TOTAL PROTEIN ASSAY
The following procedures are provided as illustrations of the determination of total protein content in pharmacopeial preparations.Other techniques,such as HPLC,are also acceptable if total protein recovery is demonstrated.Many of the total protein assay methods described below can be performed successfully using kits from commercial sources.[NOTE—Where water is required,use distilled water.] Method 1 Protein in solution absorbs UVlight at a wavelength of 280nm,due to the presence of aromatic amino acids,mainly tyrosine and tryptophan.This property is the basis of this method.Protein determination at 280nm is mainly a function of the tyrosine and tryptophan content of the protein.If the buffer used to dissolve the protein has a high absorbance relative to that of water,there is an interfering substance in the buffer.This interference can be compensated for when the spectrophotometer is adjusted to zero buffer absorbance.If the interference results in a large absorbance that challenges the limit of sensitivity of the spectrophotometer,the results may be compromised.Furthermore,at low concentrations protein can be absorbed onto the cuvette,thereby reducing the content in solution.This can be prevented by preparing samples at higher concentrations or by using a nonionic detergent in the preparation.[NOTE—Keep the Test Solution,the Standard Solution,and the buffer at the same temperature during testing.] Test Solution— Dissolve a suitable quantity of the protein under test in the appropriate buffer to obtain a solution having a concentration of 0.2to 2mg per mL. Standard Solution— Unless otherwise specified in the individual monograph prepare a solution of USP Reference Standard or reference material for the protein under test in the same buffer and at the same concentration as the Test Solution. Procedure— Concomitantly determine the absorbances of the Standard Solutionand the Test Solutionin quartz cells at a wavelength of 280nm,with a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ),using the buffer as the blank.To obtain accurate results,the response should be linear in the range of protein concentrations to be assayed. Light-Scattering— The accuracy of the UVspectroscopic determination of protein can be decreased by the scattering of light by the test specimen.If the proteins in solution exist as particles comparable in size to the wavelength of the measuring light (250to 300nm),scattering of the light beam results in an apparent increase in absorbance of the test specimen.To calculate the absorbance at 280nm due to light-scattering,determine the absorbances of the Test Solutionat wavelengths of 320,325,330,335,340,345,and 350nm.Using the linear regression method,plot the log of the observed absorbance versus the log of the wavelength,and determine the standard curve best fitting the plotted points.From the graph so obtained,extrapolate the absorbance value due to light-scattering at 280nm.Subtract the absorbance from light-scattering from the total absorbance at 280nm to obtain the absorbance value of the protein in solution.Filtration with a filter having a 0.2-µm porosity or clarification by centrifugation may be performed to reduce the effect of light-scattering,especially if the solution is noticeably turbid. Calculations— Calculate the concentration,CU,of protein in the test specimen by the formula: CS(AU/AS), in which CSis the concentration of the Standard Solution;and AUand ASare the corrected absorbances of the Test Solutionand the Standard Solution,respectively (see Spectrophotometry and Light-Scattering á851ñ). Method 2 This method,commonly referred to as the Lowry assay,is based on the reduction by protein of the phosphomolybdic-tungstic mixed acid chromogen in the Folin-Ciocalteu's phenol reagent,resulting in an absorbance maximum at 750nm.The Folin-Ciocalteu's phenol reagent reacts primarily with tyrosine residues in the protein,which can lead to variation in the response of the assay to different proteins.Because the method is sensitive to interfering substances,a procedure for precipitation of the protein from the test specimen may be used.Where separation of interfering substances from the protein in the test specimen is necessary,proceed as directed below for Interfering Substancesprior to preparation of the Test Solution.The effect of interfering substances can be minimized by dilution,provided the concentration of the protein under test remains sufficient for accurate measurement. Standard Solutions— Unless otherwise specified in the individual monograph,dissolve the USP Reference Standard or reference material for the protein under test in the buffer used to prepare