Comparison of synchronous and laser-induced fluorescence spectroscopy applied to the Eu(III)-fulvate complexation

The complexation of europium ion (Eu(III)) with a soil fulvic acid (FA) has been studied at pH 5 in 0.01 M NaClO₄ by different experimental methods, i.e. synchronous fluorescence spectroscopy (SyFS) and time resolved laser-induced fluorescence spectroscopy (TRLFS). A series of SyFS quenching spectra was obtained by increasing the Eu(III) concentration and keeping the FA concentration constant. The emission spectra and fluorescence lifetimes of the Eu(III) bound to the FA were also measured by a TRLFS system using the same solution used in the SyFS spectral measurement. From the analysis of the fluorescence data obtained by the SyFS and the TRLFS using a non-linear least-squares method, the concentration of the binding sites (C_L) of the FA accessible for the Eu(III) and the corresponding conditional stability constants (log K) were estimated. The two different methods gave rise to constants being comparable with one another. The log K and C_L values (mean ± standard deviation of three determinations) determined by the SyFS were 6.4 ± 0.2 (6.7 ± 0.1 μmol L⁻¹: by the TRLFS) and 10 ± 1 μmol L⁻¹ (7 ± 1 μmol L⁻¹: by the TRLFS), respectively. The applicability of the FA fluorescence quenching techniques for estimating the europium binding parameters was proved by the direct monitoring of the Eu(III) bound to the FA using the TRLFS system.     

Binding site and elution behavior of DNA and other large biomolecules in monolithic anion-exchange chromatography

Our previous study has shown that there is a good correlation between the number of charges of DNA (from trimer to 50-mer) and the number of binding sites B in electrostatic interaction chromatography (ion-exchange chromatography, IEC). It was also found that high salt (NaCl) concentration is needed to elute large DNAs (>0.6 M). In this paper we further performed experiments with large DNAs (up to 95-mer polyT and polyA) and charged liposome particles of different sizes (ca. 30, 50 and 100 nm) with a monolithic anion-exchange disk in order to understand the binding and elution mechanism of very large charged biomolecules or particles. The peak salt (NaCl) concentration increased with increasing DNA length. However, above 50-mer DNAs the value did not increase significantly with DNA length (ca. 0.65–0.70 M). For liposome particles of different sizes the peak salt concentration (ca. 0.62 M) was similar and slightly lower than that for large DNAs (ca. 0.65–0.70 M). The binding site values (ca. 25–30) are smaller than those for large DNAs. When arginine was used as a mobile phase modulator, the elution position of polyA and polyT became very close whereas in NaCl gradient elution polyT appeared after polyA eluted. This was mainly due to suppression of hydrophobic interaction by arginine.     

Modelling of Pb-(TAPS)x-(OH)y system and refinement of stability constants in the region of lead hydrolysis and lead hydroxide precipitation

The influence of [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS) on solutions containing lead(II) was studied by direct current polarography (DCP) and glass electrode potentiometry (GEP). The readings were taken at fixed total TAPS to total lead(II) concentration ratios and various pH values, at 25.0 ± 0.1 °C and ionic strength 0.1 M KNO₃.Due to the basic pK_a of the ligand, which occurs in the pH range where large amount of lead polynuclear species are formed, and the occurrence of ligand adsorption, that disabled the use of high concentrations of TAPS on DCP experiments, GEP and DCP experimental conditions were put to the limit in order to provide the correct Pb-TAPS-OH model and reliable stability constants.The proposed final model is: PbL, PbL₂, PbL₂(OH) and PbL₂(OH)₂ with overall stability constants values, as log β, 3.27 ± 0.06, 6.5 ± 0.1, 12.7 ± 0.1 and 17.27 ± 0.06, respectively.A comparative analysis of the strength of complexation of TAPS and a structural related buffer, 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), with lead is also discussed.     

