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Am. J. Enol. Vitic. 56:2:182-187 (2005)
Copyright © 2005 by the American Society for Enology and Viticulture.
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Technical Brief

Development of a New Procedure for Protein Recovery and Quantification in Wine

Simone Vincenzi1, Silvia Mosconi2, Gianni Zoccatelli2, Chiara Dalla Pellegrina2, Gianluca Veneri2, Roberto Chignola2, Angelo Peruffo2, Andrea Curioni1 and Corrado Rizzi2,*

1 Dipartimento di Biotecnologie Agrarie, Università di Padova, Viale dell’Università 16, 35020 Legnaro (Padova) Italy; 2 Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15–CV1, 37134, Verona, Italy.

* Corresponding author [Fax: 39 045 8027952; email: corrado.rizzi{at}univr.it];


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 Conclusion
 Literature Cited
 
In order to develop a procedure for protein recovery and quantification in wines, known amounts of bovine serum albumin (BSA) were added to samples of protein-free wine. Dialysis followed by lyophilization, precipitations with ethanol, acetone, trichloroacetic acid, and by a new method that precipitates proteins as protein-potassium dodecyl sulfate complexes (KDS method) were used to recover BSA from protein-free wine. Recovered BSA was then quantified by three colorimetric assays: Bradford, Lowry, and Smith. The KDS method followed by the Smith assay obtained the best match between actual and measured BSA quantities. When this procedure was used for quantification of wine proteins, the results were in accordance to those determined by densitometric quantification of the protein bands separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), indicating that the proposed method allows a reliable determination of the protein content of wine.

Key words: wine proteins, protein recovery, protein quantification, electrophoresis

Proteins have considerable technological importance since they affect the stability and the sensory quality of wines. Various methods for the quantification of proteins in wines have been described in the literature (Moreno-Arribas et al. 2002). However, an extreme variability of protein content, ranging from less than 1.0 mg/L to greater than 1.0 g/L, has been reported for the same wine type (Marchal et al. 1997). This variability may be due to the many compounds that can interfere with the protein measurements (Moreno-Arribas et al. 2002). The most widely used procedure to quantify wine proteins is based on the Bradford assay (Marchal et al. 1997). Waters et al. (1991) observed that this method, based on Coomassie dye-protein binding, was unsuitable for quantification of purified wine protein fractions because it can underestimate the true values by 50 to 80%. Specific interferences with the Coomassie dye by alcohol and phenols during wine protein quantification have been established (Marchal et al. 1997).

A rational approach to eliminate the interfering compounds might be to separate the proteins from the wine before proceeding with quantification, which has been done by exploiting differences in molecular size (for example, gel filtration, dialysis, or ultrafiltration) or sensitivity to certain precipitating agents such as organic solvents, trichloroacetic acid, ammonium sulfate, sulfosalicylic acid, or phosphotungstic acid (Moreno-Arribas et al. 2002). However, the presence of interfering compounds cannot be excluded. Such compounds may be associated with the proteins and/or can become insoluble in the presence of protein-precipitating agents.

Because of their low concentration in wine, proteins must be recovered in order to carry out electrophoretic analyses. Depending on the selected procedures, modification and/or degradation of wine proteins may occur (Hsu and Heatherbell 1987). In addition, most procedures for protein recovery from wine are time-consuming and cumbersome, mainly when several samples have to be analyzed simultaneously.

The potassium dodecyl sulfate (KDS) method described in this paper, which was previously developed to recover sodium dodecyl sulfate (SDS)-denaturated muscle proteins (Carraro et al. 1994, Sandri et al. 1992) in highly diluted samples, allows for rapid protein precipitation from wine by consecutive addition of SDS and potassium chloride (KCl). The KDS-protein complexes so recovered can be precisely quantified by the Smith assay (Smith et al. 1985) or loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), allowing for clear and reproducible electrophoretic patterns.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 Conclusion
 Literature Cited
 
 Wine samples.   One sample each of Incrocio Manzoni 6.0.13 and Prosecco white wines (typical wines of the Conegliano region) were collected before and after bentonite fining. Incrocio Manzoni, Prosecco, Sauvignon blanc, and Chardonnay (white wines) and Bordeaux and Valpolicella (red wines) were also purchased at a local market.

