A comparative study of the protein content of some helminths and the suitability of assay methods

The protein content of fresh homogenates and their corresponding TCA precipitated fractions of 10 different species of helminths was estimated by the methods of Lowry et al. and Spector using the Folin phenol reagent and Coomassie brilliant blue G-250 respectively. The former method gives exaggerated values as compared to the latter method. The parasite phenols, phenolic proteins and catecholamines could be responsible for interference in the Lowry's procedure. The TCA noln-precipitable moieties also give colour only with the Folin phenol assay. The pronounced intra-specific differences in the total protein content of helminths reflect their metabolic variations and adaptations. Habitat does not appear to influence the protein content of parasites, however, the effect of host variation was evident in the pouched amphistome G. crumenifer. It is concluded that the dye binding method gives more consistent results and it can be conveniently applied to crude tissue homogenates of helminths.     

Lowry protein assay using an automatic microtiter plate spectrophotometer

The method of protein determination reported by Lowry et al. (1951, J. Biol. Chem. 193, 265-275) has been adapted for use with 96-well microtiter plates and an automatic microplate spectrophotometer. The spectrophotometer has been interfaced with a computer which plots the standard curve and calculates the protein content of each sample. The adapted method offers advantages over previously reported methods in that it is more rapid and uses a smaller sample volume (100 microliters) for samples containing 3-300 micrograms/ml (0.3-30 micrograms/assay) of protein. The method of Bensadoun and Weinstein (1976, Anal. Biochem. 70, 241-252) for precipitating microgram amounts of protein away from substances which interfere with the Lowry assay has also been adapted to this microplate procedure. These techniques should be particularly useful for laboratories where large numbers of samples containing a wide range of protein concentrations are assayed.     

A comparison of five of the methods commonly used to measure protein concentrations in fish sera

The concentration of protein in the sera of rainbow trout, Salmo gairdneri , brown trout S. trutta and Atlantic salmon S. salar has been measured by six standard techniques viz refractometry, copper sulphate specific gravity, automated and manual biuret, optical density and Lowry et al. phenol reagent and the results compared. Good correlation was obtained in most cases and interconversion formulae are given between each method in the three salmonid species. The concentrations obtained with the refractometer and optical density methods were approximately one and a half times those obtained with the others.     

Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml⁻¹) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml⁻¹) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.     

A tracking marker for the first dimension of the two-dimensional gel electrophoresis of ribosomal proteins

Protein content of different subcellular fractions from chick brain is compared by using Lowry, TCA—Lowry and Bradford assay methods. Caution is urged in application of Bradford's method to general assay for protein concentration in subcellular fractions.     

Lowry protein determination on membrane preparations: need for standardization by amino acid analysis

The protein content of three membrane protein preparations has been determined by the Lowry method with bovine serum albumin as a standard and also by quantitative amino acid analysis as an absolute method. The results differ considerably, the Lowry method giving 29–42% higher values. This implies that many published data for such proteins, based on Lowry protein determinations with bovine serum albumin as the generally applied standard, are in error. Suggestions are made on how to standardize the Lowry method so that reliable values can be obtained for membrane protein.     

Modification of the Lowry assay to measure proteins and phenols in covalently bound complexes

It is well established that phenols interfere with many routine protein assays and a number of protocols have been developed to overcome this. One such method is based on the differences in response obtained with the Lowry assay in the presence and absence of copper. This assumes that the phenol response with the Lowry assay is not affected by copper. However ortho-diphenols such as catechol, methylcatechol, caffeic acid, chlorogenic acid, and phaselic acid show decreased responses in the presence of copper. Three methods of estimating protein were compared for their accuracy in measuring proteins in the presence of covalently bound ortho-diphenols; the Lowry assay, the modified Lowry assay, and a new method including a calculation to take into account differences in ortho-diphenol response in the presence and absence of copper. The ortho-diphenols were caffeic acid and phaselic acid, which were bound to bovine serum albumin and red clover protein either chemically or enzymatically. For all assays, the new method gave values within 4 to 8% of control values for protein (without bound phenols) as determined by the modified Lowry method. Values for the Lowry and modified Lowry methods varied by 20-50% from control protein values. The new method also gave a good approximation of protein-bound phenol content.     

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.     