the Test Solution.Dilute portions of this solution with the same buffer to obtain not fewer than five Standard Solutionshaving concentrations between 5and 100µg of protein per mL,the concentrations being evenly spaced. Test Solution— Dissolve a suitable quantity of the protein under test in the appropriate buffer to obtain a solution having a concentration within the range of the concentrations of the Standard Solutions.An appropriate buffer will produce a pHin the range of 10.0to 10.5. Blank— Use the buffer used for the Test Solutionand the Standard Solutions. Reagentsand Solutions— Copper Sulfate Reagent— Dissolve 100mg of cupric sulfate and 200mg of sodium tartrate in water,dilute with water to 50mL,and mix.Dissolve 10g of sodium carbonate in water to a final volume of 50mL,and mix.Slowly pour the sodium carbonate solution into the copper sulfate solution with mixing.Prepare this solution fresh daily. SDS Solution— Dissolve 5g of sodium dodecyl sulfate in water,and dilute with water to 100mL. Sodium Hydroxide Solution— Dissolve 3.2g of sodium hydroxide in water,dilute with water to 100mL,and mix. Alkaline Copper Reagent— Prepare a mixture of Copper Sulfate Reagent,SDS Solution,and Sodium Hydroxide Solution(1:2:1).This reagent may be stored at room temperature for up to 2weeks. Diluted Folin-Ciocalteu’s Phenol Reagent— Mix 10mLof Folin-Ciocalteu’s phenol TSwith 50mLof water.Store in an amber bottle,at room temperature. Procedure— To 1mLof each Standard Solution,the Test Solution,and the Blank,add 1mLof Alkaline Copper Reagent,and mix.Allow to stand at room temperature for 10minutes.Add 0.5mLof the Diluted Folin-Ciocalteu's Phenol Reagentto each solution,mix each tube immediately,and allow to stand at room temperature for 30minutes.Determine the absorbances of the solutions from the Standard Solutionsand the Test Solutionat the wavelength of maximum absorbance at 750nm,with a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ),using the solution from the Blankto set the instrument to zero. Calculations— [NOTE—The relationship of absorbance to protein concentration is nonlinear;however,if the standard curve concentration range is sufficiently small,it will approach linearity.]Using the linear regression method,plot the absorbances of the solutions from the Standard Solutionsversus the protein concentrations,and determine the standard curve best fitting the plotted points.From the standard curve so obtained and the absorbance of the Test Solution,determine the concentration of protein in the Test Solution. INTERFERING SUBSTANCES In the following procedure,deoxycholate-trichloroacetic acid is added to a test specimen to remove interfering substances by precipitation of proteins before testing.This technique also can be used to concentrate proteins from a dilute solution. Sodium Deoxycholate Reagent— Prepare a solution of sodium deoxycholate in water having a concentration of 150mg in 100mL. Trichloroacetic Acid Reagent— Prepare a solution of trichloroacetic acid in water having a concentration of 72g in 100mL. Procedure— Add 0.1mLof Sodium Deoxycholate Reagentto 1mLof a solution of the protein under test.Mix on a vortex mixer,and allow to stand at room temperature for 10minutes.Add 0.1mLof Trichloroacetic Acid Reagent,and mix on a vortex mixer.Centrifuge at 3000×gfor 30minutes,decant the liquid,and remove any residual liquid with a pipet.Redissolve the protein pellet in 1mLof Alkaline Copper Reagent.Proceed as directed for the Test Solution. NOTE—Color development reaches a maximum in 20to 30minutes during incubation at room temperature,after which there is a gradual loss of color.Most interfering substances cause a lower color yield;however,some detergents cause a slight increase in color.Ahigh salt concentration may cause a precipitate to form.Because different protein species may give different color response intensities,the standard protein and test protein should be the same. Method 3 This method,commonly referred to as the Bradford assay,is based on the absorption shift from 470nm to 595nm observed when the brilliant blue Gdye binds to protein.The brilliant blue Gdye binds most readily to arginyl and lysyl residues in the protein,which can lead to variation in the response of the assay to different proteins. Standard Solutions— Unless otherwise specified in the individual monograph,dissolve the USP Reference Standard or reference material for the protein under test in the buffer used to prepare the Test Solution.Dilute portions of this solution with the same buffer to obtain not fewer than five Standard Solutionshaving concentrations between 100µg and 1mg of protein per mL,the concentrations being evenly spaced. Test Solution— Dissolve a suitable quantity of the protein under test in the appropriate buffer to obtain a solution having a concentration within the range of the concentrations of the Standard Solutions. Blank— Use the buffer used to prepare the Test Solutionand the Standard Solutions. Coomassie Reagent— Dissolve 100mg of brilliant blue G3in 50mLof alcohol.