Continuous Spectrophotometric Method Research Articles

Lab scale fermentation studies with marine bacteria to maximize the production of bioemulsifiers for cosmetic and food industries

Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases.

Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model

ObjectiveDiabetes mellitus (DM) is known to be associated with increase of oxidative stress products. The direction of effect of any treatment on these products could therefore be a reliable measure of its efficacy on DM. So the aim of this study was to investigate the activity of insulin, Ocimum gratissimum L. (OG) and Vernonia amygdalina L. (VA) on oxidative stress products. MethodsThirty-six female Wistar rats weighing 150–200 g were randomly divided into six groups of six rats each. Thirty rats were induced for type 1 DM (DM1) with a single intraperitoneal administration of 65 mg/kg body weight of streptozotocin. Group 1 was normal control and was administered distilled water while Group 2 served as DM1 control group; Groups 3, 4, 5 and 6 were diabetic rats treated with 208 mg/kg OG (DM1 + OG), 52 mg/kg VA (DM1 + VA), 208 mg/kg OG + 52 mg/kg VA (DM1+OG +VA) and 0.16 IU insulin (DM1 + insulin) respectively. Determination of methemoglobin and sulfhemoglobin was achieved by the absorption spectrum principle. Red blood cell (RBC) catalase was assayed by continuous spectrophotometric method. ResultsThe RBC catalase concentration was significantly decreased in the DM1 and DM1+VA groups when compared with the normal control. DM1 + OG significantly increased RBC-catalase when compared to DM1. The methemoglobin concentration was significantly reduced in the DM1, DM1 + VA, DM1 + OG + VA and DM1 + insulin groups when compared to the normal control group. The sulfhemoglobin concentration was significantly increased in the diabetic control and the diabetic treated groups when compared to the normal control. DM1 + OG reduced the sulfhemoglobin concentration when compared to DM1. The blood glucose concentration of all the diabetic groups was significantly raised compared to normal control. OG, VA and insulin significantly reduced the blood glucose concentration with the efficacy of OG and VA higher than insulin. ConclusionAdverse alteration of oxidative indices were observed in type 1 DM model. Treatment with OG and insulin showed potent antioxidant activity, while the hypoglycemic efficacy of OG and VA were higher than insulin.

α-Eleostearic acid-containing triglycerides for a continuous assay to determine lipase sn-1 and sn-3 regio-preference

Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases.

Assessment of serum Lactate Dehydrogenase in preeclamptic pregnant women

Background: Evidences prove that endothelial cell and altered endothelial cell function play an important role in the pathogenesis of preeclampsia. Therefore serum lactate dehydrogenase (LDH) is useful biochemical markers reflecting the severity of the occurrence of preeclampsia. Objective: Determination of serum lactate dehydrogenase (LDH) in severe preeclamptic pregnant women. Methods: This is the case-control hospital based study carried in the Al Azhar Medical College and Super speciality Hospital, Thodupuzha. Normal pregnant women (n=55) and women with severe preeclampsia (n=63) were included in the study. Both the groups were in their third trimester and of same age and same gestational age. Serum LDH levels were measured by continuous spectrophotometric pyruvate method for both groups. Results: Higher values of serum lactate dehydrogenase (LDH) were found in severe preeclamptic women compared with normal pregnant women in third trimester. Conclusion: Elevated levels of serum LDH indicates the tissue damage and might be the cause of the occurrence of preeclampsia.

Photobiomodulation therapy reduces apoptotic factors and increases glutathione levels in a neuropathic pain model.

Neuropathic pain (NP) is caused by damage to the nervous system due to reactive oxygen spices (ROS) increase, antioxidants reduction, ATP production imbalance, and induction of apoptosis. In this investigation, we applied low-level laser 660nm (photobiomodulation therapy) as a new strategy to modulate pain. In order to study the effects of photobiomodulation therapy (660nm) on NP, chronic constriction injury (CCI) model was selected. Low-level laser of 660nm was used for 2weeks. Thermal and mechanical hyperalgesia were measured before and after surgery on days 7 and 14, respectively. Paw withdrawal thresholds were also evaluated. Expression of p2x3, Bax, and bcl2 protein was measured by western blotting. The amount of glutathione (GSH) was measured in the spinal cord by continuous spectrophotometric rate determination method. The results are presented as mean ± SD. Statistical analysis of data was carried out using SPSS 21. CCI decreased the pain threshold, 2-week photobiomodulation therapy significantly increased mechanical and thermal threshold, decreased P2X3 expression (p < 0.001), and increased bcl2 expression (p < 0.01), but it was not effective on the Bax expression. We speculated that although photobiomodulation therapy increased ROS generation, it increased antioxidants such as GSH. Increase in bcl2 is another mitochondrial protection mechanism for cell survival and that pain relief and decrease in P2X3 expression confirm it.

