CALIBRATION FEATURES

Calibration graphs for the Lowry assay are usually curved. A linear response is obtained for simple compounds (Sn^2‡, tyrosine, etc.) (14). One explanation for the nonlinear calibration graphs for proteins is that Mo^6‡/

W^6‡ is degraded at high pH with a half-life of about 8 seconds (1).

Assuming this explanation is correct, the color yield during protein analysis re¯ects a balance between Mo^6‡/W^6‡ decomposition by alkali and its reduction to form a blue complex. Inorganic reductants react rapidly with Mo^6‡/W^6‡ before its degradation. Nonlinearity was also ascribed to the declining copper/protein ratio as the concentration of protein is increased.

Attempts to improve the linearity by increasing the concentration of copper led to higher readings for the sample blank.

Readings of A750may be converted to protein concentration [P] using a nonlinear graph. This practice emphasizes data points adjacent to the unknown value. By contrast, a linear graph gives equal weighting to all the experimental points. In chemical analysis, ``linearity is next to cleanliness.'' Most analysts feel a sense of relief where calibration data conform to a linear function;

DA750ˆ F‰PŠ …10†

where F is the slope of the calibration graph. The line described by Eq. (10) passes through the origin and hence

‰PŠ ˆ DA₇₅₀=F …11†

Stauffer (17) ®tted Eq. (12) or its logarithmic form [Eq. (13)] to data from the Lowry assay.

A₇₀₀ˆ o‰PŠ^F …12†

log A700ˆ F log‰PŠ ‡ w …13†

where F and o are constants and w ˆ log o. For a protein concentration range of 4±400 mg mL ¹ a plot of log A700 versus log‰PŠ was linear. The unknown protein concentration can be determined from Eq. (14).

‰PŠ ˆ 10^{Y w†=F} …14†

Notice that Y…ˆ log A₇₅₀† is the value for the unknown sample. Coakley and Jones (18) used reagent volumes three times higher than in the standard method.* Their calibration results were described by a hyperbolic curve.

* A 0.6-mL portion of protein standard solutions was reacted with 3.0 mL of Lowry reagent C.

After standing for 10 minutes, 0.3 mL of Folin-Ciocalteu regent was added. The A750readings were taken 30 minutes later.

A750ˆ ‰ ŠP

w P‰ Š ‡ F …15†

Equation (15) applies for within-cuvette BSA concentrations of 0.01±

0.77 mg mL ¹and for A750values of 0.18±2.2.* The constants F and w were determined by calibration. The protein concentration can be found using Eq. (16).

P

‰ Š ˆ FA750

1 wA₇₅₀ …16†

For routine use, Eq. (16) can be linearized using a double-reciprocal transformation.

1 A750ˆ F

P

‰ Š‡ w …17†

Equation (17) allows a two-point calibration: (a) analyze the unknown sample with two protein standards and (b) determine the constants F and w from Eqs. (18) and (19).

F ˆy₁ y₂

x1 x2 …18†

where y₁ˆ 1/A_{750 (1)}and y₂ˆ 1/A_{750 (2)}. Likewise, x₁ˆ 1/ [P]₁and x₂ˆ [P]₂. The concentrations for the protein standards should be [P]₁ˆ 0.1 mg mL ¹ and [P]₂ˆ 5±10 mg mL ¹BSA. The intercept of Eq. (17) (w) is found from averages for y₁, y₂and x₁, x₂.

w ˆ y Fx where

y ˆ…y₁‡ y₂†

2 and x ˆ…x₁‡ x₂†

2 …19†

The previous treatment applies for a protein concentration between 0.1 and 10 mg mL ¹. With protein concentrations below 0.2 mg mL ¹, a straightfor-ward calibration graph can be used with little loss of accuracy.

* Allowing for the 6.5 times dilution during the Lowry assay, a protein stock solution of 0.065±

5 mg mL ¹leads to a ``within-cuvette'' concentration of 0.01±0.77 mg mL ¹. Investigators are liable to cite either of these protein concentrations in their work.