 Preparation of BSA in protein-free wine.   All wine samples were subjected to ultrafiltration to remove endogenous proteins. The liquid eluted from the ultrafiltration chamber (1 kDa cut-off) (protein-free wine) was collected and used to prepare standard solutions containing different amounts of BSA (50 to 200 µg/mL).

 Standard methods of protein recovery.   Protein precipitation with ethanol, acetone, or trichloroacetic acid (TCA) was performed at 0°C by adding three volumes of the organic solvents or one volume of 20% TCA solution to 1.0 mL of wine samples. After 30 min, samples were centrifuged at 14,000 g for 15 min at 4°C. The pellets obtained with ethanol and acetone were dried at 37°C, whereas those of the TCA-treated samples were washed once with acetone and then dried. Protein recovery by ultrafiltration was carried out on 1.0 kDa cut-off Amicon membrane (Millipore, Milano, Italy). Retentates were exhaustively dialyzed against distilled water in a dialysis bag with a 3.5 kDa cut-off membrane, frozen, and lyophilized.

 Protein recovery by KDS.   Protein precipitation by the potassium dodecyl sulfate (KDS) method was performed as described previously (Carraro et al. 1994, Sandri et al. 1992). However, to adapt the method to wine proteins, different experimental conditions were assayed using a sample of Incrocio Manzoni wine. Sodium dodecyl sulfate (SDS) (Bio-Rad, Milano, Italy) from a 10% stock solution was added to wine to final concentrations ranging from 0.025 to 0.2% under vigorous vortexing. Samples were gently mixed for 60 min at room temperature or heated in a boiling water bath for 5 min. Potassium chloride (KCl) (1 M) was then added to reach final concentrations ranging from 25 to 400 mM. Samples were gently mixed for further 30 min, and KDS-protein pellets were recovered by centrifugation at 14,000 g for 15 min at 4°C. Pellets were used for SDS-PAGE analysis or washed with 1 M KCl for protein quantification.

 Electrophoretic and densitometric analyses.   Electrophoretic analyses were performed by SDS-PAGE according to Laemmli (1970). Protein recovered from 1.0 mL of Incrocio Manzoni wine by the procedures described above were solubilized with 200 µL of 62.5 mM Tris HCl buffer pH 6.8, containing 5% (w/v) 2-mercaptoethanol, 1.3% (w/v) SDS, and 10% (w/v) glycerol. Samples were then heated at 100°C for 5 min and 30 µL loaded onto SDS-14% PAGE. Two BSA standards (2 and 4 µg), solubilized as described above, were also electrophoresed in parallel. SDS-PAGE was carried out in a Mini Protean III apparatus (Bio-Rad) at 35 mA until the tracking dye bromophenol blue ran off the gel. Gels were stained with 0.05% (w/v) Coomassie Brilliant Blue R-250, 5% (w/v) TCA, 17% (v/v) methanol, and 6% (v/v) acetic acid, and destained in 7% (v/v) acetic acid.

Densitometric titration curves were obtained using data from five different gels in which increasing amounts of BSA (1 to 4 µg) were loaded. All experimental conditions, including staining (16 hr) and destaining (6 hr) steps, were the same as those used for wine protein analysis. Digitalized images of the SDS-PAGE patterns were acquired with a Gel Doc 2000 apparatus (Bio-Rad) and analyzed with Scion Image software (Frederick, MD).

 Colorimetric quantification of recovered proteins.   Proteins were recovered either from BSA containing protein-free wines or from wine samples. For protein quantification in solution, the pellets obtained through acetone and TCA precipitation were washed three times with acetone; those obtained through ethanol precipitation were washed with ethanol. In all cases, pellets were then resolubilized in 1.0 mL of distilled water. The following colorimetric assays were carried out following manufacturers’ procedures: Bradford (Bradford 1976), with the Bio-Rad Protein Assay kit in the microassay format; Lowry (Lowry et al. 1951), with the Bio-Rad DC Protein Assay kit in the microassay format; and Smith (Smith et al. 1985), with the Micro BCA Protein Assay kit (Pierce, Rockford, IL). Calibration curves were obtained by using known concentrations of BSA dissolved in distilled water. Each experiment was repeated at least five times and given measures are the average of three replicates.


