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Dulal Borthakur - One of the best experts on this subject based on the ideXlab platform.
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Mimosine facilitates metallic cation uptake by plants through formation of Mimosine cation complexes
Plant Molecular Biology, 2020Co-Authors: Michael D H Honda, Dulal BorthakurAbstract:Iron deficiency conditions as well as iron supplied as a Fe(III)–Mimosine complex induced a number of strategy I and strategy II genes for iron uptake in leucaena. Leucaena leucocephala (leucaena) is a tree-legume that can grow in alkaline soils, where metal-cofactors like Fe(III) are sparingly available. Mimosine, a known chelator of Fe(III), may facilitate Fe(III) uptake in leucaena by serving as a phytosiderophore. To test if Mimosine can serve as a phytosiderophore, three sets of experiments were carried out. First, the binding properties and solubility of metal–Mimosine complexes were assessed through spectrophotometry. Second, to study Mimosine uptake in plants, pole bean, common bean, and tomato plants were supplied with Mimosine alone and metal–Mimosine complexes. Third, the expression of strategy I (S1) and strategy II (S2) genes for iron uptake from the soil was studied in leucaena plants exposed to different Fe(III) complexes. The results of this study show that (i) Mimosine has high binding affinity for metallic cations at alkaline pH, Fe(III)–Mimosine complexes are water soluble at alkaline pH, and that Mimosine can bind soil iron under alkaline pH; (ii) pole bean, common bean, and tomato plants can uptake Mimosine and transport it throughout the plant; and (iii) a number of S1 and S2 genes were upregulated in leucaena under iron-deficiency condition or when Fe(III) was supplied as a Fe(III)–Mimosine complex. These findings suggest that leucaena may utilize both S1 and S2 strategies for iron uptake; and Mimosine may play an important role in both strategies.
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Mimosine accumulation in leucaena leucocephala in response to stress signaling molecules and acute uv exposure
Plant Physiology and Biochemistry, 2019Co-Authors: Kelly Cristine Da Silva Rodriguescorrea, Dulal Borthakur, Michael D H Honda, Arthur Germano FettnetoAbstract:Mimosine is a non-protein amino acid of Fabaceae, such as Leucaena spp. and Mimosa spp. Several relevant biological activities have been described for this molecule, including cell cycle blocker, anticancer, antifungal, antimicrobial, herbivore deterrent and allelopathic activities, raising increased economic interest in its production. In addition, information on Mimosine dynamics in planta remains limited. In order to address this topic and propose strategies to increase Mimosine production aiming at economic uses, the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on Mimosine accumulation in growth room-cultivated seedlings of Leucaena leucocephala spp. glabrata. Mimosine concentration was not significantly affected by 10 ppm salicylic acid (SA) treatment, but increased in roots and shoots of seedlings treated with 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound), and in shoots treated with UV-C radiation. Quantification of Mimosine amidohydrolase (mimosinase) gene expression showed that ethephon yielded variable effect over time, whereas JA and UV-C did not show significant impact. Considering the strong induction of Mimosine accumulation by acute UV-C exposure, additional in situ ROS localization, as well as in vitro antioxidant assays were performed, suggesting that, akin to several secondary metabolites, Mimosine may be involved in general oxidative stress modulation, acting as a hydrogen peroxide and superoxide anion quencher.
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An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase
Applied Biochemistry and Biotechnology, 2014Co-Authors: Jannai T. Yafuso, Jonpaul Bingham, Vishal Singh Negi, Dulal BorthakurAbstract:In plants, the final step of cysteine formation is catalyzed by O -acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, Mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a Mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the K _m was calculated to be 1,850 ± 414 μM sulfide and the V _max was 200.6 ± 19.92 μM cysteine min^−1. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for Mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not Mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.
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a carbon nitrogen lyase from leucaena leucocephala catalyzes the first step of Mimosine degradation
Plant Physiology, 2014Co-Authors: Vishal Singh Negi, Jonpaul Bingham, Dulal BorthakurAbstract:The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, Mimosine, and also an enzyme, mimosinase, for Mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for Mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in Mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing Mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced Mimosine content in the future.
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midd encoded rhizomimosinase from rhizobium sp strain tal1145 is a c n lyase that catabolizes l Mimosine into 3 hydroxy 4 pyridone pyruvate and ammonia
Amino Acids, 2013Co-Authors: Vishal Singh Negi, Jonpaul Bingham, Dulal BorthakurAbstract:Rhizobium sp. strain TAL1145 catabolizes Mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in Mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using Mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10−4 mol, 4.96 × 10−5 mol s−1 mg−1, and 2,256.05 s−1, respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5′-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to Mimosine, and the presence of its structural analogs including l-tyrosine, l-tryptophan and l-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of Mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C–N lyase.
