THE UDY METHOD

The Udy assay using Acid Orange 12 is described here (Method 1). Information in the public domain deals with milk and dairy products. Other applications developed by Udy Corporation during the 1960s were probably commercially sensitive and not published (Table 1). The Udy method is the basis of Udy Protein Systems2_.

2.1. Protein Determination Using Acid Orange 12

Method 1 is equivalent to AOAC Method 967.12 for milk protein analysis (19). Instrument requirements include a spectrophotometer, short-path- length ¯ow cuvette, and automatic pipettes. For small-scale analysis, a degree of improvisation is possible. For large numbers of samples the required accessories include the Udy calorimeter, a 40-mL dye regent

FIGURE1 Basic amino acid concentration in a range of proteins as measured by

Orange G binding and by titration. Units of the Y-axis are 6 10 4_moles

dispenser, and a React-R-Shaker* for highly ef®cient mixing of powdered samples with dye solution.

Method 1

Acid Orange 12 dye binding (20±22). Reagents{

1. Acetic acid (glacial) 2. Acid Orange 12 3. Oxalic acid

4. Potassium dihydrogen phosphate Procedure

Puri®cation of Acid Orange 12. Dissolve 400 g of dye in 400 mL of boiling water. Add 400 mL of reagent-grade ethyl alcohol. Cool to room temperature and refrigerate at 0±58C overnight. Vacuum ®lter dye solids using a Buchner ¯ask-®ltration unit ®tted with a polypropylene ®lter. Wash with cold ethyl alcohol and dry the resulting solid in an oven at 1258C.

TABLE1 A Range of Commodities Analyzed by the Udy Method

Alfafa Gaines burger Oats Soybean

Barley Gram Oat groats Soybean hulls

Beans Grass peas Peanut meal Soybean meals

Bermuda grass Lentils Peas Sun¯ower meal

Caseinate Linseed meal Pigeon peas Triticale

Cheese (hard) Malted barley Rapeseed meal Urd beans

Chickpeas Meat Rice Wheat

Corn silage MilkЯuid Rye Wheat germ

Cottonseed meal MilkÐpowders Saf¯ower meal WheatÐgluten

Cowpeas Mung beans Sesame meal WheyÐdelactose

Fish meal Mustard meal Sorghum (milo) WheyÐfresh

WheyÐpowdered

Source: Adapted from Udy Corporation advertising literature.

* Udy and React-R-Shaker are trademark terms for the Udy Corporation, 201 Rome Court, Fort Collins, CO 80524. Fax: 1-970-482-2067. Telephone: 1-970-482-2060. Internet address: http://www.udycorp.thomasregister.com

{ Other requirements include 2-oz polyethylene bottles or 125-mL conical ¯asks, automatic pipettes for dispersion of 40 mL of reagent, syringe pipettes (2±5 mL), sample mill, and ®ltration equipment or a low-speed centrifuge.

Phosphate buffer (0.05 M, pH 1.8±1.9). Dissolve potassium dihydro- gen phosphate (3.4 g) and oxalic acid (2 g) in 100 mL of warm water. Add to 800 mL of water containing phosphoric acid (3.4 mL), acetic acid (60 mL), and propionic acid (1 mL) and dilute the mixture to 1 L.

Working Acid Orange 12 dye reagent (0.13% w/v). Dissolve 1.3 g of Acid Orange 12 in 100 mL of warm phosphate buffer. Allow to cool and dilute to 1 L with phosphate buffer.

Reference dye solution (0.06% w/v). Dilute the working dye reagent with phosphate buffer. Prepare further dilutions and produce a calibration curve of free dye concentration versus A480readings.

Performing a dye-binding assay. Place 1.5±2.4 mL of liquid sample (or 0.25±0.5 g of solid) in a 2-oz plastic polyethylene bottle. Add 40.44 g (40 mL) of working dye solution and shake vigorously for 30 seconds. Solid samples may be shaken for 5 minutes.

Centrifuge at 3500 rpm for 30 minutes or ®lter to remove insoluble dye-protein complex. Dilute* the supernatant 100-fold with phosphate buffer and record A480 readings versus an appropriate

buffer blank. Determine the amount of free dye from the calibration graph.

Calibration for protein determination. Determine dye binding for samples with known amounts of crude protein (%N 6 6.25). Establish the regression equation relating Dbversus crude protein.