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
Conclusion
 Literature Cited
 
A major difficulty in the validation of protein recovery and quantification in wine is that the exact amount of proteins in a given wine sample cannot be known beforehand. This problem was circumvented by using protein-free wine samples to which known amounts of BSA were added. These standard protein-containing wine samples have then been subjected to different combinations of protein recovery and quantitation methods to produce reliable results in typical wines.

 Wine protein recovery by KDS.   The KDS method involves the complexation of proteins with the detergent dodecyl sulfate (DS) (added as Na-DS) and insolubilization of the protein-DS complexes by addition of potassium ions (added as KCl) (Carraro et al. 1994, Sandri et al. 1992). Optimal experimental conditions were established using Incrocio Manzoni wine treated with varying SDS and KCl concentrations. The resulting precipitated proteins were quantified by the Smith assay. The obtained results indicated that concentrations of KCl greater than 200 mM in the samples with a SDS concentration >0.1% gave the best results in terms of protein precipitation (Figure 1Go). However, at the highest SDS and KCl concentrations used (0.2% SDS and 400 mM KCl), the obtained protein pellets did not pack properly and showed poor electrophoretic patterns when separated by SDS-PAGE (not shown). In contrast to what was found when the original protocol was developed (Carraro et al. 1994, Sandri et al. 1992), acidification of the SDS-treated sample did not improve protein precipitation (data not shown), which is probably due to the effect of the acidic pH conditions of the wine.



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  		Figure 1 Protein recovery from 250 µL of Incrocio Manzoni by the KDS procedure as a function of SDS and KCl concentrations. Precipitated protein was quantified by the Smith assay after resolubilization in 500 µL of distilled water.

 
 Colorimetric quantification of BSA.   Three colorimetric assays commonly used for protein measurements were evaluated for their ability to quantitate wine proteins using known concentrations of BSA dissolved in protein-free Incrocio Manzoni wine. These standard solutions were considered a good model for proteins in wine because BSA is similar to wine proteins in terms of functional properties (Dupin et al. 2000, Waters et al. 1991). BSA was recovered from the wine by the different methods and, after resolubilization, quantified by Bradford, Lowry, and Smith assays. The effectiveness of the different recovery and quantification procedures was evaluated by comparing the measured BSA concentrations with that actually present in the solutions.

The Bradford assay failed to provide correct estimates of BSA recovered from protein-free wine irrespective of the recovery procedure (Figure 2, A–EGo). The high background observed when the Bradford assay was applied to the BSA sample obtained by the KDS method may be due to an interference of the precipitating agent itself. Both KCl and SDS are claimed to interfere with the Bradford assay (Bio-Rad Protein Assay instruction manual). On the contrary, the low absorbance values obtained with the other recovery methods may be related to the presence of phenolics coprecipitated with the protein (Marchal et al. 1997). Although the Smith assay gave an overestimation of the BSA concentration in most cases (Figure 2, K, M, NGo), very high accuracy (that is, providing values that almost match the true ones) was observed when BSA was recovered by the KDS method (Figure 2, LGo).



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  		Figure 2 Concentration of BSA (expressed as µg/mL on y axes) measured with Bradford (A–E), Lowry (F–J), and Smith (K–O) assays versus that dissolved in protein-free wine (expressed as µg/mL on x axes). BSA was recovered by dialysis followed by lyophilization (A, F, K), by KDS (B, G, L), and by precipitation with acetone (C, H, M), ethanol (D, I, N), and TCA (E, J, O). In each panel, data (mean ± SD of eight replicates) were fitted with a first-order polynomial (solid line) and estimated parameters b(0) (intercept of solid line with y axis), b(1) (slope of solid line), and R2 are given. Dashed lines indicate theoretical dose/response values.