Joyce L. Hamlin - One of the best experts on this subject based on the ideXlab platform.
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the dual effect of Mimosine on dna replication
Experimental Cell Research, 1997Co-Authors: Robert F Kalejta, Joyce L. HamlinAbstract:Abstract The plant amino acid, Mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells, but the mechanism by which this inhibition is achieved is unknown. The drug has been proposed either to inhibit initiation at origins of replication or to inhibit chain elongation by lowering nucleotide pool levels. In an attempt to determine which mode of action is correct, we have analyzed its effects on SV40 DNA replication. Using a two-dimensional gel replicon mapping technique, we show that Mimosine completely inhibits incorporation of [ 3 H]thymidine into viral DNA, but only after ∼4 h. Qualitative analysis of replication intermediates during this interval suggests that the drug partially inhibits both initiation and elongation, and pulse–chase experiments support this contention. The drug has no effect when added directly to an SV40 in vitro replication extract. However, extracts prepared from cells pretreated with Mimosine are compromised in their ability to support replication in vitro in the presence of a full complement of nucleotides. Thus, although Mimosine may alter nucleotide pool levels in vivo, it also appears to affect one or more essential replication proteins.
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Mimosine Targets Serine Hydroxymethyltransferase
The Journal of biological chemistry, 1996Co-Authors: Hong-bo Lin, Rocco Falchetto, Paul J. Mosca, Jeffrey Shabanowitz, Donald F. Hunt, Joyce L. HamlinAbstract:The plant amino acid, Mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G1/S boundary, and blocks DNA replication when added to S phase cells after a lag of approximately 2.5 h. We have shown previously that [3H]Mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [3H]Mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a Mimosine-resistant CHO cell line and in a CHO gly- cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly- cell line is still sensitive to Mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.
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Mimosine a novel inhibitor of dna replication binds to a 50 kda protein in chinese hamster cells
Nucleic Acids Research, 1995Co-Authors: Paul J. Mosca, H B Lin, Joyce L. HamlinAbstract:Abstract We recently demonstrated that the plant amino acid, Mimosine, is an extremely efficacious inhibitor of DNA replication in mammalian cells [P. A. Dijkwel and J. L. Hamlin (1992) Mol. Cell. Biol. 12, 3715-3722; P. J. Mosca et al. (1992) Mol. Cell. Biol. 12, 4375-4383]. Several of its properties further suggested that Mimosine might target initiation at origins of replication, which would make it a unique and very useful inhibitor for studying the regulation of DNA synthesis. However, Mimosine is known to chelate iron, a cofactor for ribonucleotide reductase. Thus, the possibility arose that Mimosine functions in vivo simply by lowering intracellular deoxyribonucleotide pools. In the present study, we show that, in fact, it is possible to override Mimosine inhibition in vivo by adding excess iron; however, copper, which is not a substitute for iron in ribonucleotide reductase, is equally effective. Evidence is presented that Mimosine functions instead by binding to an intracellular protein. We show that radiolabeled Mimosine can be specifically cross-linked to a 50 kDa polypeptide (termed p50) in vitro. Binding to p50 is virtually undetectable in CHO cells selected for resistance to 1 mM Mimosine, arguing that p50 is the biologically relevant target. p50 is not associated with the cellular membrane fraction and, hence, is probably not a channel protein. Furthermore, the binding activity does not vary markedly as a function of cell cycle position, arguing that p50 is not a cyclin. Finally, both iron and copper are able to reverse the Mimosine-p50 interaction in vitro, probably explaining why both metal ions are able to overcome Mimosine's inhibitory effect on DNA synthesis in vivo.
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the plant amino acid Mimosine may inhibit initiation at origins of replication in chinese hamster cells
Molecular and Cellular Biology, 1992Co-Authors: Paul J. Mosca, P A Dijkwel, Joyce L. HamlinAbstract:An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid Mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of Mimosine on cell cycle traverse in the Mimosine [corrected]-resistant CHO cell line CHOC 400. When administered to cultures for 14 h after reversal of a G0 block, the drug appears to arrest the population at the G1/S boundary, and upon its removal cells enter the S phase in a synchronous wave. However, when methotrexate is administered to an actively dividing asynchronous culture, cells are arrested not only at the G1/S interface but also in early and middle S phase. Most interestingly, two-dimensional gel analysis of replication intermediates in the initiation locus of the amplified dihydrofolate reductase domain suggests that Mimosine may actually inhibit initiation. Thus, this drug represents a new class of inhibitors that may open a window on regulatory events occurring at individual origins of replication.
Boyka Anachkova - One of the best experts on this subject based on the ideXlab platform.
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Treatment of mammalian cells with Mimosine generates DNA breaks.
