A microcolorimetric assay of inorganic pyrophosphatase

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction

Background

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.

Methods

Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.

Results

Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations < 10 nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.

General significance

Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1–10 nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.     

Spectral resolution of a second binding site for Nile Red on cytochrome P4503A4

Nile Red is sequentially metabolized by cytochrome P4503A4 to the N-monoethyl and N-desethyl products, which typifies the metabolism of many amine containing drugs. Sequential metabolism of a single substrate results in complex kinetics that confound predictive models of drug clearance. As a fluorescent model for drugs which undergo sequential metabolism, Nile Red provides the opportunity to monitor drug-CYP interactions wherein the fluorescent properties of Nile Red could, in principle, be exploited to determine individual rate and equilibrium constants for the individual reactions. Previously, it was shown that Nile Red binds at the active site and fluoresces (K_D ≈ 50 nM) with maximum emission at ∼620 nm, but it was unclear whether a red-shifted emission, at ∼660 nm, consisted of only free Nile Red or Nile Red bound at a second site on the protein. Here, equilibrium binding studies, including ‘reverse titrations’ spanning low ratios of CYP3A4/Nile Red, indicate two binding sites for Nile Red with a contribution to the ‘red emission’ greater than can be accounted for by free Nile Red. Singular value decomposition affords basis spectra for both spectral components and fits well to the experimentally determined concentration dependence of Nile Red emission. In addition, the red spectral component, with an apparent K_D = 2.2 μM, is selectively eliminated by titration with the known allosteric effectors of CYP3A4, α-napthoflavone and testosterone. Furthermore, the double mutant L2311F/D214E, previously demonstrated to perturb a peripheral allosteric site, lacks the red-emitting Nile Red binding site, but retains the blue-emitting site. Together these data indicate that a second Nile Red site competes with other effectors of CYP3A4 at a site that results in Nile Red emission at 660 nm.     

A modified protein assay from microgram to low nanogram levels in dilute samples

In this article, we present a modified and improved protein assay that was previously described as “amidoschwarz assay” by Schaffner and Weissmann [13]. Our improved protein assay is user-friendly and 30–40 times more sensitive than the earlier method. The assay was developed into three formats (macro-, micro-, and nanoassay) with trichloroacetic acid (TCA) as protein precipitating agent, measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots bound to nitrocellulose membrane with lowest protein measurements to 1 and 0.1 μg, respectively. On the other hand, the nanoassay, with combination staining of amido black followed by colloidal gold, can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 μg/ml prior to their biochemical analysis such as in comparative proteomics.     

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: Correlation with studies of DPH and Laurdan fluorescence

The effect of non-ionic detergents on baclofen (GABA_B-R agonist)-stimulated G-protein activity was measured as a [³⁵S]GTPγS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic — a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABA_B-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58 > Triton X-100 > Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (r_DPH) incorporated into the hydrophobic PM interior. Decrease of r_DPH, in the order of Brij58 > Triton X-100 > Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time ? paralleled by an increase of diffusion wobbling constant D_w (analysis by time-resolved fluorescence according to “wobble-in-cone” model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58 > Triton X-100 > Digitonin.     

A gas chromatographic method for quantification of detergents frequently used in membrane protein structural studies

A novel and reliable gas chromatography-flame ionization detection (GC-FID) method that can separate and quantify detergents frequently used in membrane protein structural studies has been developed. Different detergents were identified through FID peaks with different retention times. A quadratic regression curve was found to fit the integrated FID peak area against different detergent concentrations. Detergents can be quantified as low as the nanogram level: lauryl-dimethylamine-N-oxide (LDAO), 5 ng; dodecyl maltoside (DDM), 10 ng; and dodecyl phosphocholine (DPC), 50 ng. This method can be applied directly to measure detergent concentration and molar ratio of membrane protein to detergents during membrane protein extraction, purification, concentration, and crystallization.     

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.     

Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranoside detergents

A procedure was developed for the quantitation of solubilized proteins using the Bradford assay in the presence of glucopyranoside detergents. These detergents solubilized membrane-bound proteins with minimal background absorbance at 595 nm. Absorbance at 650 nm was also low, indicating that these detergents do not significantly stabilize the neutral species of Coomassie brilliant blue G-250 that produces interference in the presence of other detergents. Hexyl-beta-D-glucopyranoside produced less absorbance than did larger glucopyranosides, and the increase in its absorbance at 595 nm in the presence of dye reagent was related linearly to its concentration from 0 to 2%. Absorbance produced by membrane-bound protein was increased by the presence of up to 0.2% hexyl-beta-D-glucopyranoside (final concentration in dye reagent) and then remained stable up to 1%, indicating that these concentrations of this detergent allowed membrane-bound proteins to react completely with the dye reagent. Standard curves of several proteins were similar in the absence or presence of 0.1-0.5% hexyl-beta-D-glucopyranoside. The quantitation of both soluble and membrane-bound proteins by the Bradford assay was similar in the presence of 0.2% hexyl-, heptyl-, and octyl-beta-D-glucopyranoside. Estimates of membrane-bound protein by this assay agreed with estimates obtained with the Lowry assay and with quantitative amino acid analysis. This procedure requires no extra steps; thus, it is as rapid and convenient as the original Bradford protein assay.     