 
Overestimation of BSA was also observed with the Lowry quantification assay (Figure 2, F, H, IGo), although to a lesser extent than with the Smith assay. These results cannot be explained in terms of physicochemical associations between interfering compounds and proteins because the calculated protein quantity for the sample without added BSA was greater than zero. The compounds interfering with both the Smith and the Lowry assays seemed to be precipitated by both ethanol and acetone (Figure 2, H, I, N, MGo), suggesting that the wine interferents might be insoluble in organic solvents. More difficult to interpret are the results obtained with the Lowry and the Smith assays for the samples prepared by dialysis followed by lyophilization (Figure 2, F, KGo). The results might be due to interfering or contaminating substances in the protein-free wine. Deproteinization by ultrafiltration was carried out on filters with a 1.0 kDa cut-off and the resulting samples were dialyzed on 3.5 kDa cut-off membranes. The aggregation of small interferring compounds after ultrafiltration may explain the data shown at 0 BSA concentration (Figure 2, F, KGo).

In conclusion, results show that the best coupling between BSA recovery methods and protein quantification assays was the KDS method, followed by the Smith assay.

 KDS method/Smith assay.   The data shown above were obtained using Incrocio Manzoni protein-free wine samples to which known amounts of BSA were added and demonstrate that low molecular weight compounds can interfere with either the recovery and/or the quantification methods. To investigate the accuracy of the KDS method/ Smith assay with other wine types, the procedure was applied on various white (Incrocio Manzoni, Prosecco, Chardonnay, and Sauvignon blanc) and red (Bordeaux and Valpolicella) protein-free wines. Results (Figure 3Go) indicate that the KDS method/Smith assay is also accurate with these wine samples.



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  		Figure 3 Concentration of BSA (expressed as µg/mL on y axes) measured with the Smith assay versus that dissolved in different protein-free wines (expressed as µg/mL on x axes). BSA was recovered by the KDS method. Wines were prepared from different commercial bottled wines (A: Incrocio Manzoni 6.0.13; B–E: Prosecco; F: Chardonnay; G: Bordeaux; H: Valpolicella; I: Sauvignon blanc). In each panel, data (mean ± SD of eight replicates) were fitted with a first-order polynomial (solid line) and estimated parameters b(0) (intercept of solid line with y axis), b(1) (slope of solid line), and R2 are given. Dashed lines indicate theoretical dose/response values.

 
 Determination of wine protein content.   The procedures of protein recovery and quantification have been studied thus far on model wines (that is, protein-free wines with BSA). The KDS method coupled to the Smith assay was then applied to the protein determination of several commercial samples of Incrocio Manzoni and Prosecco wines and to the same wines taken before bentonite fining (Table 1Go). In the latter samples, the measured protein concentration was higher than that of bottled wines, with the exception of one bottled sample containing 176.1 µg/mL of protein. Bottles of this sample were found to be hazy after about one year from bottling.


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  		Table 1 Quantification by the Smith assay of protein recovered by the KDS method from Prosecco and Incrocio Manzoni 6.0.13 wine samples.
 
An independent measure of the protein content in Incrocio Manzoni sample no. 14 was obtained by densitometry of SDS-PAGE gels to validate the data of Table 1Go. SDS-PAGE is suitable to obtain a measure of protein content because the strong denaturing conditions allow the removal of the compounds noncovalently bound to proteins (such as phenolics) that might interfere with the Coomassie dye staining (Marchal et al. 1997).

Densitometric analysis of wine proteins using BSA standards (1 to 4 µg) showed strong linearity (r2 = 0.99). On this basis, two BSA standards (1 and 2 µg), electrophoresed in the same gel in which the wine proteins were fractionated (Figure 4Go), allowed us to quantify wine proteins. All procedures for protein recovery from Incrocio Manzoni wine gave similar results, the average amount of protein corresponding to 27.65 ± 2.81 µg/mL (Table 2Go). The exception was the TCA-precipitated protein sample, which gave an electrophoretic pattern (Figure 4Go, lane 7) of lower intensity and protein amount (Table 2Go). This result can be attributed to the strong denaturing effect of TCA, which can inhibit protein solubilization in the SDS-PAGE loading buffer. Consequently, the result obtained for the TCA-precipitated sample was not considered in calculating the mean reported in Table 2Go.