Mutation research, 2000Co-Authors: Ivailo Mikhailov, George Russev, Boyka AnachkovaAbstract:Exponentially growing mouse erythroleukemia (MEL) cells and quiescent human peripheral blood lymphocytes (PBL) were treated with different concentrations of the nonprotein amino acid Mimosine for 16 h. The treatment of the cycling cell population with 400 μM Mimosine caused inhibition of DNA replication, changes in the progression of the cells in the cell cycle, and apoptosis. Nucleoid sedimentation analysis and comet assay were used to monitor the appearance and accumulation of DNA breaks. The rate of break accumulation was dose-dependent, did not depend on the stage of the cell cycle and was not connected with the mechanism of DNA replication. The data indicate that the effects of Mimosine on DNA synthesis and the cell cycle may be a result of introduction of breaks into DNA.
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effect of Mimosine on dna synthesis in mammalian cells
Cancer Research, 1997Co-Authors: Lyuben Tsvetkov, George Russev, Boyka AnachkovaAbstract:Abstract We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid Mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F 4 N cells. We found out that during the first 2 h after application of 25–400 µm Mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of Mimosine was similar to that of γ-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, Mimosine inhibits the initiation of DNA replication as well.
K Lalitha - One of the best experts on this subject based on the ideXlab platform.
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Selective determination of Mimosine and its dihydroxypyridinyl derivative in plant systems
Amino Acids, 2006Co-Authors: K Lalitha, S. Rajendra KulothunganAbstract:Our observations on the growth stimulatory nature of Mimosine, (β-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both Mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of Mimosine while interference from other relevant compounds was 15–35%. The determination of Mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of Mimosine and DHP with distinctly differing R_f, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of Mimosine from 0.1 to 0.75 μM or DHP from 0.2 to 1.5 μM and with recoveries of 99.2 to 100.5%.
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determination of Mimosine by a sensitive indirect spectrophotometric method
Talanta, 2004Co-Authors: K Lalitha, Rajendra S KulothunganAbstract:A simple and sensitive indirect spectrophotometric method is described for the determination of Mimosine based on its reaction with diazotized sulfanilamide (DZSAM). DZSAM couples with N-(1-naphthyl)ethylenediamine (NEDA) forming a pink colored azodye, absorbing maximally at 540nm (epsilon(max)=27mM(-1)cm(-1)). In the present method, Mimosine was first reacted with known excess of DZSAM and the unreacted DZSAM was determined by coupling with NEDA. The reaction of Mimosine with DZSAM proceeded optimally at neutral pH. The decrease in absorbance of the DZSAM-NEDA-coupled product obeyed Beer's law in the concentration range of 0.005-0.15mugml(-1) of Mimosine. The present method was applied to estimate Mimosine in plant extracts containing lesser than 0.05mugml(-1) with recovery at 99+/-0.41%. The method described is superior to other reported methods in terms of ease of adaptability and sensitivity.
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oxidative stress during selenium deficiency in seedlings of trigonella foenum graecum and mitigation by Mimosine part i hydroperoxide metabolism
Biological Trace Element Research, 1999Co-Authors: T R Santosh, M Sreekala, K LalithaAbstract:Actaptive alterations in glutathione (GSH) metabolism were studied during oxidative stress induced by selenium (Se) deficiency in germinating seedlings ofTrigonella foenum- graecum grown for 72 h and the response to supplementation individually of Se or Mimosine was explored. Growth enhancement with improved mitochondrial efficiency was elicited by supplementation of Se at 0.5-0.75 ppm or Mimosine at 0.1-0.2 mM. Total thiol and protein levels of mitochondrial and soluble fractions, in general, did not vary significantly with supplementation of either Se or Mimosine except that the mitochondrial protein levels in Mimosine groups (0.1-0.2 mM) decreased by 20–30%. Mitochondrial glutathione peroxidase (GSH-Px) increased by twofold in activity toward H2O2, cumene hydroperoxide (CHP), and t-butyl hydroperoxide (tBHP) in Se groups, and by 50–60% increase toward H2O2 and CHP but by a twofold enhancement in enzyme activity with tBHP in Mimosine groups. Soluble GSH-Px activity increased by 30–40% only in Mimosine groups and remained unaltered in Se groups. Glutathione S-transferase activity (GST) in the soluble fraction of both Se and Mimosine groups increased dramatically by fivefold to sixfold. Distinct differences were noted in the response of the stressed seedlings toward exposure to Se or Mimosine and included a decline in glutathione reductase (GR) activity by 50–60% in both mitochondria and soluble fractions of Se groups and an increase in GR activity of the mitochondria by twofold and of the soluble enzyme activity by 30% in the Mimosine groups. Mimosine exposure resulted in a dose-dependent decrease in the γ-glutamyl transpeptidase levels, but, in contrast, a significant enhancement by 50% was noted in the Se group at 0.75 ppm. The results including the differential response of GR activity to Se or Mimosine supplementation are reflective of an effective reductive environment in Se groups and increased turnover of GSH in the presence of Mimosine.