5. INTERFERENCE COMPOUNDS

Interference compounds for the Lowry assay are either chelators or reducing agents. Chelators diminish color formation by sequestering Cu^2‡. Reducing agents react with the Folin reagent to create extra color. Some interfering compounds act as both chelators and reducing agents. Dyes having an absorbance maximum near 600±750 nm are also interfering compounds.

Peterson classi®ed interfering compounds into more than 13 classes comprising over 180 chemicals (Table 2).

Many of these compounds are encountered in raw and processed foods.* Ascorbic acid and other reducing compounds are found in foods of plant origin (fruits, juices, pastes, concentrates). Wines and certain beverages have high concentrations of phenols and related compounds.

Carbohydrates (simple sugarsÐsucrose, glucose, and fructose) and poly-saccharides (pectin or starch) occur in foods including jams, preserves, and TABLE2 Some Potential Interfering Compounds for the Lowry Assay

Biochemical classi®cation Food additives

1. Amine derivatives Acids and acidulants

2. Amino acids Amino acids

3. Buffers Colors

4. Chelating agents Dyes

5. Detergents (e.g., Triton X-100,

Tween) Sweeteners

6. Drugs Arti®cial antioxidants (BHA, BHT, etc.)

7. Hexosamines Starch

8. Lipids and fatty acids Polysaccharides 9. Miscellaneous compounds Sulfur dioxide, sul®tes

10. Cryoprotectants Uric acid

11. Polyvinylpyrrolidone Fe^2‡

12. Nucleic acids 13. Organic solvents 14. Phenols and polyphenols 15. Polysaccharides

16. Reducing agents 17. Salts

18. Sugars

* However, the analyst may not be aware of the presence of interfering compounds in a given sample.

wheat products. Synthetic reductants, acidulants, and dispersants are just some of the other additives associated with foods. Low-molecular-weight interfering substances can be removed by dialysis. Another approach is to recover the protein from the surrounding solvent medium by TCA precipitation (Method 3). Where possible, sample pretreatment should be avoided for routine and high-throughput analysis. The effects of selected interferences on the Lowry assay are discussed in the following.

5.1. Buffers, Chelating Agents, and Detergents

Peters and Fouts (19) showed that BICINE [N,N-bis- (2-hydroxethyl) glycine] and HEPES [N-(2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)] produce color in proportion to their concentration. Ethylene diamine tetraacetic acid (EDTA), tris-hydroxythethylaminomethane (Tris), TRI-CINE, citrate, and Triton X-100 also produced similar effects (20). These interfering compounds increase the absorbance value for the reagent blank.

However, they reduce the color yield from proteins. These effects are attributed to complex formation with copper. Interference by buffer components can easily be corrected using the appropriate buffer solution as the reagent blank. Sodium phosphate buffer had no effect on the Lowry assay.

5.2. Carbohydrates

Reducing sugars (mainly hexoses) react with Cu^2‡ and the Folin reagent.

Nonreducing sugars (sucrose, oligosaccharides, polysaccharides) form complexes with Cu^2‡. Tagatose, sucrose, and inulin interfered with the Lowry assay after exposure to hot alkali or acid (21). These solvents are routinely used to dissolve proteins. As well as reducing Cu^2‡to Cu^1‡, sugars reduce tungstate-molybdate. The effects of simple sugars are signi®cant at concentrations above 1 mM. The order of effectiveness is fructose >

sorbose > xylose > rhamnose > mannose > glucose (22).

Toldra (23) considered protein analysis in connection with the production of high-fructose syrup. Effective control of this process requires the determination of a-amylase and glucoamylase speci®c activity (hence protein concentration) in samples containing up to 30±40% sugar. A solution of 1% (w/v) glucose or maltose produced an A750 response equivalent to that of 18.8 or 16.6 mg mL ¹ BSA. Protein analysis is also necessary to determine the activity of fungal pectinases produced in submerged culture using high concentrations of pectin as inducer (24).