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  		Figure 4 SDS-PAGE analysis of Incrocio Manzoni proteins: (lane 1) BSA standard 4 µg, (2) BSA standard 2 µg; proteins recovered by (3) dialysis followed by lyophilization, (4) by KDS method, and by precipitation with (5) acetone, (6) ethanol, and (7) TCA; lane M: molecular weight standard proteins (kDa).

 

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  		Table 2 Quantification of the proteins recovered from Incrocio Manzoni wine with different methods by densitometry of SDS-PAGE gels and by Smith assay.
 
This mean value was further used to validate the accuracy of the Smith assay conducted on wine proteins recovered through the different methods from the Incrocio Manzoni wine sample no.14 in Table 1Go (Table 2Go). Quantification of KDS-precipitated proteins shows the best fit with the densitometric data over other recovery methods, indicating that the method removes a majority of the Smith assay interfering substances.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results and Discussion
 Conclusion
Literature Cited
 
The KDS procedure coupled to the Smith assay is simple and effective in determining protein content in wines. Moreover, the KDS procedure can be used to easily prepare wine protein samples for clear and reproducible electrophoretic patterns from SDS-PAGE.


   Footnotes
 
Acknowledgments: This study was supported by grants from the Università di Padova (Progetto di Ateneo Università di Padova 2002) and from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST 60% to C.R). This work is part of the research activities of the Dottorato in Viticoltura, Enologia e Marketing delle Imprese Vitivinicole, supported by the Provincia di Treviso. The authors acknowledge Marzio Pol for wine samples.

Manuscript submitted June 2004; revised October, December 2004


   Literature Cited
Top
 Abstract
 Materials and Methods
 Results and Discussion
 Conclusion
 Literature Cited
 
Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.[ISI][Medline]

Carraro, U., C. Rizzi, M. Sandri, and D. Doria. 1994. A new two-step precipitation method removes free-SDS and thiol reagents from diluted solutions and then allows recovery and quantitation of proteins. Biochem. Biophys. Res. Commun. 200:916–924.[ISI][Medline]

Dupin, I.V., V.J. Stockdale, P.J. Williams, G.P. Jones, A.J. Markides, and E.J. Waters. 2000. Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: Evaluation of extraction methods and immunolocalization. J. Agric. Food Chem. 48:1086–1095.[ISI][Medline]

Hsu, J.C., and D.A. Heatherbell. 1987. Isolation and characterization of soluble proteins in grapes, grape juice, and wine. Am. J. Enol. Vitic. 38:6–10.[Abstract/Free Full Text]

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.[Medline]

Lowry, O.H., J. Nira, A. Rosenbrough, L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.[Free Full Text]

Marchal, R., V. Seguin, and A. Maujean. 1997. Quantification of interferences in the direct measurement of proteins in wines from the Champagne region using the Bradford method. Am. J. Enol. Vitic. 48:303–309.[Abstract/Free Full Text]

Moreno-Arribas, M.V., E. Pueyo, and M.C. Polo. 2002. Analytical methods for the characterization of proteins and peptides in wines. Anal. Chim. Acta. 458:63–75.

Sandri, M., C. Rizzi, C. Catani, and U. Carraro. 1992. Small and large scale preparative purification of myosin light and heavy chains by selective KDS precipitation of myosin subunits: Yield by SDS PAGE and quantitative orthogonal densitometry. Basic Appl. Myol. 2:107–114.

Smith, P.K., R.I. Krohn, G.H. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goike, B.J. Olson, and D.K. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76–85.[ISI][Medline]

Waters, E.J., W. Wallace, and P.J. Williams. 1991. Heat haze characteristics of fractionated wine proteins. Am. J. Enol. Vitic. 42:123–127.[Abstract/Free Full Text]





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