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Oxidative stress during selenium deficiency in seedlings oftrigonella foenum-graecum and mitigation by Mimosine
Biological Trace Element Research, 1999Co-Authors: M Sreekala, T R Santosh, K LalithaAbstract:Oxidative stress during selenium (Se) deficiency in the seedlings of Trigonella foenum-graecum grown for 72 h was investigated and the response to supplemented levels of Se (0.5-1 ppm) and Mimosine (0.05-1 mM) was evaluated. Beneficial effects of Se was maximal at 0.75 ppm. Mimosine, a toxic amino acid, was also found to be beneficial to the growth of the seedlings exposed up to 0.2 mM. When compared to the stressed seedlings, mitochondrial oxygen uptake from seedlings of Se (0.75 ppm) group and Mimosine (0.2 mM) group exhibited threefold enhancement in state 3 respiration rate and a controlled state 4 rate, with respiratory control ratios of 5–8. Upon supplementation at the optimal levels, Superoxide dismutase (SOD) activities were enhanced fourfold with Se and eightfold with Mimosine in the mitochondria. The soluble activity in Mimosine groups increased twofold, but only by 75% in Se groups. Peroxidase activity registered a significant increase by threefold in mitochondria and fourfold in soluble fraction in both Se and Mimosine groups. Exposure to Se or Mimosine exhibited a differential response in the mitochondrial catalase and ascorbate peroxidase (Asc-Px) activities. In the Se groups, both catalase and Asc-Px in mitochondria decreased by 50–60%, which was contrasted by 60% increase in Asc-Px activity and 40% in catalase activity in Mimosine groups. Supplementation with either Se or Mimosine evoked similar responses of increases with respect to soluble catalase by twofold to threefold and Asc-Px by 90%. The results of the present study reveal (1) the Prevalence of oxidative stress in T. foenum-graecum during Se deficiency, (2) enhanced mitochondrial functional efficiency mediated by Se and Mimosine independently, and (3) an antitoxidative role for Mimosine during Se deficiency. The study demonstrates for the first time that Mimosine, a naturally occuring toxic amino acid, could be a beneficial growth factor in concentrations between 0.1 and 0.2 m M .
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spectrophotometric determination of Mimosine and 3 hydroxy 4 1h pyridone the toxic principles of leucaena leucocephala
Analytical Biochemistry, 1993Co-Authors: K Lalitha, C M Vargheese, N BalasubramanianAbstract:A sensitive and selective spectrophotometric method for the estimation of the toxic factors Mimosine and 3-hydroxy-4-(1H)-pyridone (DHP) has been developed based on the intense yellow-colored azodye formed with p-nitroaniline which showed a sharp absorption maximum at 400 nm. The method was optimized based on relative sensitivity of the reaction with various aromatic primary amino compounds and under different conditions of pH. Interference from a variety of structurally related compounds and phenols was tested and found insensitive to this method. The molar extinction coefficient at 400 nm for the azodye formed with Mimosine was 5.31 x 10(4) M-1 cm-1 and that for DHP was 1.699 x 10(4) M-1 cm-1. The applicability of the method was tested using different plant extracts and recovery was found to be at 100 +/- 0.3%. The method is suitable for accurate estimation of both Mimosine and DHP after paper chromatographic separation of extracts of different biological samples.
N Balasubramanian - One of the best experts on this subject based on the ideXlab platform.
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spectrophotometric determination of Mimosine and 3 hydroxy 4 1h pyridone the toxic principles of leucaena leucocephala
Analytical Biochemistry, 1993Co-Authors: K Lalitha, C M Vargheese, N BalasubramanianAbstract:A sensitive and selective spectrophotometric method for the estimation of the toxic factors Mimosine and 3-hydroxy-4-(1H)-pyridone (DHP) has been developed based on the intense yellow-colored azodye formed with p-nitroaniline which showed a sharp absorption maximum at 400 nm. The method was optimized based on relative sensitivity of the reaction with various aromatic primary amino compounds and under different conditions of pH. Interference from a variety of structurally related compounds and phenols was tested and found insensitive to this method. The molar extinction coefficient at 400 nm for the azodye formed with Mimosine was 5.31 x 10(4) M-1 cm-1 and that for DHP was 1.699 x 10(4) M-1 cm-1. The applicability of the method was tested using different plant extracts and recovery was found to be at 100 +/- 0.3%. The method is suitable for accurate estimation of both Mimosine and DHP after paper chromatographic separation of extracts of different biological samples.