University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Cow Serum

Classification: Biological fluid -> blood -> serum -> cow

Citations 32

"Flow Injection Determination Of Proteins Based On The Lowry Spectrophotometric Method"
Anal. Chim. Acta 1989 Volume 217, Issue 2 Pages 359-362
Hans Lüdi and Anita Bärtschi

Abstract: The flow injection system was based on the use of Folin - Ciocalteu reagent, with 1,4-dithio-DL-threitol to accelerate the formation of the colored complex, which was detected at 700 nm. The calibration graph for bovine serum albumin was rectilinear between 0.01 and 0.1 mg mL-1. The detection limit was 5 µg mL-1 and up to 0.5 mg mL-1 could be determined. The injection frequency was 20 h-1. Results obtained by the flow injection method and by the manual Lowry procedure correlated well. Egg lysozyme, alkaline phosphatase and β-lactoglobulin were also determined.
Albumin Lysozyme Enzyme, alkaline phosphatase β-Lactoglobulin Spectrophotometry Complexation Method comparison

"Off-line And Online Assay Of Membrane Protein With O-phthaldialdehyde By Flow Injection With Post-column Reaction"
Anal. Chim. Acta 1990 Volume 231, Issue 1 Pages 249-257
Samuel M. Mozersky

Abstract: Sample (325 µL) was injected into an off-line flow injection analysis system (details and diagram given) with 300 mM sucrose - 1 mM 2-mercaptoethanol (I) - 1 mM dithiothreitol - 3 mM HEPES buffer (pH ~7.4) as sample carrier solution (0.5 mL min-1) and 2% sodium dodecyl sulfate - 11.9 mM o-phthalaldehyde - 57 mM I - 4% methanol - 100 mM tetraborate buffer (pH ~10.8) as reagent solution (0.5 mL min-1). Adequate flushing was ensured by using a load volume of 1 mL to fill the injection loop. Detection was at 340 nm. A rectilinear relationship between peak height and protein concentration. was observed for 0 to 1000 mg L-1 of bovine serum albumin and 0 to 140 mg L-1 of mitochondrial protein. The off-line system was modified for online monitoring of the effluent from a field flow fractionator (the same basic design could be applied to any separation device yielding an effluent stream, e.g., a chromatographic column). The online apparatus was applied in the determination of the distribution of membrane protein in the channel effluent after fractionation of sub-cellular particle preparations containing corn root mitochondria and microsomes.
Albumin Spectrophotometry Membrane Buffer pH Post-column derivatization

"Effect Of Sample Viscosity On The Behaviour Of Thin-channel Flow-through Dialysers In Flow Injection Analysis"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 435-440
Lars Risinger and Gillis Johansson*

Abstract: An evaluation has been made of the effect of pressure variations (caused by viscous samples) in a flow injection system for the enzymatic determination of glucose. The sample of bovine serum albumin was injected into 0.1 M phosphate - 0.14 M NaCl (pH 6.5) as carrier, and the resulting solution was dialysed against a second stream of the same carrier. The acceptor stream was merged with a similar solution additionally containing 5 mM NAD+ before passing through a reactor containing glucose dehydrogenase and aldose 1-epimerase co-immobilized on porous glass to an amperometric detector for monitoring of the NADH produced. The pressure changes caused by injection of viscous samples caused pre-peaks on the recorded signal, especially with thin liquid channels in the dialyser. The problem could be counteracted by using a stiffer membrane and removing restrictions to flow on the sample side after the dialyser. Viscous samples will cause pressure changes which affect the transfer of solutes through an online dialyzer. Pressure changes can cause prepeaks in the recorded signal due to membrane movements. The flow-rate in the receiver channel increases temporarily as the pressure builds up. A pressure differential of 30 kPa forced 1% of the liquid (at 0.4 mL min-1) through the membrane to the side with the lower pressure. This hydrodynamic flow will influence the diffusionally driven transfer of solutes and may cause anal. errors. The design of a flow injection analysis (FIA) system with a flow-through dialyzer is discussed. The membrane should be stiff, with a low solvent transport rate to minimize fluctuations of the response due to changes of the differential pressure when a viscous sampler is passing the dialyzer. The system should be made with stiff components and without flow restrictors after the dialyzer on the sample side.
Albumin Glucose Amperometry Dialysis Viscosity Immobilized enzyme

"High-sensitivity Flow Method For The Determination Of Proteins With Micro-flow Plunger Pumps"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 67-74
Takashi Korenaga*, Xiaojing Zhou, Miyoko Izawa, Teruo Takahashi and Tosio Moriwake, Sumio Shinoda