[NOTE—Not all dyes have the same brilliant blue Gcontent,and different products may give different results.]Add 100mLof phosphoric acid,dilute with water to 1L,and mix.Pass the solution through filter paper (Whatman No.1or equivalent),and store the filtered reagent in an amber bottle at room temperature.[NOTE—Slow precipitation of the dye will occur during storage of the reagent.Filter the reagent before use.] Procedure— Add 5mLof the Coomassie Reagentto 100µLof each Standard Solution,the Test Solution,and the Blank,and mix by inversion.Avoid foaming,which will lead to poor reproducibility.Determine the absorbances of the solutions from the Standard Solutionsand the Test Solutionat 595nm,with a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ),using the Blankto set the instrument to zero.[NOTE—Do not use quartz (silica)spectrophotometer cells:the dye binds to this material.Because different protein species may give different color response intensities,the standard protein and test protein should be the same.] There are relatively few interfering substances,but detergents and ampholytes in the test specimen should be avoided.Highly alkaline specimens may interfere with the acidic reagent. Calculations— [NOTE—The relationship of absorbance to protein concentration is nonlinear;however,if the standard curve concentration range is sufficiently small,it will approach linearity.]Using the linear regression method,plot the absorbances of the solutions from the Standard Solutionsversus the protein concentrations,and determine the standard curve best fitting the plotted points.From the standard curve so obtained and the absorbance of the Test Solution,determine the concentration of protein in the Test Solution. Method 4 This method,commonly referred to as the bicinchoninic acid or BCAassay,is based on reduction of the cupric (Cu2+)ion to cuprous (Cu1+)ion by protein.The bicinchoninic acid reagent is used to detect the cuprous ion.The method has few interfering substances.When interfering substances are present,their effect may be minimized by dilution,provided that the concentration of the protein under test remains sufficient for accurate measurement. Standard Solutions— Unless otherwise specified in the individual monograph,dissolve the USP Reference Standard or reference material for the protein under test in the buffer used to prepare the Test Solution.Dilute portions of this solution with the same buffer to obtain not fewer than five Standard Solutionshaving concentrations between 10and 1200µg of protein per mL,the concentrations being evenly spaced. Test Solution— Dissolve a suitable quantity of the protein under test in the appropriate buffer to obtain a solution having a concentration within the range of the concentrations of the Standard Solutions. Blank— Use the buffer used to prepare the Test Solutionand the Standard Solutions. Reagents— BCA Reagent— Dissolve about 10g of bicinchoninic acid,20g of sodium carbonate monohydrate,1.6g of sodium tartrate,4g of sodium hydroxide,and 9.5g of sodium bicarbonate in water.Adjust,if necessary,with sodium hydroxide or sodium bicarbonate to a pHof 11.25.Dilute with water to 1L,and mix. Copper Sulfate Reagent— Dissolve about 2g of cupric sulfate in water to a final volume of 50mL. Copper-BCA Reagent— Mix 1mLof Copper Sulfate Reagentand 50mLof BCA Reagent. Procedure— Mix 0.1mLof each Standard Solution,the Test Solution,and the Blankwith 2mLof the Copper-BCA Reagent.Incubate the solutions at 37for 30minutes,note the time,and allow to come to room temperature.Within 60minutes following the incubation time,determine the absorbances of the solutions from the Standard Solutionsand the Test Solutionin quartz cells at 562nm,with a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ),using the Blankto set the instrument to zero.After the solutions are cooled to room temperature,the color intensity continues to increase gradually.If substances that will cause interference in the test are present,proceed as directed for Interfering Substancesunder Method 2.Because different protein species may give different color response intensities,the standard protein and test protein should be the same. Calculations— [NOTE—The relationship of absorbance to protein concentration is nonlinear;however,if the standard curve concentration