Question

you are asked to determine the protein content of ground beef, using a spectrophotometric method. Discuss your considerations to ensure sufficient protein extraction.

Answer 1

Ans.)

Materials and Methods
2.1. Chemicals and Reagents
All reagents used in the experiment were of analytical grade. Potassium chloride, disodium phosphate, monopotassium phosphate, urea, thiourea, dithiothreitol, cholamidopropyl dimethyl hydroxy propanesulfonate (CHAPS), IGEPAL® CA-630 NP 40, glycerol, and tris were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acrylamide, bis-acrylamide, ammonium persulfate (APS), N,N,N,N-Tetramethylethylenediamine (TEMED), sodium dodecyl sulfate (SDS), tris(hydroxymethyl)-aminomethane, glycine, bromophenol blue, β-mercaptoethanol, and Coomassie Brilliant Blue G-250 were purchased from Bio-Rad Laboratories (Hercules, CA).

Phosphate buffer (pH 7, 0.003 M), KCl phosphate buffer (pH 7.5), and Tris-HCl (pH 8, 20 mM) were freshly prepared. Ultrapure water was obtained in the laboratory using a Water Purification System Barnstead™ Pacific TII (ThermoFisher Scientific, USA).

2.2. Samples Collection and Preparation
Meat samples from longissimus thoracis muscle of beef and lamb; pectoralis major muscle of chicken; and dorsal white muscle of fish from sole (Solea solea), European hake (Merluccius merluccius), and sea bass (Dicentrarchus labrax) were purchased from a local market and immediately transferred under refrigeration to the laboratory. For each species, a total of fifteen animals were included in the experiment. Adipose and connective tissues were removed from meat samples, while bones, scales, and fat were discarded from fish samples. All fresh samples were finely minced prior to protein extractions.

2.3. Protein Extraction Methods
The flowchart of the extraction of muscle protein fractions from different species analyzed is shown in Figure 1. Meat and fish proteins were fractionated based on different solubility. Samples were homogenized with 0.03 M phosphate buffer (pH 7) containing a protease inhibitors cocktail (P2714, Sigma-Aldrich, St. Louis, MO) on ice for 2 min using an Ultra-Turrax T18 basic (IKA, Wilmington, Germany). The homogenate was centrifuged at 8,000 × (Eppendorf 5810R, Eppendorf AG, Hamburg, Germany) for 20 min at 4°C. After centrifugation, the supernatant (sarcoplasmic proteins) was discarded, and the extraction of myofibrillar proteins were obtained as follows.
Two different extraction methods were carried out for myofibrillar proteins using denaturing and nondenaturing solutions. The extraction of myofibrillar proteins with non-denaturing solution is based on the method reported by Hashimoto et al. [10] with the modifications reported as follows: the pellet recovered was resuspended in 10 volumes of KCl phosphate buffer pH 7.5 (0.45 M KCl, 15.6 mM Na2HPO4, 3.5 mM KH2PO4) and vortexed for 2 min. The vortexing step was introduced to optimize the homogenization of the pellet and to prevent the formation of a mellow complex. The mixture was centrifuged twice at 5,000 × (Eppendorf 5810R, Eppendorf AG, Germany) for 15 min at 4°C. After centrifugation, the supernatant containing the myofibrillar proteins was recovered, aliquoted, and frozen at −80°C.

For comparison, myofibrillar proteins were extracted using denaturing solution according to Marino et al. [11]. Briefly, the pellet was resuspended in a solution (8.3 M urea, 2 M thiourea, 64 mM dithiothreitol (DTT), CHAPS 2% (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate), IGEPAL 2%, glycerol 10%, and 20 mM Tris-HCl, pH 8) and incubated overnight at 4°C in an orbital shaker. Subsequently, samples were centrifuged at 15,000 × (Eppendorf 5810R, Eppendorf AG, Germany) for 20 min at 10°C. After centrifugation, the supernatant containing myofibrillar proteins was recovered, aliquoted, and frozen at −80°C until further protein analysis to avoid calpain protease activation.

For each species, all myofibrillar extracts obtained with the different methods were quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Absorbance was measured at 580 nm by the spectrophotometric assay (Power Wave XS, Biotek, UK), with a bovine serum albumin (BSA; >98% pure, Sigma-Aldrich) standard curve.

2.4. SDS-PAGE Analysis
The fifteen myofibrillar extracts of each species obtained by the denaturing or nondenaturing method were pooled and resolved by SDS-polyacrylamide gel electrophoresis in a gradient gel 8–18% [11]. Gels were loaded with 50 μg of proteins and run with a Protean II xi vertical slab gel unit (Bio-Rad Laboratories, Hercules, CA). Coomassie Brilliant Blue G-250 was used to visualize bands of interest. Gels were destained in an aqueous solution of acetic acid and methanol (10% v/v, and 7% v/v, respectively) and acquired by the ChemiDoc EQ system (Bio-Rad Laboratories, Hercules, CA). The relative quantity of each band was determined as percentage of the signal intensity of the defined band in a lane with the Quantity One software (Bio-Rad Laboratories, Hercules, CA). Identification of the protein molecular weight was done by comparison with the precision plus protein standard-broad range (Bio-Rad Laboratories, Hercules, CA).

2.5. Statistical Analysis
Protein concentration and electrophoretic data were analyzed using the GLM procedure of the SAS statistical software [12]. The tested effect was the extraction methods on the myofibrillar fraction of muscle proteins from beef, lamb, chicken, sole, hake, and sea bass. When significant differences were found (at ), the Student t-test was used to locate significant differences among means.

3. Results and Discussion
3.1. Protein Extractability
Solubility is an indicator of protein extractability; indeed, a solubilized protein could be easily extracted into a solution from muscle fibers or myofibrils [9]. The amount of myofibrillar proteins extracted using denaturing and nondenaturing solutions from beef, lamb, chicken, sole, European hake, and sea bass is reported in Figure 2. No differences were found in the protein extractability of beef, European hake, and sea bass when the different extraction methods were tested, evidencing that nondenaturing extraction method led to successful protein extraction as denaturing extraction method. The physical force applied by repeated centrifugations damaged the structures of myofibrillar proteins partly allowing the dissolution of myofibrillar proteins in water.