Study of binding and denaturation dynamics of IgG and anti-IgG using dual color fluorescence correlation spectroscopy

In this article, we present a systematic study on IgG and Fab fragment of anti-IgG molecules using fluorescence auto- and cross-correlation spectroscopy to investigate their diffusion characteristics, binding kinetics, and the effect of small organic molecule, urea on their binding. Through our analysis, we found that the diffusion coefficient for IgG and Fab fragment of anti-IgG molecules were 37 ± 2 μm² s⁻¹ and 56 ± 2 μm² s⁻¹, respectively. From the binding kinetics study, the respective forward (k_a) and backward (k_d) reaction rates were (5.25 ± 0.25) × 10⁶ M⁻¹ s⁻¹ and 0.08 ± 0.005 s⁻¹, respectively and the corresponding dissociation binding constant (K_D) was 15 ± 2 nM. We also found that urea inhibits the binding of these molecules at 4 M concentration due to denaturation.     

Effect of ionic strength and protein concentration on the transport of proteins through chitosan/polystyrene sulfonate multilayer membrane

The influence of ionic strength and protein concentration on the transport of bovine serum albumin (BSA), ovalbumin and lysozyme through chitosan (CHI)/polystyrenesulfonate (PSS) multilayers on polyether sulfone supports are investigated under ultrafiltration conditions. The percentage transmission and flux of BSA, ovalbumin and lysozyme were found to increase with increase in salt concentration in the protein. The percentage transmission of BSA through 9 bilayer membrane was found to increase from 5.3 to 115.6 when the salt concentration was varied from 0 to 1 M. It was observed that 0.1 M NaCl in BSA solution is capable of permeating all the BSA. When the salt concentration in BSA was further increased, a negative solute rejection (solute enrichment in permeate) was found to take place. With 9 bilayer membrane, the percentage transmission of ovalbumin was found to increase from 23.3 to 125.8 when the salt concentration in protein was increased from 0 to 0.05 M. The effect of protein concentration on protein transport is studied taking BSA as a model protein. BSA was rejected by the multilayer membrane at all the studied concentrations (0.25, 0.5, 1 and 2 mg/ml). With increase in feed concentration, maximum rejection of protein occurred at higher number of CHI/PSS bilayers. BSA solution flux was found to decrease with an increase in BSA concentration. This study indicates that it is possible to fine tune the transport properties of proteins through multilayer membranes by varying the concentration and ionic strength of protein solutions.     

Real time monitoring uracil excision using uracil-containing molecular beacons

As a highly conserved damage repair protein, UDG excises uracil bases through its glycosylase activity. We report here an alternative fluorescence method for UDG assay with high accuracy and sensitivity by applying uracil-modified molecular beacons as substrates. The detection limit of UDG is 0.005 U mL⁻¹. The K_M and k_cat are 0.89 ± 0.1 μM and 210 ± 10 min⁻¹, respectively. The method is applied to screening inhibitors and the results indicate that both of the 5-FU and cisplatin can inhibit UDG activity with the IC₅₀ values of 6.1 ± 0.52 mM and 3.2 ± 0.24 mM, respectively. Furthermore, the combination of uracil-modified molecular beacons and nuclease inhibitor makes the new method possible to specifically detect UDG activity in cell-free extracts and serum. Taken together, the simple, rapid and sensitive method has potential relevance for a variety of applications, such as molecular diagnosis and screening of UDG inhibitors.     

Dextran-grafted cation exchanger based on superporous agarose gel: Adsorption isotherms,uptake kinetics and dynamic protein adsorption performance

A novel chromatographic medium for high-capacity protein adsorption was fabricated by grafting dextran (40 kDa) onto the pore surfaces of superporous agarose (SA) beads. The bead was denoted as D-SA. D-SA, SA and homogeneous agarose (HA) beads were modified with sulfopropyl (SP) group to prepare cation exchangers, and the adsorption and uptake of lysozyme on all three cation-exchange chromatographic beads (SP-HA, SP-SA and SP-D-SA) were investigated at salt concentrations of 6–50 mmol/L. Static adsorption experiments showed that the adsorption capacity of SP-D-SA (2.24 mmol/g) was 78% higher than that of SP-SA (1.26 mmol/g) and 54% higher than that of SP-HA (1.45 mmol/g) at a salt concentration of 6 mmol/L. Moreover, salt concentration had less influence on the adsorption capacity and dissociation constant of SP-D-SA than it did on SP-HA, suggesting that dextran-grafted superporous bead is a more potent architecture for chromatographic beads. In the dynamic uptake of lysozyme to the three cation-exchange beads, the D_e/D₀ (the ratio of effective pore diffusivity to free solution diffusivity) values of 1.6–2.0 were obtained in SA-D-SA, indicating that effective pore diffusivities of SP-D-SA were about two times higher than free solution diffusivity for lysozyme. At 6 mmol/L NaCl, the D_e value in SA-D-SA (22.0 × 10⁻¹¹ m²/s) was 14.4-fold greater than that in SP-HA. Due to the superior uptake kinetics in SA-D-SA, the highest dynamic binding capacity (DBC) and adsorption efficiency (the ratio of DBC to static adsorption capacity) was likewise found in SP-D-SA. It is thus confirmed that SP-D-SA has combined the advantages of superporous matrix structure and drafted ligand chemistry in mass transport and offers a new opportunity for the development of high-performance protein chromatography.     