Relationship of Serum Lactate Dehydrogenase (LDH) Level with Severity of Preeclampsia

Preeclampsia is a pregnancy specific disease and associated with multisystem complications. This crosssectional study was carried out in the Department of Physiology, Dhaka Medical College (DMC), Dhakafrom January'14 to December'14 to assess the relationship between serum lactate dehydrogenase (LDH) levelwith the severity of the disease in preeclampsia. For this purpose, a total number of 105 pregnant women atthird trimester (28-40 weeks) with age ranging from 18 to 35 years were selected from DMC Hospital,Dhaka. Among them, 70 pregnant women with preeclampsia were selected as study group (Group B) which wasfurther subdivided as mild preeclamptic women (Group B1) and severe preeclamptic women (Group B2)consisting of 35 subjects in each subgroup. Age matched 35 healthy pregnant women were considered ascontrol group (Group A) for comparison. Serum LDH level was estimated by continuous spectrophotometricmethod. In this study, serum LDH level showed positive correlation with progressive rise of blood pressure inthe study group. The frequency of symptoms and complications of preeclampsia were more in patients with LDH&gt;600 U/L compared with those who had lower levels. The present study showed that high serum LDH levelpositively correlates with the severity of the disease in patients of preeclampsia.

Effects of 660nm Low-Level Laser Therapy on P2X3 Expression of Lumbar DRG of Adult Male Rats with Neuropathic Pain

Background: Neuropathic Pain (NP) is a serious suffering medical condition that frequently leads to disability and life style changes. Although the exact mechanisms of NP are still unknown, recently the role of reactive oxygen species (ROS) reported as an important factor for NP. Apoptosis, increase of ATP production and reduction of antioxidants are also the other factors influencing in NP. There are certain therapeutic procedures for NP among them using laser therapy newly received more attention. In the present research we studied the molecular effects of Low Level Laser Therapy (LLLT) on a rat model of NP. Methods: Thirty adult male Wistar rats (200-250 g) that randomly divided into three groups including chronic constriction injury (CCI), CCI+LLLT and control were used in this study. CCI technique was used to induce NP. Laser therapy was done by using laser beam of 660 for 14 days following CCI. After that, expression of P2X3 of the DRG, Bax and Bcl2 in lumbar spinal segments measured by Western Blotting. Level of glutathione (GSH) was also measured in lumbar spinal cord segments by Continuous Spectrophotometric Rate Determination method. For behavioral study the mechanical and thermal hyperalgesia were evaluated in days 7 and 14 after CCI. Results: LLLT for two weeks increased expression of Bcl 2 and GSH, whereas decreased Bax and P2X3 expression significantly. Comparing the results of behavioral study showed significant differences in the mechanical and thermal threshold showed between CCI and CCI+ LLLT groups. Conclusion: Based on our findings, the therapeutic effects of LLLT for NP act throughout cellular and molecular mechanisms which improve mitochondrial function that in turn improve cell function and prevent apoptosis.

A continuous spectrophotometric assay for adenosine 5′-phosphosulfate reductase activity with sulfite-selective probes

Mycobacterium tuberculosis (Mtb) adenosine 5′-phosphosulfate (APS) reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of tuberculosis (TB) infection. Despite the importance of APR to Mtb and other bacterial pathogens, current assay methods depend on the use of 35S-labeled APS or shunt adenosine 5′-monophosphate (AMP) to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here, we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or “off–on” fluorescent levulinate-based probes. Thus, the APR activity can be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michaelis–Menten kinetic constants (Km, kcat, and kcat/Km) and the apparent inhibition constant (Ki) for adenosine 5′-diphosphate (ADP), which compared favorably with values obtained in the “gold standard” radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer.

A continuous spectrophotometric assay for mitogen-activated protein kinase kinases

We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, which is then used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methyl purine ribonucleoside. Of the reaction products ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and, hence, provides a spectrophotometric signal that can be continuously followed. In the presence of excess phosphatase, the phosphorylated protein substrate molecules undergo dephosphorylation almost immediately after their formation; the steady-state use of the resultant inorganic phosphate is a reflection of the constant initial velocity of the exchange reaction. The validity of this method has been confirmed by using it to measure the activities of MEK1 (MAPK/ERK kinase 1) and MKK6 (MAPK kinase 6) toward their physiological substrates. Our findings of the MAPK kinases in the current study provide evidence that the substrate binding affinities of this subfamily of protein kinases are at the submicromolar concentration.

