Quantitative Determination Of Proteins Research Articles

Nanogram quantitation of secreted protein in a recombinant yeast fermentation using an immuno-ligand assay

a liquid-phase immuno-ligand assay has been developed for quantitative determination of recombinant tick anticoagulant protein (rTAP) secreted in yeast fermentations. A polyclonal anti-TAP antibody was labeled with biotin or fluorescein. Labelled antibodies were used in a non-competitive sandwich format to capture rTAP from solution, then reacted with urease-conjugated anti-fluorescein antibody. Detection of the immune complex was by a commercially available silicon-based potentiometric sensor which measures urease activity. Sample throughput was 90 samples per 7 h with a 2 h incubation time. The range of the standard curve was 0.1–10 ng/ml with an assay sensitivity of 0.025 ng/ml. For a mid-range concentration of 1 ng/ml, intraday method precision was determined to be 1.031 ± 0.061 and 1.077 ± 0.026 ng/ml, respectively. Typically, spiked samples of 1 μg rTAP/ml fermentation medium required dilutions of 1/1000 to generate a response in the mid-range of the standard curve. This assay provides a convenient method to quantitate product expression in multiple fermentation samples within 3 h after sampling. In addition, a modified version of the assay was developed which provided accurate results within 1 h of sample acquisition.

An Easy Preparation of Chemically Pure, High-Activity Tritium-LabelledN-Propionylampicillin for the Analysis of Penicillin-Binding Proteins

Abstract The laboratory preparation of chemically pure, 3H-labelled β-propionylampicillin suitable for studies of the mode of action of β-lactam antibiotics has been investigated. The chemical purity of the labelled antibiotic is a crucial factor for the quantitative determination of penicillin-binding proteins. Ampicillin (D-α-aminobenzylpenicillin) was N-[3H]propionylated by N-succinimidyl [3H]propionate, the labelled reaction products were analyzed by thin-layer chromatography followed by fluorography. The synthesis and the purification of the resulting N-[3H]propionylampicillin have been optimized, the best conditions for the acylation reaction were at pH 8.25 for 2 h, giving both a high yield of the labelled antibiotic and minimal amounts of unwanted by-products. The reaction mixture was fractionated by a two-step procedure, the purified radioactive antibiotic did not contain any residual reactant, especially free ampicillin, or degradation products such as D-α.-[3H]propionamidobenzylpenicilloic acid. This improved procedure allows an easy, fast and rather inexpensive preparation of a good quality radiolabeled antibiotic for studies of penicillin-interacting proteins.

Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection.

Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A0, and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A0 reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A0 for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage.

Quantitative Determination of Urinary Marker Proteins: A Model to Detect Intrarenal Bioeffects after Extracorporeal Lithotripsy

To detect the source of relevant acute intrarenal side effects after extracorporeal piezoelectric lithotripsy and its impact on repeat treatment, urinary excretion of highly specific marker proteins was determined before (day −1) and after (days 0, 1, 4, 7, 14 and 21) treatment. Marker proteins included high molecular weight α-2-macroglobulin, immunoglobulin G, albumin, α-1-microglobulin as well as the enzyme N-acetyl-β-ghicosaminidase. Of 50 patients who underwent 4,000 shock waves to caliceal stones (group 1) 15 were identically retreated after 5 (group 2) or 15 (group 3) days, respectively, to determine the shortest safe interval to repeat extracorporeal piezoelectric lithotripsy. The course of lithotripsy damage was also evaluated in 15 pre-damaged kidneys (group 4).The α-2-macroglobulin enhancement found in all groups on day 0 (p <0.005 to p <0.05) documented intrarenal bleeding from ruptured vessels. Ratios of α-2-macroglobulin/albumin greater than 2.00 on days 0 and 1 exclude a glomerular source of gross hematuria (groups 1 to 4). There was only slight acute tubular damage after extracorporeal piezoelectric lithotripsy (N-acetyl-β-glucosaminidase increase, p <0.05 for groups 1 to 4). Retreatment after 5 days did not enhance the amount of proteinuria compared to the same patients from group 1 (statistically significant at p <0.45 to p <0.10). Group 3 also showed a similar elevation of proteinuria as the identical patients pretreated 15 days previously. Thus, the data seem to suggest that early repeat sessions of extracorporeal piezoelectric lithotripsy are as safe as delayed retreatments. The course of proteinuria in group 4 did not suggest enhancement of extracorporeal piezoelectric lithotripsy damage in preinjured kidneys. The urinary marker α-2-macroglobulin detects intrarenal vessel ruptures, which are responsible for intrarenal hematomas, as evidenced by animal and human histology. A model is offered to understand and detect the most important parenchymal bioeffects to minimize the risk of injury.