range is sufficiently small,it will approach linearity.]Using the linear regression method,plot the absorbances of the solutions from the Standard Solutionsversus the protein concentrations,and determine the standard curve best fitting the plotted points.From the standard curve so obtained and the absorbance of the Test Solution,determine the concentration of protein in the Test Solution. Method 5 This method,commonly referred to as the Biuret assay,is based on the interaction of cupric (Cu2+)ion with protein in an alkaline solution and the resultant development of absorbance at 545nm. Standard Solutions— Unless otherwise specified in the individual monograph,prepare a solution of Albumin Human for which the protein content has been previously determined by nitrogen analysis (using the nitrogen-to-protein conversion factor of 6.25)or of the USP Reference Standard or reference material for the protein under test in sodium chloride solution (9in 1000).Dilute portions of this solution with sodium chloride solution (9in 1000)to obtain not fewer than three Standard Solutionshaving concentrations between 0.5and 10mg per mL,the concentrations being evenly spaced.[NOTE—Low responses may be observed if the sample under test has significantly different level of proline than that of Albumin Human.Adifferent standard protein may be employed in such cases.] Test Solution— Prepare a solution of the test protein in sodium chloride solution (9in 1000)having a concentration within the range of the concentrations of the Standard Solutions. Blank— Use sodium chloride solution (9in 1000). Biuret Reagent— Dissolve about 3.46g of cupric sulfate in 10mLof hot water,and allow to cool (Solution 1).Dissolve about 34.6g of sodium citrate dihydrate and 20.0g of sodium carbonate in 80mLof hot water,and allow to cool (Solution 2).Mix Solution 1and Solution 2,and dilute with water to 200mL.This Biuret Reagentis stable at room temperature for 6months.Do not use the reagent if it develops turbidity or contains any precipitate. Procedure— To one volume of a solution of the Test Solutionadd an equal volume of sodium hydroxide solution (6in 100),and mix.Immediately add a volume of Biuret Reagentequivalent to 0.4volume of the Test Solution,and mix.Allow to stand at a temperature between 15and 25for not less than 15minutes.Within 90minutes after the addition of the Biuret Reagent,determine the absorbances of the Standard Solutionsand the solution from the Test Solutionat the wavelength of maximum absorbance at 545nm,with a suitable spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ),using the Blankto set the instrument to zero.[NOTE—Any solution that develops turbidity or a precipitate is not acceptable for calculation of protein concentration.] Calculations— Using the least-squares linear regression method,plot the absorbances of the Standard Solutionsversus the protein concentrations,determine the standard curve best fitting the plotted points,and calculate the correlation coefficient for the line.[NOTE—Within the given range of the standards,the relationship of absorbance to protein concentration is approximately linear.]Asuitable system is one that yields a line having a correlation coefficient of not less than 0.99.From the standard curve and the absorbance of the Test Solution,determine the concentration of protein in the test specimen,making any necessary correction. Interfering Substances— To minimize the effect of interfering substances,the protein can be precipitated from the initial test specimen as follows.Add 0.1volume of 50percent trichloroacetic acid to 1volume of a solution of the test specimen,withdraw the supernatant layer,and dissolve the precipitate in a small volume of 0.5Nsodium hydroxide.Use the solution so obtained to prepare the Test Solution. Comments— This test shows minimal difference between equivalent IgGand albumin samples.Addition of the sodium hydroxide and the Biuret Reagentas a combined reagent,insufficient mixing after the addition of the sodium hydroxide,or an extended time between the addition of the sodium hydroxide solution and the addition of the Biuret Reagentwill give IgGsamples a higher response than albumin samples.The trichloroacetic acid method used to minimize the effects of interfering substances can also be used to determine the protein content in test specimens at concentrations below 500µg per mL. Method 6 This fluorometric method is based on the derivatization of the protein with o-phthalaldehyde (OPA),which reacts with the primary amines of the protein (i.e.,NH2-terminal amino acid and the e-amino group of the lysine residues).The sensitivity of the test can be increased by hydrolyzing the protein