Protein, cholesterol, acid phosphatase and aspartate aminotransaminase in the seminal plasma of turkeys (Meleagris gallopavo) producing normal white or abnormal yellow semen

Turkeys which produce yellow semen have abnormal ductuli efferentes' epithelial morphology, with blebbing of cytoplasmic material into the ductal lumen. This could possibly increase the activity or concentration of seminal plasma components. In the present study, seminal plasma from 270 Large White breeder turkeys was evaluated for protein and cholesterol concentrations and the activities of acid phosphatase and asparate aminotransaminase. In a separate experiment, protein concentrations of turkey seminal plasma were estimated by biuret or Bradford methods. Bradford estimates were 46.6% less than those obtained with the biuret assay, using bovine serum albumin as the standard. Estimates of seminal plasma protein concentration in the main study were obtained using the Bradford method, and should be adjusted accordingly when compared with other studies using the biuret technique. Abnormal yellow seminal plasma, compared to normal white seminal plasma, had elevated levels of total protein and cholesterol and increased activities of acid phosphatase and aspartate aminotransaminase. Overall means were: 14.3 mg/ml, 38.9 mg/dl, 232.6 IU/ml, 81.0 IU/ml, respectively. Correlation coefficients for cholesterol concentration, acid phosphatase and aminotransaminase activity with protein concentration were +0.65, 0.70 and 0.50 (P less than 0.0001), respectively. Specific activities of both enzymes showed a significant reduction as seminal plasma protein increased, indicating a disproportionate increase in proteins other than these enzymes in yellow seminal plasma.     

Interference of melanin in protein determination

Using several assay methods, synthetic eumelanin prepared by autooxidation of L-beta-(3,4-dihydroxyphenyl)alanine and a natural melanin isolated from dog hair melanosomes were tested in model experiments to assess their possible interference in protein determination. The degree of interference was assessed by comparing the data obtained with the melanin samples with those derived from measurements of bovine serum albumin. In the common biuret and Lowry methods melanin interferes by falsely increasing the values obtained; the addition of Folin reagent only after melanin removal, as suggested by Doezema, decreased but did not eliminate melanin interference. Methods working at acid pH such as those according to Salo and Honkavaara with Ponceau S or Sedmak and Grossberg or Spector using Coomassie blue G-250 proved much better. Although melanins adsorbed a small amount of dye from the reaction systems in these procedures, their sensitivity to proteins makes the melanin interference negligible. Such procedures can therefore be recommended for protein determination in the presence of melanin.     

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.     

Color development time of the Lowry protein assay

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

Polypeptide linkages and resulting structural features as powerful chromogenic factors in the Lowry phenol reaction. Studies on a glycoprotein containing no Lowry phenol-reactive amino acids and on its desialylated and deglycosylated products.

With bovine serum albumin as the reference standard, the armadillo salivary-gland glycoprotein, although containing no chromogenic amino acids and only small amounts of colour-yielding peptides [Chou & Goldstein (1960) Biochem. J. 75, 100-115], is highly reactive in the Lowry phenol protein assay [Wu & Pigman (1977) Biochem. J. 161, 37-47]. After desialylation and Smith degradation of the glycoprotein, the Lowry phenol value increased by 13 and 30% respectively, which suggests that both sialic acid and N-acetylhexosamine exert shielding effects in this reaction. Acid hydrolysis for 30 min decreased the Lowry phenol value by more than 45%, which indicates that the peptide linkages and steric features affect the Lowry phenol reactivity. After hydrolysis for up to 6h, the remaining Lowry phenol value of the partially hydrolysed core protein paralleled the amount of unhydrolysed peptides, inferring that both acid-sensitive and acid-resistant chromophoric peptides are fairly evenly distributed along the whole polypeptide chain. As with bovine serum albumin, more than 80% of the colour yield obtained in the Lowry phenol assay with this glycoprotein is Cu2+-dependent.     

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

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 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), 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.     

A linear Lowry--Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins

The protein method of Lowry, Rosebrough, Farr and Randall was modified to give a linear standard curve of absorbance versus μg of bovine serum albumin at 650 and 750 nm wavelengths (1 cm optical path) over the range up to 50 μg protein per ml and an absorbance of 1.1. This was achieved mainly by using a high concentration of Folin-phenol reagent, added rapidly in a volume that was large relative to the final volume. Sodium dodecyl sulfate (SDS) incorporated in the test did not change the albumin standard curve, and linear results were obtained with water-insoluble membrane proteins (myelin proteolipid and rhodopsin) solubilized by SDS.     

A simplified method of quantitating protein using the biuret and phenol reagents.

A new modification of the Lowry method of quantitating protein is introduced, whereby the protein sample is mixed first with a diluted biuret reagent and later with 2 n phenol reagent (undiluted) for color development. The method is superior to the original in (i) extremely stable color development (0.3% change from 20 min to 2 hr), (ii) good reproducibility (±2% for 50–600 μg/ml of protein), (iii) elimination of the need to mix reagents for each assay, (iv) good storability (the diluted biuret reagent is storable for months), (v) simplicity (both reagents are available commercially), and (vi) the biuret method can be immediately converted to the Lowry method if the former does not yield a sufficient absorbance. It was found that the relationship between absorbance and protein concentration is expressed by a straight line with a slope of 1 in the Hill plot.     