A procedure for total protein determination with special application to allergenic extract standardization

A method for total protein determination of allergenic extracts has been developed and evaluated. Samples were hydrolyzed with 5 M NaOH followed by colorimetric determination with ninhydrin of the released amino acids using bovine serum albumin as the standard. The entire procedure was carried out in disposable plastic tubes. Substances (glycerol, phenol and mannitol) commonly present in allergenic extracts manufactured for human use did not affect the assay results. Analyses of four different pollen extracts by the method gave good agreement with amino acid analyses. Other methods of analysis (total N, protein N unit assay, Lowry) gave more variable results compared with amino acid analysis. Analysis of the total protein content of 53 different lots of allergenic extracts gave narrow ranges of values for each species. Standardized mite extracts analyzed for total protein by US FDA-licensed manufacturers using this assay showed a good correlation of biological activity with total protein.     

Measurement of protein by dye-binding assay in the presence of metrizamide

Quantitative determination of protein using the binding of Coomassie Brilliant Blue G-250 was investigated with respect to interference with the density gradient material metrizamide, and compared with the corresponding interference using the Lowry method. The background absorption obtained with metrizamide in the absence of protein was less than 10% of that obtained with the Lowry method. In the presence of 0–4% metrizamide, parallel standard curves were obtained with 0–67 μg of protein in the samples. The curves overlapped in the range 0–40 μg of protein when metrizamide was included in the blanks. With up to 2% final concentration of metrizamide in the assay, the curves overlapped at all protein concentrations tested (0–67 μg). Correction for metrizamide interference is thus a simple procedure and a precise estimation of the metrizamide concentration is less critical than when the Lowry assay is used. The method is well suited for quantitation of protein in samples collected from metrizamide grandients.     

An evaluation of protein assays for quantitative determination of drugs

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.     

Individual mitochondrion characterization: a comparison of classical assays to capillary electrophoresis with laser-induced fluorescence detection.

A method has been developed that uses capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF) for measuring protein abundance in individual mitochondria collected from a discontinuous density gradient and labeled with Mitotracker Green. From these measurements we determined the distribution of protein content per mitochondrion and the relative abundance of mitochondrial proteins in density gradient fractions. In addition, this method is useful for counting mitochondria and, as a consequence, determining the number of mitochondria per unit volume or estimate mitochondria copy number per cell. It was determined that mitochondria accumulate in two interfaces defined by consecutive layers of 35% Metrizamide, 17% Metrizamide, and 6% Percoll. The presence of mitochondria in these interfaces was also confirmed using a modified Lowry assay that prevents interference from Metrizamide and Percoll and determines total protein content, and a succinate dehydrogenase assay that uses dichloroindophenol as an electron acceptor and that specifically indicates abundance of mitochondria. The CE-LIF analysis of mitochondrial properties, based on the individual mitochondrial determinations, has a wider scope than the average values determined by enzymatic or bulk protein assays.     

A simplification of the protein assay method of Lowry et al. which is more generally applicable

Some recent modifications of the protein assay by the method of Lowry, Rosebrough, Farr, and Randall (1951, J. Biol. Chem.193, 265–275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from interfering substances even in very dilute solutions (< 1 μg/ml of protein). SDS is added to alleviate possible nonionic and cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry et al.     

Intestinal lymph and plasma lipoproteins in the preruminant calf: partial resolution of particle heterogeneity in the 1.040-1.090 g/ml interval.

Our previous studies in the preruminant calf have provided evidence for the heterogeneity of lipoprotein particles in the 1.040-1.090 g/ml density interval in both plasma and postprandial intestinal lymph (Bauchart, D. et al., 1989. J. Lipid Res. 30: 1499-1514; and Laplaud, P. M. et al., 1990. J. Lipid Res. 31: 1781-1792). We therefore attempted to resolve this heterogeneity by use of heparin-Sepharose affinity chromatography. Experiments were performed on three calves; portal vein plasma and intestinal lymph were obtained simultaneously 10 h after a meal, i.e., at peak lipid absorption. In both fluids, the chromatographic profile presented three fractions, I, II, and III. Fraction I was characterized by the presence of cholesteryl ester-rich particles (approximately 35-37% of lipoprotein mass), which migrated electrophoretically as typical high density lipoproteins and exhibited Stokes diameters in the 130-160 A range; apoA-I was the predominant protein. In addition to this polypeptide, fraction II contained small amounts of a supplementary protein (Mr approximately 51,000), exhibiting heparin-binding properties. In the light of results reported in the literature, we suggest that this latter protein could correspond to beta 2 glycoprotein I. The chemical composition of each fraction II closely resembled that of the corresponding fraction I, while their electrophoretic migrations appeared slightly slower and their Stokes diameters slightly larger (155-165 A). Apart from the presence of small amounts of apoA-I, two high Mr proteins (Mr approx. 560,000 and 300,000) were typical of the apolipoprotein moiety of fractions III. The lower Mr form was present as a trace component only in fraction III originating from plasma; its proportion increased in lymph fraction III so as to approximately match that of the higher Mr (i.e., 560,000) protein. In both plasma and lymph, fraction III was electrophoretically heterogeneous, exhibiting a doublet of bands with migration and Stokes diameters (250 A) typical of low density lipoprotein particles. However, no evidence for the presence of a particle resembling lipoprotein[a] in fraction III could be obtained. In lymph only, fraction III contained a supplementary population of lipoproteins with migration intermediary between those of conventional low and high density lipoproteins and with Stokes diameters in the 190-200 A range. Other specific features of lymph fraction III included a sevenfold increase in its triglyceride content (8.5 +/- 3.4% vs. 1.2 +/- 1.1% in the corresponding fraction from plasma), to the detriment of cholesteryl esters, and a higher proportion of protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