Abstract: Proteins were determined by the dye association method with use of Coomassie Brilliant blue G250 (C. I. Acid Blue 90; I) in conjunction with a flow device based on that of Korenaga and Stewart (Ibid., 1988, 214, 87), which incorporated a double-plunger micro-pump. The device was operated in either flow injection or continuous-monitoring mode (manifolds illustrated). For the former, the sample solution (8 µL) was injected into a stream (0.02 mL min-1) of 0.1 M K2HPO4 - KH2PO4 (pH 7.4) which was then merged with 60 µM-I in 2.8% HClO4, also at 0.02 mL min-1, in a Ti reaction tube (20 cm x 0.5 mm i.d.) before passing through a spectrophotometric flow cell for absorbance measurement at 600 nm. In the continuous mode, the aqueous sample solution and the I reagent (each at 0.02 mL min-1) were mixed in a Ti tube (10 cm x 1 mm i.d.) before entering the flow cell. The best calibration graphs were obtained at low flow rates. Titanium reaction tubes were used because of unstable baseline absorbance obtained with PTFE tubes. For bovine serum albumin determined by the flow injection method, the calibration graph was rectilinear up to 140 µg mL-1, and the standard deviation (n = 10) was 1.5 ng mL-1 at 1.0 µg mL-1. The method was successfully applied to urine samples. To determine microamounts of proteins with low mol. diffusivity, a flow method was developed by both miniaturizing the common flow-injection apparatus and using the dye association method. A very precise double-plunger micropump, which was recently developed for this purpose, was used. The optimum conditions using bovine serum albumin (BSA) as standard and some ion-association dye reagents at very low flow-rates were examined A flow-rate of <50 µL/min was acceptable when a short, small-bore reaction tube was used. Up to 15 samples/h could be analyzed with a relatively long residence time; linear calibration graphs were obtained with both 8 µL volume loop valve injection and no-injection methods. The detection limit and relative standard deviation of the injection method were 0.003 µg/mL and 0.15%, respectively, for BSA.
Albumin Proteins Spectrophotometry Pump Optimization

"Simultaneous Spectrophotometric Determination Of Iron And Copper In Serum With 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)aniline By Flow Injection Analysis"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 197-203
Sam Woo Kang, Tadao Saki* and Noriko Ohno, Kazunori Ida

Abstract: The system described and illustrated incorporates a double-beam spectrophotometer with two flow cells. The sample is injected into 0.1 M HCl - 0.1 mM KIO4 as carrier and mixed with a 0.1 mM solution of the cited reagent in acetate buffer of pH 4.5 in a 15-cm reaction coil. The solution is passed through the first flow cell for measurement of the absorbance due to Cu(II) at 558 nm. A 10 mM solution of Na ascorbate in the buffer is introduced into the stream, which passes through a 700-cm reaction coil heated at 60°C before measurement of the absorbance at 558 nm in the second flow cell; in this instance an inverted peak for Fe(II) and a positive peak for Cu(II) are obtained. The Fe(III) present after adding the sample to the carrier does not interfere. The calibration graphs are rectilinear for 50 to 200 µg L-1 of Cu(II) or Fe(II) and the limit of detection is 2.4 µg l-1. Nickel and Co interfere at concentration. >10 µg L-1 which, however, are not encountered in serum samples. Serum is deproteinized with trichloroacetic acid at 90°C before analysis. Results on human, horse, bovine, chicken and goat serum agreed with those obtained by ICP-AES. A method for the simultaneous spectrophotometric determination of iron and copper by flow injection analysis was developed using 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N- sulfopropylamino)aniline. The flow system utilizes a double-beam spectrophotometric detector with two flow cells. The reagent forms water-sol. chelates with Cu(II) and Fe(II) in an acetate-buffered medium at pH 4.5. The molar absorptivities of the complexes are 65,000 L mol-1 cm-1 at 578 nm for Cu(II) and 87,000 L mol-1 cm-1 at 558 nm for Fe(II). In one reaction coil Fe(II) is oxidized to Fe(III) by KIO4, so the Fe(II) chelate is not formed and only the colored complexes with Cu(II) are monitored in the first flow cell. In a second reaction coil, the solution merges with sodium ascorbate solution to reduce Cu(II) and Fe(III) to Cu(I) and Fe(II). Thus only the Fe(II) complex is measured in the second flow cell. Cu(II) and Fe(II) in the range 50-200 µg L-1 are determined at 558 nm. The sample throughput is 30 h-1 with a precision of 1.2%.
Iron Copper Spectrophotometry Simultaneous analysis Method comparison Buffer Complexation Interferences

"Spectrophotometric Measurement Of PH Gradients In Continuous-flow Systems"
Anal. Chim. Acta 1992 Volume 268, Issue 1 Pages 29-38
S. Sagrado Vives, M. J. Medina Hern&aacute;ndez, J. L. Mart&iacute;n Herrera and G. Ramis Ramos*

Abstract: The use of acid - base indicators to monitor pH changes during flow injection analyzes was investigated. The flow injection system incorporated an injection valve, a flow cell (18 µL), a coil reactor made from a PTFE tube and 0.5 mm i.d. PTFE tubing; a single-bead string reactor following the coil reactor was used for experiments involving bovine serum albumin. A pH meter and a fast-scanning diode-array spectrophotometer linked to a computer completed the assembly. To illustrate the system, in addition to HCl - NaOH systems containing pH indicators, experiments were carried out with a sample flow solution of aqueous bovine serum albumin (25 µM) at several pH values containing 10 µM-thymol blue; the carrier solution contained phthalaldehyde - N-acetyl-L-cysteine - H3BO3 - borate buffer of pH 9.5. The flow rate was 1.9 mL min-1. The pH and the absorbance were continuously monitored, one spectrum per s being recorded. The method allows effective monitoring of pH gradients within the spectrophotometric cell at the same time as the flow injection analysis. In the presence of an acid-base indicator and using fast diode-array spectrophotometric scans, pH gradients in the spectrophotometric cell can be measured at the same time as any other colorimetric flow injection (FI) determination is performed. The effects of refractive index changes, adsorption-desorption processes at the tube walls and association of the indicator species with the system to be investigated are considered. Effective correction for the first two effects is demonstrated. Indicators with different mol. structures and polarities gave similar pH gradients, suggesting adequate accuracy for most potential applications. Application to the detection and correction of systematic and random errors in pH-sensitive FI procedures is demonstrated using the o-phthalaldehyde-N-acetyl-L- cysteine method for bovine serum albumin. In the presence of an acid-base indicator and using fast diode-array spectrophotometric scans, pH gradients in the spectrophotometric cell can be measured at the same time as any other colorimetric flow injection (FI) determination is performed. The effects of refractive index changes, adsorption-desorption processes at the tube walls and association of the indicator species with the system to be investigated are considered. Effective correction for the first two effects is demonstrated. Indicators with different molecular structures and polarities gave similar pH gradients, suggesting adequate accuracy for most potential applications. Application to the detection and correction of systematic and random errors in pH-sensitive FI procedures is demonstrated using the o-phthalaldehyde-N-acetyl-L-cysteine method for bovine serum albumin.
Albumin pH Spectrophotometry Computer Single bead string reactor Refractive index Gradient technique pH gradient