Sacrificial spacer and non-covalent routes toward the molecular imprinting of “poorly-functionalized” N-heterocycles

A comparison of three different methods for the imprinting of small aromatic heterocycles containing only a single nitrogen atom, for the preparation of specific analytical phases, was carried out. A conventional non-covalent approach to the imprinting of pyridine using methacrylic acid as the functional monomer was compared with two sacrificial spacer methods, in which heterocycles were imprinted as covalent template analogues. The results of binding experiments showed that discrimination based on ligand size was possible when polymers were prepared using a silyl ester-based template. The most selective polymer was able to bind pyridine in preference to quinoline or acridine which is opposite to the trend predicted by the pK_HB values for the three ligands. Curve fitting of the isotherm for pyridine binding to this polymer to the Langmuir model gave an approximate K_d of 1.1±0.1 mM and a binding site concentration of 57±2 mmol g⁻¹. Acridine binding did not show saturation behaviour and was non-specific and cooperative in nature.     

Molecular sensing of 3-chloro-1,2-propanediol by molecular imprinting

A covalent interaction-based molecularly imprinted polymer (MIP) material for 3-chloro-1,2-propanediol (3-MCPD), a post-testicular anti-fertility agent and possible carcinogen and mutagen in food products containing acid-hydrolyzed vegetable proteins, has been successfully fabricated using 4-vinylphenylboronic acid as the functional monomer. Rebinding assay revealed that the binding constant, K_B, for the receptor sites and non-specific sites are 1.93±0.1×10⁴ and 2.74±0.7×10² M⁻¹, respectively. The estimated number of receptor site, B_max, imprinted is 123.3±3 μmol/g of MIP. The MIP material is able to act as a potentiometric chemosensor for 3-MCPD via increase in Lewis acidity of the receptor sites upon reaction of the arylboronic acid with 3-MCPD to form the more acidic arylboronic acid esters. A simple pH glass electrode is sufficient to monitor the analyte-specific rebinding. In unbuffered aqueous media, linear potentiometric response from 0 to 350 ppm of 3-MCPD can be achieved. The MIP-based chemosensing in a soya sauce matrix has also been attempted. It is found that the dynamic range of the potentiometric chemosensing response of the MIP material is much reduced, probably due to the blocking or deactivation of receptor sites by interferents in soya sauces. Nevertheless, the present work demonstrated the feasibility of using MIP-based chemosensors as semi-quantitative analytical tools for screening purposes in quality control of food products.     

Enthalpy change and mechanism of oxidation of o-phenylenediamine by hydrogen peroxide catalyzed by horseradish peroxidase

The hydrogen peroxide-oxidation of o-phenylenediamine (OPD) catalyzed by horseradish peroxidase (HRP) at 37 °C in 50 mM phosphate buffer (pH 7.0) was studied by calorimetry. The apparent molar reaction enthalpy with respect to OPD and hydrogen peroxide were −447 ± 8 kJ mol⁻¹ and −298 ± 9 kJ mol⁻¹, respectively. Oxidation of OPD by H₂O₂ catalyzed by HRP (1.25 nM) at pH 7.0 and 37 °C follows a ping-pong mechanism. The maximum rate V_max (0.91 ± 0.05 μM s⁻¹), Michaelis constant for OPD K_m,S (51 ± 3 μM), Michaelis constant for hydrogen peroxide Km,H₂O₂ (136 ± 8 μM), the catalytic constant k_cat (364 ± 18 s⁻¹) and the second-order rate constants k₊₁ = (2.7 ± 0.3) × 10⁶ M⁻¹ s⁻¹ and k₊₅ = (7.1 ± 0.8) × 10⁶ M⁻¹ s⁻¹ were obtained by the initial rate method.     