Quantitation of electrophoretic eluted proteins

Quantitation of stained, electroeluted proteins by the classical Lowry and Bradford protein assay is not possible because of some different interferences. In particular we have found that the substance interfering in the Lowry method cannot be removed by trichloroacetic acid precipitation nor can be compensated for by the appropriate blank. Interferences in the Bradford protein assay are due to detergents and pH of the protein buffer as well as to Coomassie brilliant blue R250 electroeluted with the protein sample. However, while these interferences can be compensated for by appropriate blank and standard curves, others (probably due to acrylamide fines) cannot be corrected. All these problems can be overcome by concentration and dialysis of electroeluted samples which permit the removal of interfering substances and the use of Bradford and Lowry protein assay in the 1-20 micrograms range, respectively. Successful applications are described for electroeluted bovine serum albumin, human hexokinase and phosphoglucomutase.     

Optimization of Folin-Ciocalteu reagent concentration in an automated Lowry protein assay

The Lowry method (G. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951, J. Biol. Chem.193, 265–275) for protein concentration measurement has been automated to permit assay of samples with concentrations from 1 to 400 μg/ml. Calibration with solutions of bovine serum albumin resulted in a nonlinear (quadratic) curve. The quantity of color developed in the assay was found to be strongly dependent on the concentration of the Folin-Ciocalteu phenol reagent. Color yield peaked sharply at a reagent concentration 40% lower than that used in the Lowry procedure. Optimization of the reagent concentration is necessary to obtain maximum sensitivity from the Lowry assay.     

Development and calibration of a standard for the protein content of granulocyte colony-stimulating factor products

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 μg protein per ampoule (95% C.I: 212.407-218.486 μg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.     

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.     

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.     

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.     

Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estimation of protein in allergen extracts and influence of different parameters on the modified Lowry assay

Protein values of dialysed allergen extracts determined by Lowry, modified Lowry (trichloroacetic acid precipitation of the proteins) and dye-binding assay were compared. The influence of different parameters on the modified Lowry was examined. The reproducibility of the modified Lowry was checked with three independent measurements. For the examination of recovery a constant amount of 6-grass pollen allergen proteins was added to the samples of the standardized human serum albumin prepared for the calibration curve. The samples were measured by modified Lowry. The mean of the ratio between the protein values of the dialysed allergen extracts obtained by modified Lowry and those obtained by classical Lowry was 3.59 (coefficient of variation Cv = 45%). The mean of the ratio between the protein values of the allergen extracts obtained by modified Lowry and dye-binding assay was 1:0.71 (Cv = 31%). Phenol interfered with the modified Lowry. Phenolic allergen extracts showed higher "protein values" than non-phenolic allergen extracts. This influence could be reduced by a second precipitation of the dissolved precipitate. The precipitation of non-phenolic dialysed aqueous allergen extracts was complete after the first trichloroacetic acid precipitation. By incubating samples with the Folin-Ciocalteu's reagent at 55 degrees C in a waterbath, the time necessary for developing the colour could be reduced from 45 min to 5 min. Protein measurements by modified Lowry of a 6-grass pollen allergen extract in three different laboratories showed good reproducibility. For these extract 785 micrograms protein/ml (Cv = 4%) could be measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Variable sensitivity in the microbiuret assay of protein

Microbiuret methods have been introduced which have sensitivity similar to that of the method of Lowry et al. (1) and are claimed to be less subject to interference (2–5). Each method has three steps: (I) formation of the protein-copper biuret complex in alkaline solution, (II) separation of excess copper reagent from protein-bound copper, and (III) colorimetric assay of the latter with diethyldithiocarbamate. They differ chiefly in steps I and II. However, the concentration of dry bovine serum albumin (BSA) reported to give absorbance 1.0 in these methods ranges from 64 to 99 μg/ml of final coloured solution. In preliminary studies in this laboratory these variations were confirmed and in one case [the method of Westley and Lambeth (3)] linearity and sensitivity were significantly improved by addition of detergents in step III. This was surprising since albumin is added in step III of that procedure to prevent precipitation of the poorly soluble copper complex. This report shows that absorbance and stability of the copper-diethyldithiocarbamate complex is markedly affected both by the concentration of alkali and by the presence or absence of detergents. The method of Klungsöyr (4), in which excess alkaline copper tartrate reagent is removed by adsorption on a Sephadex column, is probably the simplest for multiple assays and a modified procedure giving a reproducible high sensitivity of absorbance 1.0 for 51–53 μg of dry BSA/ml of colour is described.     

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in culture media

A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglass. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cpm) and protein concentration was found. Intra- and interest reproducibility was excellent (C.V.'s less than 7%). By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally defined medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer.     

The measurement of insoluble proteins using a modified Bradford assay

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.