"Fluorescence-based Flow Injection Determination Of Biotin And Biotinylated Compouds"
Anal. Chim. Acta 1993 Volume 279, Issue 2 Pages 287-292
Truis Smith-Palmer, Minas S. Barbarakis, Tadeausz Cynkowski and Leonidas G. Bachas*

Abstract: The assay described was based on the enhancement of the emission intensity of the fluorescein-labelled conjugate of streptavidin by the biotin moiety. A merging-zone FIA system was used in which 70 µL of streptavidin-fluorescein isothiocyanate reagent (1.0 mg/l) was injected into one buffer stream and 70 µL of sample was injected into a second buffer stream. Both streams consisted of 50 mM phosphate buffer of pH 8 (1 ml/min). The streams were merged and passed through a 2-m knitted open-tubular reactor coil into the detection cell were the emission signal at 518 nm was recorded (excitation at 495 nm). The calibration graphs for biocytin, biotin, biotin-thyroxine and biotinylated BSA were linear at low concentration. (0.4 µM) but the gradients varied considerably. The detection limit was 2 nM-biotin.
Biotin Biocytin Biotin thyroxine Albumin, biotinylated Fluorescence Merging zones Buffer Knotted reactor

"Dynamic Analysis Of The Binding Process Of Bovine Serum Albumin On Glutaraldehyde-activated Controlled Pore Glass"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 261-268
Hiroyuki Ukeda*, Tohru Ishii, Masayoshi Sawamura and Hirozo Kusunose

Abstract: Twenty microlitre volumes of BSA solution (5 mg/ml) were repeatedly injected into a column (5 cm x 1.68 mm i.d.) packed with aminopropyl-controlled pore glass activated with glutaraldehyde (GA-CPG). The elution profile of BSA was recorded using phosphate buffer eluents (0.5 ml/min) and detection at 280 nm. The results were analyzed using a model based on the assumption that two modes are involved in binding BSA to GA-CPG. Binding process parameters such as bound amounts and binding rate constants were estimated by a curve fitting method. An increase in ionic strength of the carrier solution resulted in a reduction in the total amount of BSA bound. The maximum bound amount occurred at pH 6 with low ionic strength carriers and at pH 7 for higher ionic strength carriers. The reduction of GA-CPG with sodium borohydride reduced the bound amount while blocking treatment with amine had no effect.
Albumin Spectrophotometry Controlled pore glass pH Optimization

"Pulsed Electrochemical Detection Of Proteins Using Conducting Polymer Based Sensors"
Anal. Chim. Acta 1995 Volume 315, Issue 1-2 Pages 27-32
W. Lu, H. Zhao and G. G. Wallace*

Abstract: These studies reveal that sensitive responses for proteins can be generated at selected conducting polymer electrodes using flow-injection analysis (FIA) and pulsed electrochemical detection. The responses observed are dependent on the nature of the polymer, the nature of the electrolyte anion/cation as well as on the electrolyte concentration and pH. These factors can be used to modify selectivity. Detection limits in the order of nanomolar have been estimated.
Albumin Myoglobin Ovalbumin α-Lactalbumin Voltammetry Sensor

"A Novel Thermal Biosensor: Evaluation For Determination Of Urea In Serum"
Anal. Chim. Acta 1998 Volume 372, Issue 3 Pages 341-348
Bjarni Bjarnason*, Peter Johansson and Gillis Johansson

Abstract: A novel thermal biosensor has been made based on heat conduction through a BiTe-thermocouple on alumina plates. The sensing element was in close thermal contact with a gold tube enzyme reactor in a flow injection system. It was successfully applied to bovine serum samples where urea could be determined from a calibration graph or by standard addition. The linear range of the system was 0.5-15 mM with a detection limit of 0.5 mM. A throughput of up to 60 samples per hour was achieved.
Urea Sensor Thermistor Standard additions calibration

"Some Observations On The Automation By Flow Injection Analysis Of The Spectrophotometric Determination Of Amino-acids And Proteins With Phthalaldehyde"
Microchim. Acta 1992 Volume 108, Issue 3-6 Pages 293-298
M. J. Medina Hern&aacute;ndez, S. Sagrado Vives and M. C. Garc&iacute;a Alvarez-Coque