Evidence for the expression of morphological and biochemical characteristics of C3‐photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C4 carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged.Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent Km (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose‐6‐phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C3 plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C4 form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves.NAD‐malate dehydrogenase (NAD‐MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP‐malic enzyme (NADP‐ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C3‐like metabolism.

High-performance liquid chromatographic determination of fatty acid binding proteins in rat liver with fluorescence detection.

A highly sensitive, simple and reproducible method for the quantitative determination of fatty acid binding protein in rat liver by post-column high-performance liquid chromatography with fluorescence detection is presented. Fatty acid binding protein in rat liver cytosols is separated by gel-permeation column chromatography, which is followed by fluorescence detection. The detection makes use of the fluorescence enhancement observed when a fluorescent fatty acid probe, dansylundecanoic acid, binds to fatty acid binding protein. The method is rapid, simple to perform and highly sensitive. The method was applied to the determination of fatty acid binding protein in liver from control and hypolipidaemic drug treated rats.

Microgravimetric determination of precipitable antigens and antibodies in native biological fluids

A method for the exact and simultaneous measurements of immunoprecipitable antigens and antibodies in native biological fluids is described. It is based solely on the microgravimetric analysis of at least two immunoprecipitates of different stoichiometric compositions. The volume ratios of antigen and antibody solutions for the preparation of the immunoprecipitates at equivalence are determined by the two-cross immunodiffusion technique. The proposed procedure has advantages over other methods for quantitative determination of proteins, since the separation, purification and labelling of immune components, as well as the use of standards, are not required. The method was tested by using human serum albumin (HSA) and three rabbit antiHSA immune sera of various avidities. Immunoprecipitates of different stoichiometric compositions were prepared at pH 5.0, 5.5, 7.0 and 8.6. The minimal precision of the method was±0.2 μg of protein. The coefficients of variation were 0.14 to 0.38% for HSA and 0.03 to 0.08% for antiHSA antibody.

A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays

A simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 +/- 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p less than 0.001). Also, a similar correlation (r = 0.93, p less than 0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

Differentiation of hematuria by quantitative determination of urinary marker proteins

Hematuria caused by prerenal, glomerular, postglomerular, and postrenal causes is usually differentiated by a number of noninvasive and invasive diagnostic procedures. In the present study we have applied a new analytical strategy based on observations that the various forms of hematuria can be classified by their typical protein pattern. When analyzed by quantitative turbidimetric assays, urines from postrenal hematurias contained high-molecular-weight proteins (alpha 2-macroglobulin and IgG) in proportions found in plasma. Relating excretion rates (mg/mg) of these proteins to those of albumin, ratios for alpha 2-macroglobulin/albumin and IgG/albumin were 2.0-31 x 10(-2) and 20.0-180 x 10(-2), respectively. In contrast, glomerular hematurias exhibited ratios of 0.01-2.0 x 10(-2) (alpha 2-macroglobulin/albumin) and 2.0-20 x 10(-2) (IgG/albumin). Additional determination of alpha 1-microglobulin allowed us to differentiate postglomerular hematurias caused by interstitial nephropathies from glomerular and postrenal diseases. Critical evaluation of 93 cases diagnosed by independent clinical examination including histology, sonography, and cystoscopy revealed that the criteria derived from protein measurements resulted in correct classification when urine albumin exceeds 100 mg/l. This noninvasive procedure is expected to be of considerable help in the primary care of patients with unexplained hematuria.