Unexplored capabilities of chemiluminescence and thermoanalytical methods in characterization of intact and degraded hyaluronans

Three intact and four degraded hyaluronans were investigated by using chemiluminometry, differential scanning calorimetry, and thermogravimetry. Degradation of hyaluronan was induced by a system containing H₂O₂ alone (882 mM); 55 mM H₂O₂plus 1.25 μM CuCl₂; NaOCl alone (10 mM); and NaOCl plus CuCl₂ and ascorbic acid (10 mM, 0.1 μM, and 100 μM, respectively). The four different oxidative systems yielded biopolymer fragments represented by similar viscosity characteristics. The results obtained by using chemiluminescence and thermoanalytical methods indicate that hyaluronans of similar rheological properties could be distinguished from each other.     

Direct Analysis of Proteins from Solutions with High Salt Concentration Using Laser Electrospray Mass Spectrometry

The detection of lysozyme, or a mixture of lysozyme, cytochrome c, and myoglobin, from solutions with varying salt concentrations (0.1 to 250 mM NaCl) is compared using laser electrospray mass spectrometry (LEMS) and electrospray ionization-mass spectrometry (ESI-MS). Protonated protein peaks were observed up to a concentration of 250 mM NaCl in the case of LEMS. In the case of ESI-MS, a protein solution with salt concentration >?0.5 mM resulted in predominantly salt-adducted features, with suppression of the protonated protein ions. The constituents in the mixture of proteins were assignable up to 250 mM NaCl for LEMS and were not assignable above a NaCl concentration of 0.5 mM for ESI. The average sodium adducts (< n >) bound to the 7+ charge state of lysozyme for LEMS measurements from salt concentrations of 2.5, 25, 50, and 100 mM NaCl are 1.71, 5.23, 5.26, and 5.11, respectively. The conventional electrospray measurements for lysozyme solution containing salt concentrations of 0.1, 1, 2, and 5 mM NaCl resulted in < n > of 2.65, 6.44, 7.57, and 8.48, respectively. LEMS displays an approximately two orders of magnitude higher salt tolerance in comparison with conventional ESI-MS. The non-equilibrium partitioning of proteins on the surface of the charged droplets is proposed as the mechanism for the high salt tolerance phenomena observed in the LEMS measurements.

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Graphical Abstract ?

     

Kinetic determination of cysteine and thiosulphate by inhibition of Hg(II) catalyzed ligand substitution reaction

The sulphur containing inhibitors (I), cysteine (Cys) and sodium thiosulphate (THS), have been found to inhibit Hg(II) catalyzed exchange of cyanide in hexacyanoferrate(II) by nitroso-R-salt (NRS). The inhibitory effect of both the ligands are attributed to their binding tendencies with Hg(II) leading to the formation of catalyst-inhibitor (C-I) complex. The reactions have been followed spectrophotometrically in aqueous medium at 720 nm by noting the increase in absorbance of the green colour product, [Fe(CN)₅NRS]³⁻ at pH 6.50 ± 0.02, temp 25.0 ± 0.1 °C and ionic strength (μ) 0.1 M (KNO₃). A most plausible mechanistic scheme involving the role of analytes (inhibitors) has been proposed. The values of equilibrium constants for complex formation between catalyst-inhibitor (K_CI), catalyst-substrate (K_S) and Mechaelis-Menton constant (K_m) have been computed from the kinetic data. The linear calibration curves have been established between absorbance and inhibitor concentrations under specified conditions. Cys and THS have been determined in the range 1-5 × 10^− 7 M and 4.9-16.9 × 10^− 7 M respectively. The detection limits have been computed to be 1 × 10^− 7 M and 4.9 × 10^− 7 M for Cys and THS, respectively.     