Abstract: An improved flow injection system for the determination of total free amino-acids and protein is described. The procedure is based on the rapid reaction of α- and ε-amino-groups in amino-acids and proteins with phthalaldehyde in the presence of N-acetyl-L-cysteine at room temperature and pH 8.5 to 9.5. The absorbance due to the reaction products is monitored at 336 nm. For the determination of free amino-acids the system is calibrated with 0.5 to 9 mM leucine; the detection limit is 10 µM. For the determination of proteins the system is modified to include a longer reaction period. Calibration graphs are prepared with bovine serum albumin, casein and β-lactoglobulin over the range 2 to 20 µM; the detection limit is 1 µM.
Amino Acids Proteins Albumin Enzyme, casein β-Lactoglobulin Spectrophotometry Automation

"Microdetermination Of Proteins With The 1,10-phenanthroline-H2O2-cetyltrimethylammonium Bromide-Cu(II) Chemiluminescence System"
Microchem. J. 1998 Volume 60, Issue 3 Pages 217-223
Li Zheng Ping, Li Ke An and Tong Shen Yang

Abstract: Proteins can enhance the chemiluminescence (CL) intensity of the 1,10-phenanthroline-H2O2- cetyltrimethylammonium bromide (CTMAB)-Cu(II) system because unsaturated complexes of Cu(II) with protein have a much stronger catalytic effect on the CL reaction than does Cu(II). On this basis, a new flow injection analysis method for detection of some proteins was established. The method gives Linear responses over two orders of magnitude and detection limits at the 0.02-0.05 µg mL-1 level for bovine serum albumin, human serum albumin, γ-globulin, and egg albumin. The method was used for determination of proteins in human serum with satisfactory results.
Proteins Albumin Chemiluminescence

"Lowry Protein Determination By Automated Flow Injection Analysis For Bovine Serum Albumin And Hepatitis B Surface Antigen"
Anal. Biochem. 1985 Volume 151, Issue 2 Pages 309-314
Ronald A. Salerno, Cathy Odell, Nancy Cyanovich, Bernard P. Bubnis, Wayne Morges and Alan Gray

Abstract: The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90°C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyzes (90 samples/h), small sample volume (30 µL, 100 µL), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 µg), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing. Bovine serum albumin(I) or hepatitis B surface antigen(II) was determined by the Lowry method adapted for use with flow injection analysis. The sample was mixed with the alkaline tartrate copper reagent and heated at 90°C in a reaction coil (100 cm x 0.7 mm). The solution was mixed with Folin - Ciocalteu reagent in a second reaction coil (60 cm x 0.5 mm) and the absorbance of the mixture was measured at 600 nm. The pH was 10.5. The calibration graphs were rectilinear for 10 to 80 and 10 to 40 µg mL-1 of I and II, respectively, and the limit of detection was 5 µg mL-1; the within- and between-assay coefficient of variation were 2 and 3 to 6%, respectively. The results correlated well (r = 0.998) with those obtained by the manual method.
Proteins Albumin Spectrophotometry Heated reaction Optimization Tecator

"Amino-acid Analysis By High Performance Liquid Chromatography Of A Single Stained Protein Band From A Polyacrylamide Gel"
Anal. Biochem. 1987 Volume 160, Issue 2 Pages 362-367
Yoko Hashimoto, Sadako Yamagata* and Taro Hayakawa*

Abstract: A single stained band containing ~5 µg of protein was cut from a polyacrylamide gel and hydrolyzed in the presence of mercaptoacetic acid. The hydrolysate was analyzed by HPLC at 55°C on a column (15 cm x 4 mm) of sulfonated polystyrene cation-exchange resin (7 µm) with a pre-column (25 cm x 4 mm) of Dowex 50W-16 resin and step gradient elution with 0.2 M Na citrate (pH 3.15) - ethanol (93:7), 0.6 M Na citrate (pH 10.0) and 0.2 M NaOH at 0.3 mL min-1. Fluorimetric detection was at 450 nm (excitation at 348 nm) after post-column derivatization with phthalaldehyde and NaClO. Ammonia did not interfere. The recovery of methionine was inconsistent and recoveries of tryptophan, isoleucine, threonine and serine were low. Bovine serum albumin, ribonuclease B, ovalbumin, pepsin and chymotrypsinogen A were analyzed by this method with good agreement with theoretical values.
Amino Acids Methionine Tryptophan Isoleucine Threonine Serine Albumin Enzyme, ribonuclease b Ovalbumin Pepsin Chymotrypsinogen, A HPLC Fluorescence Interferences Post-column derivatization

"Direct Nanogram Quantitation Of Nucleic Acid And Protein With A Continuous-flow Microcell"
Anal. Biochem. 1988 Volume 169, Issue 1 Pages 138-141
Jingdong Zhu, Abbas Rashidbaigi and Sidney Pestka

Abstract: The samples were injected into the post-column flow stream of an HPLC system as the stream was pumped past a diode-array detector set at 260 nm for nucleic acids and 280 nm for proteins. Response was rectilinear between 3 ng and 7 µg of DNA, 10 ng and 10 µg of RNA and 10 ng and 80 µg of bovine serum albumin. The method was simple, required only 5 to 10 µL of sample solution, and was rapid (2 samples min-1).
Nucleic acids, ribo Nucleic acids, deoxyribo Albumin Spectrophotometry Post-column derivatization

"Separation Of Free Amino-acids By Reversed-phase Ion-pair Chromatography With Column Switching And Isocratic Elution"
J. Chromatogr. A 1990 Volume 507, Issue 1 Pages 95-101
Mitsuko Hirukawa, Masako Maeda and Akio Tsuji, Toshihiko Hanai