Concentration of 0.1±1.0% (w/v) pectin interfered with both the classical and modi®ed Lowry assays. Calibration graphs showed a decrease in the

slope and increased intercept. Assay sensitivity decreased while the LLD increased with increasing pectin concentration (Chapter 1). The color with protein-free samples increased linearly with the amount of added pectin.

These results imply complex formation between pectin and Cu^2‡. The Bradford method (Chapters 6 and 7) coped better with samples containing

< 0.5% (w/v) pectin. In our experience, pectin forms a gelatinous mass during the Lowry assay. To avoid this problem, samples are exposed 0.1 M CaCl2and then centrifuged before protein analysis. Berg (25) warned that

``anyone attempting to determine protein contamination in polysaccharide preparations by the Lowry procedure could be misled by positive results which may be attributed to the carbohydrate.''

5.3. Chlorophyll

Leaves, stems, and green fruit contain chlorophyll. Addition of chlorophyll (100 mg mL ¹) to BSA (300 mg mL ¹) increased A750 values by 400% (26).

For accurate analysis, leaf protein must be separated from chlorophyll.*

5.4. Cryoprotectants, Sucrose, Glycerol, and Polyhydroxyalcohols

Ethylene glycol, polyvinylpyrrolidone (PVP), and dimethyl sulfoxide (DMSO) are used to protect proteins against freeze damage. Glycerol, probably acting in a manner similar to sucrose, inhibits color formation. In the absence of copper, PVP reacts with Mo^6‡/W^6‡, leading to a blue coloration (27). Glycerol (2±5%) interferes with the Lowry procedure (28) but DMSO has no effect.

Many foods and condiments contain very high concentrations of sucrose. Sucrose is also used for gradient ultracentrifugation. Sucrose by itself gives a positive response with the Lowry assay (29). However, protein analysis in the presence of sucrose leads to a reduced color yield. The interference becomes progressively worse with 0±65% sucrose. Effects can be reduced to tolerable levels (< 5% analytical error) by (a) diluting samples to

< 10% sucrose or (b) doubling the concentration of copper in Lowry reagent B. Quadrupling the copper concentration led to an unstable reagent with a tendency to form colloidal copper (30).

* Leaf protein can be extracted with 0.1 N alkali. Next, precipitate the protein with 10% (w/v) TCA. A second approach is to treat freeze-dried leaf powder with acetone. This decolorized powder can be stored before analysis (this work is discussed in detail later).

Sucrose (and other polyhydroxy compounds) appear to complex with Cu^2‡when monitored by ultraviolet absorbance spectrophotometry. In the absence of copper, sucrose or glycerol does not interfere with the tyrosine reaction with Folin-Ciocalteu reagent. Hydrolysis of sucrose by heat, alkali, or invertase generates fructose and glucose, which affect the Lowry assay as reducing sugars (31). Ficoll (synthetic polymer of sucrose) yields an intense blue color when analyzed by the Lowry procedure (32) (Fig. 2).

Concentrations of 2±36% (w/v) ®coll are used for density gradient centrifugation of cell fractions. Because of its high average molecular mass (*400 kDa), it is not removed from samples by dialysis. Addition of the Folin-Ciocalteu reagent to ®coll leads to formation of color with a time course of over 18 hours at room temperature. Ficoll had no effect on the yield of color when added to protein samples after the Folin-Ciocalteu reagent. In the presence of ®xed concentrations (0.21±1.88%) of ®coll, the calibration graph of standard BSA solutions had progressively lower slopes while the intercept increased. Ficoll forms a UV-detectable complex with copper.

FIGURE 2 Effect of increasing ®coll concentrations on the Lowry assay. Boxed legend shows Ficoll concentrations. (Drawn from data from Ref. 32.)