Analyses of 73 whole milk or 34 spray-dried milk samples by Orange G binding led to a highly signi®cant correlation between crude protein (%N 6 6.38) and the amount of free dye (23);

cP ˆ 100…V1D V_kEm2Df† …2†

where k (226 g mole 1_{) is the dye equivalent weight,{ E (moles g} 1_{) is the}

DBC expressed as equivalents of dye bound, and m is the weight of sample in grams. For a solid sample V1& V2(i.e., volume of sample & volume of

* Colorimetric measurements are possible without dilution when using a very short (0.3 mm) path-length ¯ow-through cuvette.

{ The equivalent weight (k) for an ionic species (g mole 1_{) is the molecular weight divided by the}

number of charges per molecule; k ˆ 226 (g mole 1_{) assuming that this dye has two positive}

sample ‡ dye) and hence

cP ˆ 100V1…D D_kEm f† …3†

Rearranging Equation (2) leads to Eq. (4) or Eq. (5) for liquid or solid samples, respectively. Df ˆV_V1 2D cPEkm V2 …4† Df ˆ D cPEkm_V 1 …5†

Therefore a graph of Df versus cP yields a straight line with a slope

of mkE. The parameter E was 0.792 (mEq g 1_{) for spray-dried milk and}

0.805 (mEq g 1_{) for fresh milk. See Section 6 for further discussions of milk}

protein analysis.

2.2. Protein Determination Using Orange-G

Fraenkel-Conrat and Cooper (7) employed Orange-G in their seminal study of 1944. Udy (8) also used Orange-G for protein determination and later changed to Acid Orange 12 in 1963 (22). The color change for Acid Orange 12 was apparently 100% greater than that obtained with Orange-G.* The former dye is also less hygroscopic and more easily puri®ed. These days, high-grade samples of Orange-G are readily available. The use of Orange-G is described further in Refs. 8,9,24,25, and 26.

2.3. Protein Determination Using Amido Black 10B

Milk protein analysis using Amido Black 10B is important in both North America and Europe (27±30). Commercial instruments such as the ProMilk Mark II or ProMilk PMA (manufactured by Foss Food Technology Corp.) use Amido Black 10B. Several investigators reported dif®culties with Amido Black 10B staining of plant proteins;some Amido Black 10B samples may contain impurities with different af®nities for plant proteins (31). [Method 2 is adapted from Sherbon (32,33).]

* This view is incorrect (Section 3.2). Proteins bind equal amounts of Orange-G and Acid Orange 12. The molar extinction coef®cients for Orange-G and Acid Orange 12 are also similar.

Method 2

Protein analysis using Amido Black 10B dye binding* (32,33). Reagents

1. Amido Black 10B 2. Citric acid

3. Disodium hydrogen phosphate 4. Thymol blue (optional preservative) Procedure

0.05M Citrate±0.01 M phosphate buffer. Dissolve citric acid (52.6 g), sodium dihydrogen phosphate (3.3 g), and thymol blue (1 g) in 660 mL of water.

Working dye solution (0.075% w/v). Dissolve Amido Black 10B (3 g) in 1 L of water by heating to 708C. Mix with citrate-phosphate buffer and add 3.33 kg of water.

Reference dye solution. Dilute the working dye solution (1 volume) with 2.5 volumes of distilled water.

Add 1 mL of sample to 20 mL of dye solution. Mix for 0.5±3 minutes. Filter to remove insoluble dye-protein complex. Dilute the super- natant 100-fold with phosphate buffer. Record A620 readings.

Determine the amount of free dye from a calibration graph of A620

plotted against reference dye solutions.{

For calibration analyze at least 10 samples of known protein concentration in duplicate.

Methods 1 and 2 were readily scaled down by mixing 50±100 mL of sample with 1 mL of dye reagent in a polyethylene microcentrifuge tube. The protein-dye precipitate was then removed by microcentrifugation (13,000 rpm;5 minutes). Absorbance readings were recorded after diluting the dye- supernatant solution 100-fold. For strongly colored samples, the dry weight for the protein-dye complex was recorded. These micro-dye-binding assays led to signi®cant savings of reagent and improved convenience (34). 3. SOLID-PHASE DYE-BINDING ASSAYS

Protein is adsorbed on a ®lter support such as nitrocellulose, ®lter paper, or a glass ®ber ®lter. Sometimes, the adsorbed protein is ``®xed'' by treating

* Amido Black 10B dye binding is also called the ProMilk method.