before testing.Hydrolysis makes the a-amino group of the constituent amino acids of the protein available for reaction with the o-phthalaldehyde reagent.The method requires very small quantities of the protein. Primary amines,such as tris(hydroxymethyl)aminomethane and amino acid buffers,react with o-phthalaldehyde and must be avoided or removed.Ammonia at high concentrations will react with o-phthalaldehyde as well.The fluorescence obtained when amine reacts with o-phthalaldehyde can be unstable.The use of automated procedures to standardize this procedure may improve the accuracy and precision of the test. Standard Solutions— Unless otherwise specified in the individual monograph,dissolve the USP Reference Standard or reference material for the protein under test in the buffer used to prepare the Test Solution.Dilute portions of this solution with the same buffer to obtain not fewer than five Standard Solutionshaving concentrations between 10and 200µg of protein per mL,the concentrations being evenly spaced. Test Solution— Dissolve a suitable quantity of the protein under test in the appropriate buffer to obtain a solution having a concentration within the range of the concentrations of the Standard Solutions. Blank— Use the buffer used to prepare the Test Solutionand the Standard Solutions. Reagents— Borate Buffer— Dissolve about 61.83g of boric acid in water,and adjust with potassium hydroxide to a pHof 10.4.Dilute with water to 1L,and mix. Stock OPA Reagent— Dissolve about 120mg of o-phthalaldehyde in 1.5mLof methanol,add 100mLof Borate Buffer,and mix.Add 0.6mLof polyoxyethylene (23)lauryl ether,and mix.This solution is stable at room temperature for at least 3weeks. OPA Reagent— To 5mLof Stock OPA Reagentadd 15µLof 2-mercaptoethanol.Prepare at least 30minutes prior to use.This reagent is stable for one day. Procedure— Adjust each of the Standard Solutionsand the Test Solutionto a pHbetween 8and 10.5.Mix 10µLof the Test Solutionand each of the Standard Solutionswith 100µLof OPA Reagent,and allow to stand at room temperature for 15minutes.Add 3mLof 0.5Nsodium hydroxide,and mix.Using a suitable fluorometer (see Spectrophotometry and Light-Scattering á851ñ),determine the fluorescent intensities of solutions from the Standard Solutionsand the Test Solutionat an excitation wavelength of 340nm and an emission wavelength between 440and 455nm.[NOTE—The fluorescence of an individual specimen is read only once because irradiation decreases the fluorescent intensity.] Calculations— The relationship of fluorescence to protein concentration is linear.Using the linear regression method,plot the fluorescent intensities of the solutions from the Standard Solutionsversus the protein concentrations,and determine the standard curve best fitting the plotted points.From the standard curve so obtained and the fluorescent intensity of the Test Solution,determine the concentration of protein in the test specimen. Method 7 This method is based on nitrogen analysis as a means of protein determination.Interference caused by the presence of other nitrogen-containing substances in the test specimen can affect the determination of protein by this method.Nitrogen analysis techniques destroy the protein under test but are not limited to protein presentation in an aqueous environment. Procedure 1— Determine the nitrogen content of the protein under test as directed under Nitrogen Determination á461ñ.Commercial instrumentation is available for the Kjeldahl nitrogen assay. Procedure 2— Commercial instrumentation is available for nitrogen analysis.Most nitrogen analysis instruments use pyrolysis (i.e.,combustion of the sample in oxygen at temperatures approaching 1000),which produces nitric oxide (NO)and similar oxides of nitrogen (NOx)from the nitrogen present in the test protein.Some instruments convert the nitric oxides to nitrogen gas,which is quantified with a thermal conductivity detector.Other instruments mix nitric oxide (NO)with ozone (O3)to produce excited nitrogen dioxide (NO2),which emits light when it decays and can be quantified with a chemiluminescence detector.Aprotein reference material or reference standard that is relatively pure and is similar in composition to the test proteins is used to optimize the injection and pyrolysis parameters and to evaluate consistency in the analysis. Calculations— The protein concentration is calculated by dividing the nitrogen content of the sample by the known nitrogen content of the protein.The known nitrogen content of the protein can be determined from the chemical composition of the protein or by comparison with the nitrogen content of the USP Reference Standard or reference material.