Abstract: Seventeen free amino-acids were separated in 35 min by ion-pair chromatography using two columns: an Inertsil phenyl column (5 cm x 4.6 mm) for hydrophobic and basic amino-acids, and an Inertsil ODS-2 column (25 cm x 4.6 mm) for polar acidic and small amino-acids. Two eluents, viz. 50 mM Na phosphate buffer (pH 2.7) containing 6.5% of ethanol, 6.5% of acetonitrile and 0.11 M NaCl and the same buffer containing 25.6 mM Na dodecyl sulfate plus 12% of methanol and 94 mM NaCl, were used isocratically at 1 mL min-1. After post-column derivatization with phthalaldehyde, the amino-acids were determined by fluorescence at 450 nm (excitation at 340 nm); detection limits were 2 to 5 pmol for those with shorter retention times. Calibration graphs were rectilinear up to 500 pmol. The method was applied to bovine serum albumin hydrolysate, and results agreed well with literature values with results from LC.
Amino Acids HPLC Fluorescence Column Buffer Detection limit Post-column derivatization Method comparison

"Electrogenerated Chemiluminescence Detector For Flow Injection Analysis"
Anal. Sci. 1986 Volume 2, Issue 6 Pages 529-533

Abstract: A flow-through electrolytic cell was modified to allow measurement of electrogenerated chemiluminescence (ECL). The quartz optical window was set in front of the platinum plate working electrode. The observed luminescence intensity was amplified by using a lock-in amplification technique. The system was tested by using luminol, which exhibits strong ECL. Samples injected into a carrier solution of 0.1 M Na2CO3 gave luminescence peaks. Non-luminescent compounds could be detected by pre-labelling with luminol. The technique was applied to oligopeptides and bovine serum albumin. The method has the advantage of determining luminescence intensity and electrolytic current simultaneously.
Albumin Luminol Oligopeptides Chemiluminescence Electrode Detector

"Reconstitution Of Purified GABAA Receptors: Ligand Binding And Chloride Transporting Properties"
Biochemistry 1994 Volume 33, Issue 3 Pages 755-763
Susan M. J. Dunn and Rick P. Thuynsma

Abstract: GABAA receptors have been solubilized from bovine brain membranes and, following purification by benzodiazepine affinity chromatography, have been reconstituted into phospholipid vesicles. Reconstituted vesicles were about 120 nm in diameter, and, on average, each vesicle contained fewer than one GABAA receptor which was reconstituted in an outside-out orientation. These preparations have been used in parallel studies of radiolabeled ligand binding and chloride flux, the latter being measured by following the fluorescence changes of a chloride-sensitive probe which was trapped within the vesicles at the time of reconstitution. The benzodiazepine [3H]flunitrazepam binds to an apparently homogeneous population of sites in these preparations (Kd of 5 nM) whereas the GABA analogue [3H]muscimol binds to both high- and low-affinity sites (KdS of 10 nM and 0.27 µM). Muscimol stimulated chloride flux with an EC50 of 0.2 µM and, at similar concentrations (EC50 = 0.16 µM), potentiated [3H]flunitrazepam binding, suggesting that occupancy of the low-affinity sites may be important for these effects. Diazepam shifted the dose-response curve for muscimol- stimulated flux to about 4-fold lower concentrations without affecting the maximum response. Diazepam did not, however, alter the equilibrium binding of [3H]muscimol. The purified receptor showed densensitization since flux responses were abolished by prior exposure to muscimol. The competitive antagonist bicuculline and the channel blocker picrotoxin completely inhibited ion flux mediated by 3 µM muscimol with EC50 values of 5.3 and 2.5 µM, respectively. These results are discussed in terms of possible mechanisms for activation, inhibition, and modulation of GABAA receptors.
Chloride Flunitrazepam LC

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 1. The Flow Injection Analysis Of Protein"
Bull. Chem. Soc. Jpn. 1983 Volume 56, Issue 5 Pages 1382-1387
Tadashi Hara,Motohiro Toriyama and Kazuhiko Tsukagoshi

Abstract: A new method in which luminol-hydrogen peroxide luminescent system is used has been proposed for the determination of the presence of protein. Since the catalytic activity of copper(II) for the chemiluminescent reaction between luminol and hydrogen peroxide decreased when copper(II) interacted with polypeptide linkage, this phenomenon was applied to the determination of protein. Determination of protein was carried out by a flow-injection method. The effects of reagent concentration, flow-rate, and reaction time on the analytical value were examined and the conditions for the determination of protein were established. Similar calibration curves were obtained for human serum albumin, bovine serum albumin, bovine serum α-globulin, and bovine serum γ-globulin. According to the present flow-injection method using chemiluminescent reaction, a small amount of protein could be conveniently and economically determined over a wide range of concentration, 7 x 10^-4 - 7 x 10^-2 g dm-;3, with the detection limit of 0.2 µg and at the rate of about 30 samples per hour. The present method was applicable to the determination of protein in serum.
Albumin Proteins γ-Globulin Clinical analysis Chemiluminescence Catalysis Optimization

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 5. An Iron - Dyestuff Complex As A Catalyst"
Bull. Chem. Soc. Jpn. 1985 Volume 58, Issue 7 Pages 2135-2136
Tadashi Hara,Motohiro Toriyama,Kouichi Kitamura and Masakatsu Imaki