Quantification of folate in fruits and vegetables: A fluorescence-based homogeneous assay

A high-throughput, homogeneous, fluorescence polarization, and fluorescence intensity assay has been developed for the measurement of folate in fruits and vegetables. This assay is based on the competitive displacement of the fluorescent folate ligands Alexa Fluor (Alexa) 594-folate and Alexa 660-folate from bovine milk folate-binding protein by folates in fruit and vegetable extracts. These fluorescent ligands are employed because their excitation and emission maxima are in regions of the spectrum with minimal autofluorescence in many extracts. Folate-binding protein and Alexa-folate were typically used at concentrations of 0.5 μg/ml and 5 nM, respectively, in 20-μl volumes in 384-well microplates. The assay is complete within 100 min. The folate estimate is unaffected by the heterogeneity of polyglutamyl residues that complicates the liquid chromatography-mass spectrometry (LC-MS)-based methods of quantification. In this assay, folic acid had an apparent affinity 2.5-fold greater than 5-methyltetrahydrofolate (5MTHF); therefore, it cannot be used to quantify folate when both natural and synthetic folate are present. 5MTHF-equivalent values were measured in broccoli (240 μg/100 g), strawberry (113 μg/100 g), white grape (32 μg/100 g), orange (44 μg/100 g), tomato (12 μg/100 g), raspberry (31 μg/100 g), banana (29 μg/g), and kiwifruit (36 μg/100 g). These data are similar to published values. However, the assay will not detect 5-formyltetrahydrofolate which is a significant constituent of the total folate in lettuce, spinach, carrot, and peppers.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9_β1) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α + β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9_α + β1). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9_β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9_α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.     

Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24 h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1 h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000 Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.     

Interaction of proteins with detergents: Binding of cationic detergents with lysozyme

Binding studies of cationic detergents such as cetyl trimethylammonium bromide, Cetylpyridinium bromide and dodecyl trimethylammonium bromide with lysozyme were carried out by equilibrium dialysis, ultraviolet difference and circular dichroism techniques at 25 C. Binding isotherms at pH 5·0, 7·0 and 9·0 show cooperative binding at all concentrations of detergents and the number of available binding sites in lysozyme increases with pH. Gibbs free energy of binding calculated on the basis of Wymans’ binding potential concept increases with pH indicating increased binding strength at higher pH. The ultraviolet difference spectra of the detergent complexes with lysozyme at pH 7·0 and 9·0 in the region of 250–300 nm indicate the involvement of aromatic amino acid residues as probable binding sites and also the carboxylate groups since the binding is cooperative. The circular dichroism spectra also indicate the involvement of aromatic amino acid residues in the binding of these detergents. This is substantiated by the decrease in the intensity of the aromatic positive bands in the near ultraviolet region. The increase in the magnitude of [θ]_{222 nm} values in the far ultraviolet region with the increase in the concentration of the detergent in the complex indicates conformational changes resulting in an increase of α-helical content producing a more ordered structure of lysozyme.These binding studies show that at pH 7·0 and 9·0, hydrophobic interactions play a major role, while at pH 5·0 only electrostatic interactions play prominent role in the binding of these detergents. Paper presented at the International Symposium on Biomolecular Structure and Interactions held at the Molecular Biophysics Unit, Indian Institute of Science, Bangalore, during 17–22, December 1984.     

The ferrous–dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: The role of proximal histidine on its stability

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O₂ dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790–16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O₂ are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25 °C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576 nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O₂ at a rate of ~ 10 s^− 1. To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~ 30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate = 6.1 s^− 1). Decomposition of the oxygenated intermediate is independent of O₂ concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.     

Interference of biogenic amines with the measurement of proteins using bicinchoninic acid

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.     

Color development time of the Lowry protein assay

Color development of the Lowry protein assay was tracked over time for bovine serum albumin (BSA) concentrations ranging from 40 to 600 μg/ml. The time interval between 2 and 4 h produced the most stable readings. This time frame also improved linearity of the standard curve.     

A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K_d of 26 ± 2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z′ value of 0.73 ± 0.06 in a 384-well format.     

Homogeneous noncompetitive assay of protein via Förster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor

Homogeneous noncompetitive assay of a protein in biological samples based on Förster-resonance-energy-transfer (FRET) was proposed by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor that was defined as an analytical FRET probe. Conjugate of a suitable fluorophore, which should have an excitation peak around 340 nm but an excitation valley around 280 nm, with a moiety binding to a protein of interest gave an analytical FRET probe to the protein. To test this method, N-biotinyl-N′-(1-naphthyl)-ethylenediamine (BNEDA) was used as an analytical FRET probe for homogeneous noncompetitive assay of streptavidin (SAV). The occurrence of FRET between the bound BNEDA and tryptophan residues was supported by the modeled geometry of the complex. By excitation at 280 nm, free BNEDA produced negligible fluorescence at 430 nm, but the bound BNEDA produced much higher stable fluorescence at 430 nm after 2 min of binding reaction. The competitive binding between BNEDA and biotin gave the dissociation constant of (16 ± 3) fM for BNEDA (n = 3). By excitation at 280 nm, fluorescence at 430 nm of reaction mixtures containing 32.0 nM BNEDA responded linearly to SAV subunit concentrations ranging from 0.40 to 30.0 nM with the desirable resistance to common interferences in biological samples. Therefore, by using tryptophan residue(s) in a protein of interest as intrinsic donor(s) and its fluorescent ligand as the corresponding FRET acceptor, this homogeneous noncompetitive assay of the protein in biological samples was effective and advantageous.