A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay—the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg 2+ and is not enhanced by other divalent metal ions (Zn 2+ and Mn 2+), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

The enzyme deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and PPi thus controlling the incorporation of uracil into DNA genomes. In Campylobacter jejuni dUTPase exhibits structural properties of dimeric proteins characteristic of protozoa of the Kinetoplastidae family. In the present study we perform a kinetic analysis of Campylobacter dUTPase using the continuous spectrophotometric method and show that the enzyme is highly specific for deoxyuridine nucleotides. The Michaelis-Menten constant for dUTP was 0.66 μM while the kcat was 12.3 s− 1. dUDP was also efficiently hydrolysed although the specificity constant, kcat/Km, was five fold lower than for dUTP. The reaction product and the non hydrolysable analogue α,β imido dUDP are potent inhibitors of the enzyme while several analogues of dUMP with substituents at the 3′- and 5′-positions active against trimeric dUTPases, show poor inhibitory activity. Apparent structural and kinetic differences with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against campylobacteriosis.

A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of α-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340 nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca 2+ concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.

A Continuous Spectrophotometric Assay for NADPH-cytochrome P450 Reductase Activity Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome b5, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of A610. MTT reduction followed classical Michaelis-Menten kinetics (K(m)= 20 microM, k(cat)= 1,910 min(-1)). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.

A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase.

Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.

A continuous spectrophotometric assay for phospholipase A 2 activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A 2 (PLA 2) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A 2 and lipoxygenase were established. PLA 2 activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis–Menten kinetics ( V m =1.8 μ M/min, K m=4.5 μ M, V m/ K m =0.4 min −1). This assay also allows PLA 2 inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A 2. It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A 2.

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl] PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (Vm = 9.8 x 10(-3) micromol/min x microg protein, Km = 7.8 microM, Vm/Km = 1.3 min(-1) x microg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.

A direct continuous pH-spectrophotometric assay for arginine kinase activity.

A direct and continuous spectrophotometric method was developed for determining arginine kinase (phosphoarginine synthesis) activity. Protons are produced during the phosphoarginine synthesis course, so adding the complex acid-base indicator to this solution and monitoring the decrease of absorbance of the solution at 575 nm will follow the arginine kinase activity. For this condition, one activity unit of arginine kinase was defined as 1 micromol H+ produced in 1 minute in the enzymatic reaction catalyzed by arginine kinase.

Macro minerals deficiencies in pregnant Mauritian women

During pregnancy there is usually a fluctuation in the levels of trace elements in the body. The level of copper for example may rise whilst that of zinc may fall. Plasma zinc and copper levels were measured in pregnant and non-pregnant women (mean age 25±6 and 23±5 years respectively). A commercially available kit was used for the direct determination of zinc while copper assay was performed by atomic absorption spectrophotometry. Total plasma proteins, albumin and globulin levels were also measured by colorimetric method using commercially available kits. Plasma zinc and copper levels in pregnant women were found to be 99.3 ± 10.8 g/dl and 180 ± 31.4 g/dl respectively compared to mean values of 111.8 ± 7.2g/dl for zinc and 153.2 ± 15.2 for copper g/dl in the non-pregnant group. Plasma proteins were also significantly lower (p<0.05) in the pregnant individuals. Mean values of 63 ± 8.7g/l for total plasma proteins, 37.2 ± 4.7 g/l for albumin and 25.8 ± 10.7g/l for globulin were obtained for the pregnant women. In the controls, total protein level was 79.6± 3.8 g/l, albumin level was 43.4 ±2.6 g/l and globulin 36.1 ± 5 g/l. In conclusions, our findings indicate that high level of copper during pregnancy may be responsible for the lowering of zinc level since these two metals are known antagonists. In addition, lower levels of albumin and globulin that are all carriers of zinc in the body may also be implicated in the lower zinc level during pregnancy. Key words : zinc, copper, total proteins, pregnancy, albumin, globulin.

Synthesis and evaluation of novel dipeptide-bound 1,2,4-Thiadiazoles as irreversible inhibitors of guinea pig liver transglutaminase

Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz- l-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 μM −1 min −1.

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Leishmania major.

Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low K(m) value for dUTP (2.11 microM), a k(cat) of 49 s(-1), strict Michaelis-Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a competitive inhibitor. Discrimination is achieved for the base and sugar moiety showing specificity constants for different dNTPs similar to those of bacterial, viral, and human enzymes. In the alkaline range, the K(m) for dUTP increases with the dissociation of ionizable groups showing pK(a) values of 8.8, identified as the uracil moiety of dUTP and 10, whereas in the acidic range, K(m) is regulated by an enzyme residue exhibiting a pK(a) of 7.1. Activity is strongly inhibited by the nucleoside triphosphate analog alpha-beta-imido-dUTP, indicating that the enzyme can bind triphosphate analogs. The existence of specific inhibition and the apparent structural and kinetic differences (reflected in different binding strength of dNTPs) with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against leishmaniasis.