{ Absorbance measurements can be taken without dilution if using the purpose-made 0.3 mm path length cuvette. Speci®c instructions are given for use with commercial instruments.

with dilute TCA. Exposure to dye solution is followed by a destaining solution to remove excess dye. The dyed protein spot is then excised and placed in a test tube with elution solvent, which dissolves the protein-dye complex. Absorbance measurements are then recorded as before. The advantages of the solid-phase assay include (a) increased sensitivity and (b) improved resistance to interfering compounds. In the best cases, an LLD of 0.25 mg is attainable with a linear range extending to 200 mg of protein. 3.1. Nitrocellulose and Cellulose Acetate Membranes

Kuno and Kihara (35) employed nitrocellulose membranes for solid-phase dye-binding assays.* The linear range for analysis was 10±50 mg protein. The ef®ciency of protein binding was >97%. Compared with the Lowry assay, there was increased resistance to interference from tyrosine or Tris. Heil and Zillig (36) analyzed protein (0.5±2.5 mg) using cellulose acetate membranes. Staining was with Amido Black 10B (0.24% w/w) dissolved in (10:45:45) acetic acid±methanol±water solvent. The same solvent was used for destaining. Protein spots were air dried, excised, and eluted with 0.5 mL of solvent (glacial acetic acid, formic acid, water, TCA).

Schaffner and Weissmann (37) treated protein samples with 10% (w/w) TCA and recovered the resulting precipitate with a Millipore (HAWP 025CO) membrane ®lter. The rest of their methodology is essentially as already described. The linear dynamic range was 5±30 mg with a sensitivity of 0.027 DA630mg 1BSA. The LLD was 1.5 mg + 5% for a sample volume

of 2 mL. The following compounds did not interfere: dextran (100 mg mL 1_{), polyethylene glycol (0.5 mg mL} 1_{), glycogen (0.5 mg mL} 1_),

RNA or DNA (30 mg mL 1_{), NaCl (>1 M), ammonium sulfate (2.5 M),}

magnesium chloride (>0.1 M), EDTA (>100 mM), 2-mecaptoethanol (>100 mM), SDS (1%), and sucrose (20%).

The Schaffner-Weissmann method was modi®ed for protein analysis in the presence of 1000-fold excess lipid (38).BSA (20 mg) was accurately determined in the presence of 20 mg phospholipid (43% phosphatidylcho- line, 30% phosphatidylethanolamine, 27% unidenti®ed lipids). The linear dynamic range (2±24 mg BSA) was unaffected by added phospholipid. Assay sensitivity was also unaffected by the presence of lipid. The A630was 0.744

(+ 0.054) and 0.775 (+ 0.048) for 20 mg of BSA without and with 20 mg of

* Protein (5±50 mg) dissolved in 0.2 M magnesium chloride was ®ltered with a nitrocellulose membrane under suction. Each membrane was stained with 2 mL of Amido Black 10B

(4 mg mL 1_{in acetic acid±methanol±water (1:5:4) solvent). After destaining with 5 mL of acetic}

acid (1% w/w), membranes were eluted with 3.5 mL of 10 mM NaOH. The released dye was

lipid, respectively. There was compatibility with a great many buffer salts (200 mM): NaCl, KCl, Na phosphate, K phosphate, HEPES, Tris-HCl, MES, MOPS. However, protein-protein variations in the dye response were evident. The proceding technique was apparently more accurate than the modi®ed Lowry assay or biuret method.

The speed for solid-phase analysis was increased by Nakamura and co-workers (39). They used micro®ltration apparatus* to apply multiple samples to nitrocellulose membranes. Stained protein spots were measured directly via a densitometer. The linear dynamic range for analysis was 1±10 mg. Sensitivity using Amido Black 10B staining was twofold higher than with Ponceau Red. For both dyes the order of increasing sensitivity was trypsin < lysozyme < cytochrome c < bovine serum albumin < human serum albumin < concanavalin A < histone II < human g-globulin. Assay results were unaffected at pH 3.6±9. There was no interference from a range of salts, sugars, amino acids, nucleotides, polyols or EDTA. Detergents (SDS, Triton X-100, Tweens) or high concentrations of denaturants reduced protein binding to the nitrocellulose membrane.

3.2. Whatman Paper and Glass Membrane Filters

Protein samples (5±200 mg) were dried on to Whatman No. 42 paper followed by treatment with 7.5% (w/v) TCA (40). McKnight (41) spotted 100 mL of protein (0.5±5 mg) on glass ®ber ®lters (Whatman GF/C), dried the liquid using hot air, and then ®xed the protein with 20% (w/v) TCA. Esen (42) and Almand and Saleemuddin (43) dried protein solutions (5 mL; 1±4 mg mL 1_{) on Whatman No. 1 with no TCA ®xation step. The ®lter}

paper±bound protein was stained with Amido Black 10B or Coomassie Brilliant Blue G250 (see Chapter 7).

4. THE CHEMISTRY OF DYE-BINDING PROTEIN ASSAYS