Fluorescence detection of cytosine/guanine transversion based on a hydrogen bond forming ligand

In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate potential use of a hydrogen bond forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), for the fluorescence detection of the cytosine (C)/guanine (G) mutation sequence of the cancer repression gene p53. Our method is based on construction of the AP site in ODN duplexes, which allows small synthetic ligands to bind to target nucleobases accompanied by fluorescence signaling: an AP site-containing ODN is hybridized with a target ODN so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleobases through hydrogen-bonding. In 10 mM sodium cacodylate buffer solutions (pH, 7.0) containing 100 mM NaCl and 1.0 mM EDTA, AMND is found to strongly bind to C (K_d=1.5×10⁻⁶ M) in the target ODN while the binding affinity for G is relatively moderate (K_d=50×10⁻⁶ M). Significant fluorescence quenching of AMND is observed only when binding to C, making it possible to judge the C/G transversion with the naked eye.     

Separation of hypoviral double-stranded RNA on monolithic chromatographic supports

A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.     

Interaction of uranium(VI) with nitrogen containing model ligands studied by laser-induced fluorescence spectroscopy

The complexation of uranium(VI) with two nitrogen containing organic ligands, representing model substances for humic acid building blocks, has been investigated at pH values between 1.5 and 4.5 and an ionic strength of 0.1 M (NaClO₄). Using two independent fluorescence spectroscopic methods, time-resolved laser-induced fluorescence spectroscopy (TRLFS) and TRLFS with ultrafast pulses (fs-TRLFS), the complex formation of uranium(VI) with anthranilic and nicotinic acid in aqueous solution was studied. In both systems a decrease in the luminescence intensity was observed with increasing ligand or metal ion concentration. Uranium(VI) complexes of the type M_xL_yH_z were identified. Anthranilic acid forms a 1:1 complex under the given experimental conditions with a stability constant of log β₁₁₁ = 8.00 ± 0.31. For the uranium(VI) nicotinate system 1:1 and 1:2 complexes could be identified. The corresponding formation constants were calculated to be log β₁₁₁ = 8.59 ± 0.17 and log β₁₂₂ = 17.18 ± 0.35.     

A ratiometric fluorescent probe for magnesium employing excited state intramolecular proton transfer

A simple fluorescent sensor has been developed for the ratiometric recognition of Mg²⁺ in semi-aqueous solution at pH 7.0. The sensor, a Schiff base, undergoes Excited State Intramolecular Proton Transfer (ESIPT) to generate a keto tautomer with proficient Mg²⁺ binding capability. The sensor displays good selectivity over other metal ions including alkali/alkali earth ions and can measure Mg²⁺ ion concentration between 2.0 and 30.0 μM. The binding stoichiometry was established as 2:1 (host:guest) with an association constant (K₂₁) of (1.4 ± 0.1) × 10⁴ M⁻². The sensor could potentially be used to detect conditions such as hypermagnesaemia.     

Study on superabsorbent composite XVI. Synthesis, characterization and swelling behaviors of poly(sodium acrylate)/vermiculite superabsorbent composites

A series of superabsorbent composites were synthesized by copolymerization reaction of partially neutralized acrylic acid on unexpanded vermiculite (UVMT) micropowder using N,N′-methylenebisacrylamide (MBA) as a crosslinker and ammonium persulfate (APS) as an initiator in aqueous solution. And the samples were further characterized by means of fourier-transform spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). The effects of vermiculite content on water absorbency were studied. Swelling behaviors of the superabsorbent composites in various cationic salt solutions (NaCl, CaCl₂ and FeCl₃), anionic salt solutions (NaCl, Na₂SO₄ and Na₃PO₄) and pH solutions were also systematically investigated. Results obtained showed that the equilibrium water absorbency increased with increasing UVMT content and the concentration of 20 wt.% clay gave the best absorption (1232 g/g in distilled water and 89 g/g in 0.9 wt.% NaCl). Data achieved also suggested that the water absorbency in various saline solutions decreased with an increase in the ionic strengths of these solutions. And it was found that at a higher ionic strength (>1 × 10⁻³ M), the water absorbency in monovalent cationic solutions was higher than those in multivalent cationic solutions. However, at the same ionic strength (>1 × 10⁻³ M), the effect of three anionic salt solutions on the swelling has the following order: NaCl < Na₂SO₄ < Na₃PO₄.     