Abstract: The catalytic activity of the iron(III)-α,β,γ,δ-tetraphenylporphinetrisulfonic acid complex for the chemiluminescence reaction between luminol an H2O2 has been found to decrease in the presence of protein. On the basis of this phenomenon, 2 x 10^-5 - 2 x 10^-3 g L-1 bovine serum albumin was determined with the detection limit of 4 ng, lower than that in the previous paper.
Proteins Albumin Chemiluminescence Catalysis

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 6. The Flow Injection Analysis Of Protein Using A 1,10-phenanthroline Hydrogen Peroxide System"
Bull. Chem. Soc. Jpn. 1986 Volume 59, Issue 6 Pages 1833-1838
Tadashi Hara,Takashi Ebuchi,Akihiro Arai and Masakatsu Imaki

Abstract: The method is based on the inhibition by protein of the catalysis by Cu(II) of the chemiluminescent reaction between 1,10-phenanthroline and H2O2. Calibration graphs of log. photomultiplier peak area vs. log. protein concentration. were rectilinear for 20 µg L-1 to 0.1 g L-1 of bovine serum albumin(I) and 20 µg L-1 of bovine γ-globulin. The detection limit was 1 ng of I, but this could be improved to 250 pg (with improvement in the limit of determination to 5 µg l-1) by inclusion of arginine in the Cu(II) solution, which represented a fortyfold improvement with respect to that with the luminol - H2O2 reaction as described in Part (IV) (Ibid., 1985, 58, 109). (For Part V see Anal. Abstr., 1986, 48, 1D189).
Proteins Albumin γ-Globulin Chemiluminescence Catalysis

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 8. Effect Of Heating On The Determination Of Protein"
Bull. Chem. Soc. Jpn. 1986 Volume 59, Issue 11 Pages 3681-3683
Tadashi Hara,Kazuhiko Tsukagoshi,Akihiro Arai and Takeshi Iharada

Abstract: To the sample solution, containing bovine or human serum albumin, was added Cu(II) solution and, after heating at 95°C, the solution was injected into a flow injection apparatus and mixed with buffer solution, H2O2 solution and 1,10-phenanthroline solution The chemiluminescence intensity was measured by a photon counter. The calibration graph was rectilinear from 1 to 100 nM-Cu(II). The apparent coupling constants of the Cu(II) - protein complexes suggest that heating stabilizes the complex and improves the concentration. range for the determination of the protein.
Proteins Albumin Chemiluminescence Catalysis Heated reaction

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 10. Determination Of Protein Using A 1,10-phenanthroline - Hydrogen Peroxide - Ruthenium(III) System"
Bull. Chem. Soc. Jpn. 1987 Volume 60, Issue 4 Pages 1537-1539
Tadashi Hara,Kazuhiko Tsukagoshi and Masakatsu Imaki

Abstract: Bovine serum albumin(I), as model protein analyte, was determined in the concentration. range 50 µg L-1 to 0.1 g l-1, with a detection limit of ~1 ng, by measurement of its inhibition of the catalysis by Ru(III) of the chemiluminescence reaction between H2O2 and 1,10-phenanthroline(II). A flow injection analysis system (cf. Part VIII, Ibid., 1986, 59, 3681) was used, with a 90-cm reaction tube operated at 95°C and with solution of 0.12 mM II, 5% H2O2, 3 µM-Ru(III) and 5 mM I (average mol. wt. 66,000). Flow rates were 0.7, 0.7 and 2.3 mL min-1 for II, H2O2 and buffer solution, respectively, with a reaction time of 45 min. The coefficient of variation for chemiluminescence measurements (n = 10) in the presence and absence of 1 mg L-1 of I were 1.27% and 2.03%, respectively.
Albumin Protein Chemiluminescence Catalysis Heated reaction Indirect

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 11. Determination Of Protein Using A 1,10-phenanthroline - Hydrogen Peroxide - Osmium(VIII) System"
Bull. Chem. Soc. Jpn. 1987 Volume 60, Issue 6 Pages 2031-2035
Tadashi Hara and Kazuhiko Tsukagoshi

Abstract: Formation of a complex between the analyte protein and Os(VIII) present in known excess was used to lessen the amount of Os available to catalyse the formation of chemiluminescence in the 1,10-phenanthroline - H2O2 system. Reagent solution were 0.12 mM 1,10-phenanthroline, 5% H2O2 and 1.3 mM Os(VIII) and the flow injection system was as previously described (Ibid., 1986, 59, 3681). Calibration graphs were established for the determination of bovine and human serum albumins and human serum γ-globulin under optimum conditions. From 5 µg L-1 to 1 mg L-1 of protein could be determined with a detection limit of 0.25 ng and a coefficient of variation of 2.9% at 0.1 mg L-1 (n = 10).
Albumin γ-Globulin Proteins Chemiluminescence Optimization

"Rapid And Sensitive Determination Of Proteins In Biological Samples By Flow Injection Analysis"
Bunseki Kagaku 1987 Volume 36, Issue 4 Pages 256-260
Shuto, T.;Koga, M.;Tanaka, I.;Akiyama, T.;Igisu, H.

