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.     

Calibration of stopped-flow spectrophotometers using a two-step disulfide exchange reaction

A coupled two-step reaction of Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)) with excess thioglycerol produces a progress curve composed of two superimposed exponentials. The ratio of the two pseudo-first-order rate constants equals 22.5 and does not vary appreciably with ehanges of either pH value or temperature. Because the ratio of the two amplitudes is defined by the ratio of the rate constants, the reaction can be used to estimate both the apparent zero time of mixing (or the dead time) and the detector linearity of a stopped-flow instrument with a single mixing experiment. The reaction is used as a standard of performance for both absorbance and fluorescence measurements with a stopped-flow spectrophotometer.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.     

Adaptation of the Nelson-Somogyi reducing-sugar assay to a microassay using microtiter plates

The Nelson-Somogyi assay for reducing sugars was adapted to microtiter plates. The primary advantages of this modified assay are (i) smaller sample and reagent volumes, (ii) elimination of boiling and filtration steps, (iii) automated measurement with a dual-wavelength scanning TLC densitometer, (iv) increased range and reproducibility, and (v) automated colorimetric readings by reflectance rather than absorbance.     

Coupling of glucose oxidase and Fenton's reaction for a simple and inexpensive assay of β-glucosidase

A simple and inexpensive assay for β-glucosidase, based on the coupling of glucose oxidase and Fenton's reagent has been described. Hydrogen peroxide formed as a result of the action of glucose oxidase on glucose (derived from the action of β-glucosidase on cellobiose) oxidizes ferrous sulphate, resulting in an increase in absorbance. The oxidation products produced a peak of maximum absorbance at 340 nm. Using this assay system, a linear relationship between glucose concentration in the range 5.55–27.78 mmol l^?1(100–500 mg dl^?1) and absorbance was obtained, indicating conformity to Beer's law. The preciseness of the glucose oxidase/Fenton's reagent for the assay of glucose was shown to be satisfactory. β-Glucosidase was assayed using the hexokinase assay reagent and the glucose oxidase/ferrous sulphate reagent. The values obtained using both reagents did not differ significantly. Although 2.6 times less sensitive than the hexokinase reagent when absorbance is measured at 340 nm, the glucose oxidase/Fenton's reagent is 10 times cheaper and could be used satisfactorily for routine assays of β-glucosidase and other carbohydrases including cellulase and amylase. In this respect, fructose, mannose, xylose, sucrose and cellobiose did not affect the sensitivity of the reagent. Of several metals tested, only aluminium interfered with the reagent, decreasing its sensitivity.     

A colorimetric assay for 7-dehydrocholesterol with potential application to screening for Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS; MIM 270400) is a genetic disorder characterized by hypocholesterolemia and elevated 7-dehydrocholesterol (7DHC) levels resulting from mutations affecting 7-dehydrocholesterol reductase. We describe a colorimetric assay for 7DHC with potential application to large-scale screening for SLOS. Reaction of 7DHC and its esters with the Liebermann-Burchard reagent resulted in a brief initial absorbance at 510 nm (pink color) followed by an absorbance at 620 nm (blue color) after 2 min, while cholesterol samples were essentially colorless. The assay could identify typical SLOS blood samples by their pink color and increased absorbance at 620 nm after 2 min. Colorimetric identification of mild SLOS cases requires monitoring of the transient absorbance at 510 nm, which must be detected immediately after rapid, consistent mixing of the reagents. The need for special mixing devices and rigorous validation precludes sporadic use of the assay for diagnosing suspected SLOS cases. We also studied the stability of 7DHC in dried SLOS blood spots on Guthrie cards, which are widely used for archiving neonatal blood. Decomposition of 7DHC was effectively retarded by storage at low temperature and by precoating of the cards with antioxidants. The combined results provide a foundation for development of a simple, automated test for SLOS screening.     

A heated biuret-Folin protein assay which gives equal absorbance with different proteins

This protein assay requires heating the sample with biuret reagent for 100 min at 100°C before addition of Folin. Different proteins give almost identical optical densities on a nitrogen basis with this procedure. The absorbance per microgram-atom of protein nitrogin is linear at or below 0.400. A convenient final concentration of protein in this assay is about 0.2 μg-atom of protein nitrogen per ml. Most of the absorbance is due to amino acid combinations other than tyrosine and trytophan in the protein. The specificity appears similar to that of the bluret where few compounds of biological importance interfere significantly in the assay at normal cellular concentrations.     

Measurement of a glycoprotein with blood group A activity by light scattering

A light scattering method for quantifying glycoproteins is presented. Conditions of ethanol concentration, wavelength, time of reading, and sample and reagent volumes have been examined. The definitive assay involves addition of 9 vol absolute ethanol to 1 vol sample, mixing, and readings at 105 degrees angle at the interval between 10 and 13 min after placing the cell into the photometer. The obtained values are temperature-dependent. Thus, temperature control is essential. In this assay, a concentration range of 10-60 micrograms glycoprotein has been used. As described, the method is independent of the chemical composition of the different glycoproteins and is barely influenced by size and shape of glycoproteins.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu²⁺ in the BCA Kit’s reagent B (4% cupric sulfate) in a manner similar to that of the protein.     