Quantitation of protein A in human plasma is possible after heat inactivation of the samples

Quantitative determination of staphylococcal protein A in plasma is often hampered by the interaction between protein A and immunoglobulins. In human plasma, these interactions may not only involve the non-immune binding to the Fc or Fab regions of Ig but also antigen/antibody interaction by specific antibodies directed against protein A. In this paper we describe a method which can be used to quantitate nanogram amounts of protein A in the presence of human plasma. The ELISA used for the quantification of protein A is based on a double antibody solid-phase assay utilizing chicken anti-protein A as both capture and detector antibody. Protein A may be measured down to 5 ng/ml in plasma and 0.5 ng/ml in buffer. The plasma samples were heat-inactivated before analysis and this eliminated interference in the assay caused by interaction of protein A with an excess of immunoglobulins. This assay provides a reliable and convenient method for the detection and quatitation of soluble protein A in human plasma.

A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents

A method is described for quantiation of protein in the presence of reducing agents, detergents, and other substances which often interfere with assays of protein in solution. The proteins are applied to Whatman No. 1 filter paper, air-dried, washed with methanol, and then stained with Coomassie brilliant blue G. Following destaining, the paper is air-dried and the protein-bound dye is extracted. Sample absorbance measurements are made in a 96-well plate using an automated microplate reader (600–405 nm) or in a cuvette at 610 nm. This filter paper assay is useful for determining 100 ng to 20 μg of protein in the presence of ammonium sulfate, urea, thiol-reducing agents, amino acids, DNA, ionic and nonionic detergents, and acid or base.

Quantitative determination of the amyloid A4 precursor protein in cerebrospinal fluid

Quantitative determination of the amyloid A4 precursor protein in cerebrospinal fluid

The stoichiometry of the tightly bound NAD+ in urocanase. Separation and characterization of fully active and inhibited forms of the enzyme.

1. Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979) J. Biol. Chem. 254, 843-851] from Pseudomonas putida was submitted to high-performance liquid chromatography on a TSK-DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively. 2. The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm. 3. Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK-DEAE column confirmed their partial transformation into peak A. 4. Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+ released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8-1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7-14C]nicotinate into urocanase using a nicotinate auxotrophic mutant of P. putida. 5. Form A of urocanase showed, after treatment with NaBH4 up to 50% inhibition, an elution pattern (TSK-DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated. 6. We conclude that form A of the urocanase dimer contains two intact NAD+ molecules. In form B one of the two subunits contains an NAD+-nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+ in both subunits. Intact NAD+ can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.

Protein C activity levels in endotoxin-induced disseminated intravascular coagulation in a dog model

The levels of protein C (PC) and other coagulation factors were monitored during endotoxin-induced disseminated intravascular coagulation (DIC) in the dog. Initial evaluation of the effectiveness of intradermal administration of bolus endotoxin quantities into the dog, demonstrated induction of DIC in the canine, without the severe side effects associated with bacterial sepsis. Quantitative determination of canine plasma protein C levels were performed using a multiple step amidolytic assay, that included a specific precipitation of the vitamin K-dependent proteins from citrated plasma, followed by thrombin activation (and neutralization) and subsequent measurement of the activated protein C (APC) by chromogen hydrolysis. This investigation demonstrated, that over a twenty-four hour interval, intradermal administration of endotoxin produces a gradual decrease in the PC activity levels, concomitant with a significant reduction in the Factor V, Factor VIII and fibrinogen levels and platelet count, and a prolongation of the Prothrombin Time and Partial Thromboplastin Time. During the first 6 hours, protein C levels fell below the pre-levels and remained significantly lower in the surviving dogs. Thus, this endotoxin-induced DIC animal model permits evaluation of various hemostatic parameters, yet diminishes the severe clinical findings associated with DIC.

Factors influencing the quantitative determination of tear proteins by high performance liquid chromatography.

HPLC analysis of human tears allows tear protein profiles to be obtained within ten minutes. A tear protein profile normally consists of 4 major peaks: IgA, lactoferrin, protein G and lysozyme. Although it is a rapid method, the use of High Performance Liquid Chromatography in the (quantitative) determination of proteins in tears is influenced by various factors. The day to day variability of the quantitative use, ranges between 7 and 9% for the various tear proteins. Combining the HPLC method with a convenient collection method such as sponges or Schirmer strips, showed that the sponges and some of the Schirmer strips used in this study eluted significant material absorbing light at 280 nm. No statistical difference was observed in the HPLC protein profiles of tears collected with Schirmer strips or with sponges. Using sponges has the advantage that they can absorb almost twice as much tears in a same period of time as Schirmer strips. HPLC analysis of human tear proteins is not accurate when there is albumin leakage as in traumatic sampling with Schirmer strips or in inflammatory states. The I-125 column which was used in our study is not able to separate lactoferrin and albumin, which may cause an overestimation of lactoferrin in inflammatory conditions. The study presented here indicates that for quantitative use of HPLC in epidemiological tear protein research better separating protein gel filtration columns are needed.