Abstract: The sample was injected into a carrier stream of aqueous potassium hydrogen phosphates of pH 7.4 and then mixed with a reagent stream of 60 µg mL-1 of Coomassie Brilliant Blue G250 (C. I. Acid Blue 90) in aqueous 2.7% HClO4 before passing through the reaction coil (10 cm x 1 mm) followed by absorbance measurement at 600 nm. For determination of bovine serum albumin(I) as test protein, rectilinear calibration was obtained from 40 ng (detection limit) to 1 µg, with coefficient of variation of 0.2% (n = 10) at 1.0 µg of I. Up to 60 samples h-1 could be analyzed. The method was applied to the determination of proteins in rat liver microsomes.
Albumin Proteins Spectrophotometry

"Sensitive Determination Of Proteins By FIA With Coulometric Detection"
Bunseki Kagaku 1989 Volume 38, Issue 9 Pages 454-457
Kubo, H.;Huang, Y.S.;Kinoshita, T.;Nakazawa, H.

Abstract: Sample solution (5 ml) was injected into carrier/reagent solution comprising 0.3 M Na2HPO4 - 1 mM CuSO4 - 20 mM NH3 (pH 12) and passed to a reaction coil (5 m x 0.5 mm) operated at 95°C, and then to a cooling coil (1 m x 0.5 mm) operated at 10°C. Coulometric detection was carried out at 0.7 V. Calibration graphs were rectilinear for 10 to 100 ng of bovine serum albumin (I), human serum albumin, human γ-globulins and ovalbumin. The coefficient of variation (n = 10) at 10 and 25 ng of I were 3.5 and 3.1%, respectively. The detection limit for I was 2.5 ng. Histidine, cysteine, tryptophan and tyrosine were sensitively detected.
Albumin γ-Globulin Ovalbumin Cysteine Histidine Tryptophan Tyrosine Coulometry Heated reaction

"Development Of Capillary Stream Sensor Based On FIA For The Determination Of Proteins In Biological Samples With High Precision And Sensitivity"
Bunseki Kagaku 1991 Volume 40, Issue 9 Pages 481-487
Korenaga, T.;Izawa, M.;Takahashi, T.;Stewart, K.K.

Abstract: A FIA-type capillary stream sensor was developed for the determination of micro-amounts of proteins in biological samples. Samples (8 µL) were injected into an aqueous carrier stream (0.02 mL min-1) which was merged with a reagent stream containing bromocresol green (0.02 mL min-1) and passed through a reaction tube (20 cm x 0.5 mm) before the absorbance of the solution was measured at 620 nm. With use of bovine serum albumin, the detection limit was 0.3 µg and coefficient of variation was 0.2%.
Proteins Spectrophotometry Sensor Precision Sensitivity

"High-sensitivity Flow Injection Analysis Of Proteins Coupled With Online Alkaline Hydrolysis And The Fluorescent Derivatization Method"
Chem. Pharm. Bull. 1989 Volume 37, Issue 11 Pages 3155-3157
Ichiba, H.;Niwayama, H.;Yajima, T.

Abstract: Protein solution (20 µL) was injected into a carrier stream (0.2 mL min-1) of 0.5 M KOH - glycerol, which then passed through a Ti hydrolysis coil at 160°C and subsequently through an alkali-proof polymer tube (25 cm) packed with ceramic powder (which generated a pressure of ~200 kg cm-2) to simulate the conditions in sealed-tube hydrolysis. The carrier stream was then mixed with phthalaldehyde solution in 0.4 M H3BO3 (0.6 mL min-1) and passed through a heating coil at 55°C, and the fluorescence was measured at 450 nm (excitation at 340 nm). When equimolar solution of seven standard proteins were analyzed, the fluorescence intensity increased rectilinearly with increasing mol. wt. of the protein. For bovine serum albumin, the calibration graph was rectilinear for 0.05 to 2 µg injected; the coefficient of variation (n = 5) were 1.3 to 5.5%, and the detection limit was 25 ng. A single determination took 18 min; 5 to 6 samples could be analyzed per hour.
Proteins Albumin Fluorescence Heated reaction

"Online Measurement Of Extracellular Proteins In The Continuous Cellulase Production By Flow Injection Analysis"
Enzyme Microb. Technol. 1989 Volume 11, Issue 2 Pages 96-105
Wolfgang W. Stamm, Gunnar Pommerening, Christian Wandrey and Maria-Regina Kula

Abstract: The BCA protein assay was adapted to flow injection analysis to monitor the production of extracellular cellulases of Trichoderma species in batch and continuously operated fermentations. Response of the assay to protein (bovine serum albumin) was linear in the range of 0.1-2.0 g L-1; standard deviation of the calibration graph was 0.86%, accuracy and standard deviation were < +1.5% and 0.9% in the upper range, < +8% and < 2% in the lower range, respectively. Interferences from technical fermentation media were taken into consideration and can be calculated. The slow fermentation process was measured with a frequency of 0.5 samples h-1 over a period of 380 h. In each measuring cycle, recalibration was carried out. The necessary cell-free sample stream for the analytical system was obtained by installing the BIOPEMR, a steam-sterilizable sampling device which showed excellent performance even for long-term fermentations; membranes had to be changed after about 10 days only to keep the protein transmission constant (60-95%, depending on the special protein).
Proteins Albumin Enzyme Tecator

"Acoustic Waves And The Real-time Study Of Biochemical Macromolecules At The Liquid/solid Interface"
Faraday Discuss. 1997 Volume 107, Issue 1 Pages 159-176
B. A. Cavic, F. L. Chu, L. M. Furtado, S. Ghafouri, G. L. Hayward, D. P. Mack, M. E. McGovern, H. Su and M. Thompson