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK_a to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.     

Development of a capillary electrophoresis assay based on free sulfate determination for the direct monitoring of sulfoesterase activity.

A capillary electrophoresis assay of sulfoesterase activity was developed that overcomes the main drawbacks encountered with the usual methods for sulfate determination in complex biological medium. Conditions are described allowing direct measurement of inorganic sulfate that is enzymatically produced in the reaction mixture. The main features of this method are electrokinetic sample introduction, which allows selective extraction of sulfate from the matrix into the separation capillary, counter-electroosmotic flow migration mode, indirect absorbance detection and use of an internal standard for quantitative performances. Likewise, perfect linearity was obtained for concentrations of sulfate up to 40 ppm. The limits of detection and quantification were 0.2 and 0.6 ppm, respectively. The run-to-run and day-to-day precision are 1 and 4.5%, respectively, for sulfate concentrations varying from 35 ppm down to 1 ppm. The accuracy was established for the synthetic p-nitrocatechol sulfate substrate by comparison with the classical spectrophotometric assay. The method was applied to the kinetic monitoring of the activity of a sulfoesterase extracted from the marine mollusc Pecten maximus on fucoidan, a bioactive sulfated fucose-based polysaccharide derived from brown algae. For the first time, a sulfoesterase activity was shown to be effective on such sulfated polysaccharides.     

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.     

Phosphate determination by enzymatic colorimetric assay

A direct colorimetric assay for inorganic phosphate in serum is described. The system is based on utilization of the enzymes, purine-nucleoside phosphorylase and xanthine oxidase, to generate superoxide ions. The superoxide is measured in the presence of an electron mediator compound with 3-(4',5'-dimethyl-2-thiazolyl)-2,4-diphenyl-2H-tetrazolium bromide as the chromogen. The high absorbance of this chromogen between 550 and 660 nm affords useful results with a sample/reagent volume ratio as low as 1:100. A single working reagent is used, and the reaction is complete in 15 min at room temperature. The standard curve is linear for inorganic phosphate concentrations as high as 4.9 mmol/liter. Analytical recovery of phosphate in human sera averages 100%. Within-run precision study gives CV less than or equal to 1.0%. The results of this method compare closely (r greater than 0.99) with those obtained by the semidine method (recommended standard). The method lends itself to automation.     

Interaction between hydrogen peroxide and ferrous sulfate as a basis for glucose determinations

Summary The interaction between hydrogen peroxide and ferrous sulfate (Fenton's reaction) results in an increase in absorbance between the wavelengths of 240–400 nm. The greatest increase in absorbance occurs at 260 nm. Addition of glucose to a solution of glucose oxidase and ferrous sulfate results in an instantaneous increase in absorbance at 260 nm which is dependent upon glucose concentration and pH, with greatest increase occurring at pH 4.0. A solution of glucose oxidase and ferrous sulfate could be used as a reagent for manual glucose determinations.Operated by Martin Marietta Energy Systems, Inc., for the U.S. Department of Energy under Contract No. DE-ACO5-84OR21400.     

A method of quantitative analysis of gentamicin sulfate

A procedure for quantitative assay of gentamicin sulfate was developed. It is based on formation of an ionic associate with bromothymol blue followed by chloroform extraction and the photometric determination at 420-422 nm. The relative error does not exceed 0.94 and 0.98 per cent in regard to the substance and its dosage form (gynecological suppositories), respectively. Optimal conditions for the assay were elaborated i.e. the ratio of the reagent and extraction agent, the time and the number of the extractions and pH of the medium. The process obeys to the main law of light absorption within the concentration of gentamicin sulfate in aqueous solution equal to 8-128 micrograms/ml.     

Protein measurement using bicinchoninic acid: elimination of interfering substances

Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminish the protein-induced color development and shift the wave-length of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70,241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.     

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.     

A rapid and simple spectrophotometric assay of angiotensin-converting enzyme.

A rapid, simple, and accurate method for the chemical assay of anglotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensin-converting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a colorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-l-histidyl-l-leucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mm, and at HHL concentration of 3 mm, when the 5000g supernatant fluid of the rat lung was used.     

Selective assay for endotoxin using poly(ε-lysine)-immobilized Cellufine and Limulus amoebocyte lysate (LAL)

We developed a selective endotoxin (lipopolysaccharide; LPS) assay using poly(ε-lysine)-immobilized cellulose beads (PL-Cellufine) and Limulus amoebocyte lysate (LAL). First, LPS was selectively adsorbed on the beads in a solution containing various LAL-inhibiting or LAL-enhancing compounds (e.g., amino acids, enzymes) and the LPS adsorbed on the beads was separated from the compounds by centrifugation. Second, the LPS adsorbed on the beads directly reacted with the LAL reagent, and the LPS concentration was determined by a turbidimetric time assay. The accuracy of the adsorption method with PL-Cellufine was high compared with that of a common solution method. Apparent recovery of LPS from compound solution was 88-120%.