH 5.5, and was stable above pH 6.5 and at temperatures up to 65 degrees C for at least 90 min. Human jejunal brush-border pteroylpolyglutamate hydrolase is an exopeptidase which liberated [14C]Glu as the sole labeled product of PteGlu2[14C]Glue (where PteGlun represents pteroylpolyglutamate), failed to liberate a radioactive product from PteGlu2[14C]GluLeu2, and released all possible labeled PteGlun products during incubation with Pte[14C]GluGlu6 with the accumulation of Pte[14C]Glu. PteGlu2, PteGlu3, and PteGlu7 were substrates, each with Km = 0.6 microM, whereas PteGlu was a weak inhibitor of the hydrolysis of PteGlu3 with Ki = 20 microM. Components of the pteroyl moiety, Glu, and short chain Glun in alpha or gamma linkages were not inhibitory. The enzyme was activated by Zn2+ or Co2+. The properties of brush-border pteroylpolyglutamate hydrolase are different from those described for the soluble intracellular pteroylpolyglutamate hydrolase in other species and in human mucosa, yet are consistent with previous data on the process of hydrolysis of PteGlun in the intact human intestine.

Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method.

A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 microM for folate, 8.0 microM for methotrexate and 34.0 microM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.

A mechanism for the addition of multiple moles of glutamate by folylpolyglutamate synthetase.

The role of the alpha-carboxyl group in methotrexate (MeAPA-Glu) and the gamma-glutamate derivative of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) has been investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analogue lacking an alpha-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analogue of MeAPA-Glu-Glu that lacked the alpha-carboxyl of the terminal glutamic acid. However, MeAPA-Gaba-Glu, the analogue of MeAPA-Glu-Glu without an alpha-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. These results suggest that the alpha-carboxyl is essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. Moreover, the binding of the terminal alpha-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the alpha-carboxyl of folyl monoglutamates would allow correct positioning of the terminal gamma-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogues.

Isolation and characterization of pteroylpolyglutamate hydrolase from rat intestinal mucosa.

Pteroylpolyglutamate hydrolase was isolated from rat intestinal mucosa and purified with the aid of affinity chromatography. The affinity ligand was poly-gamma-glutamic acid (Mr approximately 12,000) derived from Bacillus subtilis. The specific enzymatic activity was increased 2,000-fold over the 100,000 X g supernatant of the mucosal homogenate with a yield of 20%. Sephadex G-200 gel filtration yielded an estimated molecular mass of 80,000 daltons. The isoelectric point was pH 8.2. The pH optimum in acetate buffer containing 1 mM zinc was 4.5. The KM values for pteroylheptaglutamate and pteroyltriglutamate were 0.21 and 0.67 microM, respectively. Polyanionic compounds, poly-gamma-glutamic acid, dextran sulfate, and heparin were noncompetitive inhibitors. Studies of the time course of hydrolysis of synthetic [3H]pteroylheptaglutamate by three separate techniques demonstrated the appearance of [3H]pteroylmonoglutamate, synchronous with substrate cleavage. Intermediate pteroyloligoglutamates were not detected. An endopeptidase-like mode of hydrolysis was further established by identification of a hexaglutamyl peptide as the other reaction product.

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography.

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated forms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on mu Bondapak C18 coupled with uv detection allows direct quantitation of the folates in the nanogram range.