Abstract: The adsorption of the proteins, bovine serum albumin, fibrinogen, avidin and neutravidin (non-glycosylated form of avidin) to a variety of surfaces imposed on thickness shear mode sensors in examined in a flow injection analysis format. In all cases, adsorption of these moieties was essentially irreversible, although the magnitude of adsorption was dependent on surface free energy and functional group chemistry. Also described is the direct, real-time detection of the binding of peptides to HIV-1 TAR RNA bound on a thickness-shear mode (TSM) sensor surface. The results clearly indicate that responses are discriminatory for two different peptides. In order to provide a theoretical backcloth for the experimental measurements, a new model for the operation of the TSM in liquids is presented.
Proteins Albumin Fibrinogen Avidin Neutravidin Sensor Solid liquid interface

"Determination Of Zinc In Serum, Blood, And Ultrafiltrate Fluid From Patients On Haemofiltration By Graphite-furnace - Atomic Absorption Spectroscopy Or Flow Injection Analysis - Atomic Absorption Spectroscopy"
J. AOAC Int. 1994 Volume 77, Issue 3 Pages 722-727
Jimenez De Blas, O.;Seisdedos Rodriguez, R.;Hernandez Mendez, J.;Sanchez Tomero, J.A.;De Leon Gomez, B.;Vicente Gonzalez, S.

Abstract: Two methods were optimized for the determination of zinc in samples of blood, serum, and ultrafiltrate fluid from patients with chronic renal impairment undergoing hemofiltration. In the first procedure, after acid digestion of the samples, Zn in blood and serum is determined by a system coupled to flow injection analysis and atomic absorption spectroscopy. The method is rapid, automated, simple, needs small amounts of sample, and has acceptable analytical characteristics. The analytical characteristics obtained were as follows: determination range of method, 0.05-2.0 ppm of Zn; precision as coefficient of variation (CV), 5.3%; recovery, 95-105%; and detection limit (DL), 0.02 ppm. The second method is optimized for ultrafiltrate fluid because the sensitivity of the first procedure is not suitable for the levels of Zn (ppb or ng/mL) in these samples. The technique chosen was atomic absorption spectroscopy with electrothermal atomization in a graphite furnace. The analytical characteristics obtained were as follows: determination range of method, 0.3-2.0 ppb Zn; CV, 5.7%; recovery, 93-107%; and DL, 0.12 ppb. The methods were used to determine zinc in samples of blood, serum, and ultrafiltrate fluid from 5 patients with chronic renal impairment undergoing hemofiltration to discover whether there were significant differences in the zinc contents of blood, serum, and ultrafiltrate fluid after the hemofiltration process. An analysis of variance of the experimental data obtained from a randomly selected group of 5 patients showed that zinc concentrations in the ultrafiltrate fluid, venous blood, and venous serum do not vary during hemofiltration (p < 0.05), whereas in arterial blood and serum, the time factor has a significant effect. Blood (5 g) was digested with 4 mL of 65% HNO3 and 1 mL of 96% H2SO4 at 130-150°C for ~1 h. After cooling, the solution was heated with 2 mL of 65% HNO3 and 1 mL of 65% HClO4. The mixture was again cooled, treated dropwise with 1 mL of 30% H2O2 and re-heated to 180°C. The residue (2 ml) was made up to 10 mL with water. The sample was diluted 1:5 with water after acid digestion. Serum was treated similarly except that H2O2 was not used and the volumes of acid were: 3 mL of 65% HNO3 and 1 mL of 96% H2SO4 in the first addition, and 1 mL of 65% HNO3 in the second. The samples were not further diluted. For analysis of ultrafiltrate, the dilution was 1:95. Sample was then mixed with water with an air intake to ensure nebulization. Analysis was by electrothermal AAS (operating conditions tabulated) with an acetylene-air flame and measurement at 213.9 nm. A graphite-furnace AAS method was optimized for the ultrafiltrate (details given). Calibration ranges were 0.05-2 and 0.3-2 ppb of Zn in blood and serum, and ultrafiltrate, respectively with corresponding detection limit of 0.02 and 12 ppb. Corresponding recoveries and RSD were 93-105 and 93-107%, and 5.3 and 5.7%.
Zinc Spectrophotometry Sample preparation Sensitivity Optimization

"Protein Band Dispersion In Axial And Radial Flow Chromatography"
J. Liq. Chromatogr. Relat. Technol. 1995 Volume 18, Issue 1 Pages 39-49
J. P. Tharakan; M. Belizaire

Abstract: Radial flow (RF) column configurations have been developed for larger scale production liquid chromatography, with the primary purpose of increasing throughput rates and decreasing trans-bed pressure drops in comparison to conventional axial flow (AF) columns. The RF columns have been quite successful in attaining these two objectives. In this work, we investigated the nature of protein band dispersion in both axial and radial flow chromatography column configurations, utilizing S-200 Sephacryl gel filtration media with bovine serum albumin as the applied protein. The effects of input feed flow rate as well as input feed albumin concentration on the nature of protein band dispersion were studied. Protein band dispersion was quantified by examining the shape of the eluting protein peak and measuring peak height-to-width (HTW) ratio's for the various flow rates and feed concentrations used in the study. Our results indicate that protein dispersion was larger in RF columns as indicated by smaller peak HTW ratio's than those obtained for AF columns, operated at the same flow rates and feed protein concentrations. We also found that moderately increasing the flow rate increased peak HTW ratio's and resulted in sharper peaks for both AF and RF columns.
Albumin LC Dispersion