Hydridic reactivity of W(CO)(H)(NO)(PMe3)3 - Dihydrogen bonding and H2 formation with protic donors

The hydridic reactivity of the complex W(CO)(H)(NO)(PMe₃)₃ (1) was investigated applying a variety of protic donors. Formation of organyloxide complexes W(CO)(NO)(PMe₃)₃(OR) (R = C₆H₅ (2), 3,4,5-Me₃C₆H₂ (3), CF₃CH₂ (4), C₆H₅CH₂ (5), Me (6) and iPr (7)) and H₂ evolution was observed. The reactions of 1 accelerated with increasing acidity of the protic donor: Me₂CHOH (pK_a = 17) < MeOH (pK_a = 15.5) < C₆H₅CH₂OH (pK_a = 15) < CF₃CH₂OH (pK_a = 12.4) < C₆H₂Me₃OH (pK_a = 10.6) < C₆H₅OH (pK_a = 10).Regioselective hydrogen bonding of 1 was probed with two of the protic donors furnishing equilibrium formation of the dihydrogen bonded complexes ROH···HW(CO)(NO)(PMe₃)₃ (R = 3,4,5-Me₃C₆H_2,3a and iPr, 7a) and the O_NO hydrogen bonded species ROH···ONW(CO)(H)(PMe₃)₃ (R = C₆H₂Me_3,3b and iPr, 7b) which were studied in hexane and d₈-toluene solutions using variable temperature IR and NMR spectroscopy. Quantitative IR experiments at low temperatures using 3,4,5-trimethylphenol (TMP) confirmed the two types of competitive equilibria: dihydrogen bonding to give 3a (ΔH₁ = −5.8 ± 0.4 kcal/mol and ΔS₁ = −15.3 ± 1.4 e.u.) and hydrogen bonding to give 3b (ΔH₂ = −2.8 ± 0.1 kcal/mol and ΔS₂ = −5.8 ± 0.3 e.u.). Additional data for the hydrogen bonded complexes 3a,b and 7a,b were determined via NMR titrations in d₈-toluene from the equilibrium constants K_(Δδ) and K_(ΔR1) measuring either changes in the chemical shifts of H_W(_Δδ) or the excess relaxation rates of H_W (ΔR₁) (3a,b: ΔH_(Δδ) = −0.8 ± 0.1 kcal/mol; ΔS_(Δδ) = −1.4 ± 0.3 e.u. and ΔH_(ΔR1) = −5.8 ± 0.4 kcal/mol; ΔS_(ΔR1) = −22.9 ± 1.9 e.u) (7a,b: ΔH_(Δδ) = −2.3 ± 0.2 kcal/mol; ΔS_(Δδ) = −11.7 ± 0.9 e.u. and ΔH_(ΔR1) = −2.9 ± 0.2 kcal/mol; ΔS_(ΔR1) = −14.6 ± 1.0 e.u). Dihydrogen bonding distances of 1.9 Å and 2.1 Å were derived for 3a and 7a from the NMR excess relaxation rate measurements of H_W in d₈-toluene. An X-ray diffraction study was carried out on compound 2.     

The measurement of cyclic nucleotide phosphodiesterase 4 activities via the quantification of inorganic phosphate with malachite green

A spectrometric method was investigated to measure the activities of recombinant human cyclic nucleotide phosphodiesterase 4 (PDE4), based on the use of malachite green (MLG) to quantify phosphate released from adenosine-5′-monophosphate (AMP) by the action of calf intestinal alkaline phosphatase (CIAP). Glycerol at 2% stabilized the complex between MLG and phosphomolybdate, whose absorbance at 630 nm was proportional to phosphate concentrations with resistance to common substances in PDE4 reaction mixtures except papaverine. CIAP had the Michaelis-Menten constant (K_m) of (12.0 ± 2.1) μM (n = 3) for AMP at pH 7.4, and was resistant to EDTA below 0.20 mM. By the coupled end-point assay at 30.0 U L⁻¹ CIAP with reaction durations within 30 min, the rates to release phosphate in PDE4 reaction mixtures containing 10.0 mM MgCl₂ and 0.10 mM EDTA linearly responded to the amounts of PDE4 over wide ranges. Meanwhile, K_m of PDE4 was (8.8 ± 0.2) μM (n = 2), zinc ion inhibited PDE4 and rolipram had the inhibition constant about 10 nM. These results supported that by the coupled end-point assay, this method was promising to screen of PDE inhibitors that had no interference with the MLG assay of phosphate.