5.5. Lipids

Lipids increase sample turbidity and A750 values. The products of lipid autoxidation also react directly with the Folin reagent. Heating lipids with alkali (a common method of protein solubilization) leads to Lowry-positive products. The problem has been examined with arachidonic acid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (33). Samples may be delipidated by shaking with chloroform or petroleum ether and then redissolved with 0.5 M NaOH by storing at 378C overnight or by heating at 1008C for 30 minutes. Solvent extraction will not be wholly successful if the protein and proteolipid complexes dissolve within an organic solvent phase. Emulsion formation can also lead to apparent loss of protein from the sample.

Lees and Paxman (34) modi®ed the Lowry assay for proteolipids and lipoproteins. Their method is fast and does not require heating.* Markwell et al. (35) added 1% SDS directly to Lowry reagent C to dissolve membranes and other lipid. The concentration of copper sulfate was also increased from 0.5 to 4% (w/w) to reduce interferences from sucrose and EDTA. The modi®ed method gave results identical to those obtained with lipoprotein samples delipidated using petroleum ether. However, the new method allowed higher rates of analysis and greater convenience. There was no interference from 40±200 mM sucrose. SDS also features in Method 3, which is therefore able to deal with membrane lipids routinely. According to Kirazov et al. (36), treating membrane-containing fractions with 1 M NaOH or 0.5% SDS reduced the stability of A750readings and did not reduce the interference from lipids.

5.6. Sulfhydryl Agents and Other Reducing Compounds

The effects of various SH compounds are also related to their standard electrode potential (E8) measured against a normal hydrogen electrode (NHE) or a calomel electrode. At concentrations below 1 M, the redox potential is described by the Nernst equation:

E ˆ E^‡RT

nF ln C …20†

where n is the number of electrons transferred, E the observed redox potential corresponding to an activity of C (mol L ¹), F the Faraday

* Treat protein samples (20±70 mg) with 0.5 mL of alkaline SDS solution (5% SDS, 0.5 M NaOH), vortex, and let stand at room temperature for 3 hours. Add 2.5 mL of Lowry reagent C followed 10 minutes later with 250 mL of Folin (1 N) reagent. Take A695reading after 45 minutes.

constant, and R the gas constant. For dilute solutions, C becomes equal to concentration. Therefore, the effect of reducing compounds on the Lowry assay is related to the ``intrinsic'' redox properties (E8 and n) and the concentration of reducing agent.

Dithiothreitol (DTT), 2-mercaptoethanol (2ME), reduced glutathione (GSH), and oxidized gluthathione (GSSG) produce color with the Lowry assay (37). Reducing compounds react directly with the Folin-Ciocalteu reagent. Table 3 shows the degree of color formation with some SH compounds. As the underlying reaction is a redox process, calibration graphs for SH compounds will be nonlinear. For each SH compound De (M ¹cm ¹) was determined from the steepest slope in Fig. 3. Values for De determined in this manner (Table 3, top half) agree with results from Ref.

14. DTT is one of the most effective interfering SH compounds. Samples with > 0.2 mM DTT have high A₇₅₀ high background absorbances.

TABLE3 The Color Yield from the Lowry Assay of Some Reducing Compounds Reducing agent
" (M ¹cm ¹)^a n^b E_1/2(mV)^c

Tyrosine 12800 2 (4) 0.398

Indole 13000 2 (4) 0.253

Tryptophan 13200 2 (2) 0.205

aThe De (M ¹cm ¹) values in the top half of Table 3 are calculated from the maximum slope in Fig. 3. Data in the bottom half of Table 3 from Ref. 14.

bn ˆ electrons transferred to Mo^6‡/W^6‡from one molecule of reducing compound. Values for n were determined by colorimetry,^cHalf-wave electrode potentials were determined by cyclic voltammetry, using standard calomel electrode (i.e., ‡ 0.242 V vs. NHE), from Ref. 78. Note that for dilute solutions E1/2ˆ E8 Eref.

Reduction of the Mo^6‡/W^6‡produces an absorbance change of 3200 units (M ¹cm ¹) per mole of electrons transferred (see Table 1).