A method of measuring protein dry weight in solutions of unknown salt concentration and an application to lipoproteins

The dialysis bag dry weight method was developed for the measurement of dry weights of lipoproteins subfractionated on density gradients where total recovery of lipoprotein in the gradient was to be determined. Use of the conventional method for dry weight determination was precluded because of inconsistent concentration changes which would occur in the dialysis step due to differences in both the lipoprotein and salt concentration among gradient fractions. The method described consists of transfer of measured undialyzed samples into previously weighed bags followed by dialysis against water, lyophilization of the protein-bag combination and calculation of the protein dry weight as the difference between the bag weight and the total weight.Since the method described incorporates dialysis in the assay, it is capable of giving an accurate protein dry weight measure of a non-predialyzed sample, whereas the conventional dry weight method gives an accurate value only of a previously dialyzed sample. The increase in the standard deviation of the overall dialysis bag method was shown to be less than double that of the conventional method for a sample of known salt concentration and there is no distinguishable difference in the central values obtained by the two methods.The particular usefulness of this method for lipoprotein solutions was presented.     

Quantitative assay for submicrogram amounts of protein

An ultrasensitive protein assay, linear between 0.01 and 0.2 μg protein, is described. In this assay, copper is complexed to protein, excess copper is removed by adsorption to a small Sephadex column, and the copper-protein complex is destroyed by digestion with hydroperoxide. Phenol and chloramine-T are added to the reaction mixture and the copper catalyzes the production of a color-producing reaction between the two compounds. The assay is not affected by low levels of phosphate, Tris, metals, or a tenfold excess of nucleic acid over protein. Reducing agents, sucrose, certain anions, high salt concentrations, and EDTA seriously interfere. The method is about 500 times as sensitive as that of Lowry et al. [Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem.193, 265]. Like the biuret procedure, the assay measures copper complexed to the peptide bonds but is probably influenced by other factors, since equivalent amounts of different proteins give similar but not identical amounts of color.     

Protein assay by Coomassie brilliant blue G-250-binding method is unsuitable for plant tissues rich in phenols and phenolases

Protein estimation in crude homogenates of plant tissues rich in phenols and phenolases was carried out by the dye-binding and, with recommended cautions, by the Lowry et al. methods and the two were compared. The dye-binding method gave grossly erroneous results with a high degree of variation when the homogenizing media differed; this was not due either to the interference by the components of the homogenizing media or to any shift in the absorbance maximum. While the reduced form of the "derived" polyphenolic compounds, generated during tissue homogenization, appeared to enhance dye binding with bovine serum albumin, their influence on the protein assay directly in crude homogenates was extremely diverse. Tissue homogenization in the absence of a reducing agent results in polyquinone-protein complexes which prevent optimal dye binding, resulting in low protein values, while the endogenous phenolics in a homogenate prepared in a mixture of cysteine and NaCl appear to suppress dye-protein complex formation. It is therefore our opinion that the dye-binding method is unsuitable for protein assay in phenol- and phenolase-rich plant tissues.     

Prediction of human acute toxicity by the hep G2/24-hour/total protein assay, with protein measurement by the CBQCA method

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)- quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r(2) = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method (r(2) = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.     

Measurement of protein using bicinchoninic acid

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.