Quantitative determination of human protein C evaluation of a “fast” functional assay in comparison to a “traditional” functional and an immunological assay

A fast functional assay for protein C was evaluated and compared with a traditional functional and an enzyme linked immunosorbent assay in parallel for the same plasma samples derived from 43 healthy subjects, 12 patients with severe hepatic dysfunction, and 23 patients under stable oral anticoagulation. By all three test systems significantly lower levels of protein C were obtained in both groups of patients compared with normal subjects (p<0.0001). No significant between - assay differences were found in normal subjects and in patiens with hepatic dysfunction; by correlation analysis coefficients higher than 0.8 were calculated between the measurements of the three tests. In patients under stable oral anticoagulation, however, the immunologic test yielded higher values than the traditional (p<0.05) and, more pronounced, the fast functional assay (p<0.0001); no or only borderline significant correlations between the results were found. In these patients protein C levels measured with the traditional functional assay were in the same range as the activity levels of factors II, VII, IX, and X, whereas the fast functional test yielded significantly lower levels. The presented results indicate that very similar protein C levels were obtained with both functional and the immunologic assay except in patients under oral anticoagulation.

A quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D 28k) using nitrocellulose filters

A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D 28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D 28k (1:500 dilution) and 125I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D 28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 ± 0.5 μg/mg protein) and cerebellum (22.1 ± 1.4 μg/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 μg liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and 125I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous 125I immunobinding assays which were not quantitative but were used as antigen “spot tests” or which required iodination of the antibody.

Diagnostic audit of C-reactive protein in neonatal infection

A prospective study of 250 consecutive neonatal admissions to a regional perinatal referral centre and of 10 additional consecutive cases with culture-proven neonatal septicaemia was undertaken. Quantitative C-reactive protein (CRP) determination, white cell count and differential were performed on blood samples obtained from all babies on admission, as well as 10-14 h and 22-26 h later. Using clinical signs, chest X-rays, blood cultures, tracheal aspirates obtained within 4 h of delivery and an abnormal immature/total neutrophil ratio (I/T), infected babies were defined as belonging to one of the following groups: culture-proven septicaemia (n = 19); clinical septicaemia (n = 35); congenital pneumonia (n = 28). The sensitivity, specificity, positive and negative predictive value of CRP were calculated for each sampling time and patient group. No baby had a rise in CRP (greater than 6 mg/l) before an abnormal I/T ratio was first detected. A delayed rise in CRP concentration in the majority of infected babies occurred approximately 12-24 h after the abnormal I/T ratio was first detected. The overall specificity of a CRP level of greater than or equal to 10 mg/l remained approximately constant (97%-94%) while sensitivity increased from 22%-61% with increasing time after admission. The same pattern emerged if each patient group was considered separately. The positive predictive value for a CRP level of greater than or equal to 10 mg/l 22-26 h after admission was 83% and the negative predictive value 82%. CRP had no value in the early diagnosis of neonatal infection. Its main role lies rather in the exclusion or confirmation of infection 24 h after the first clinical suspicion.

An Improvement of the Protein Determination in Plant Tissues with the Dye-binding Method According to {scBradford}

Summary A modified {scBradford} method for determination of plant protein is proposed using 0.003% sodium dodecyl sulfate and 0.005% Acid blue 90 (Serva blue G). The method exhibits high specificity and simple applicability but allows also quantitative protein determination nearly independently of the nature of protein.

Application of quantitative determination of urinary proteins in the diagnosis of acute rejection following renal transplantation.

Eight kinds of urinary proteins were determined after renal transplantation. The results demonstrated that the concentrations of 8 urinary proteins were markedly increased when acute rejection occurred. Increase in urinary proteins was noticed 0–7 days prior to establishment of clinical diagnosis. Of the 8 urinary proteins IgG and transferrin appeared first. The positive rates of IgG and C3, both being 93 %, were the highest among them. Provided the antirejection treatment was effective, the concentration of urinary proteins would drop rapidly, otherwise it would remain at high levels.