Protein concentrates modified

Tran, T.H., Von Moltke, L.L., Venkatakrishnan, K., Granda, B.W., Gibbs, M.A., Obach, R.S., Harmatz, J.S. and Greenblatt, D.J. (2002) Microsomal protein concentration modifies the apparent inhibitory potency of CYP3A inhibitors. Drug Metabolism and Disposition, 30 (12), 1441-1445. 

Fig. 17. Cyclic voltammogram of the water-soluble Rieske fragment from the bci complex of Paracoccus denitrificans (ISFpd) at the nitric acid modified glassy carbon electrode. Protein concentration, 1 mg/ml in 50 mM NaCl, 10 mM MOPS, 5 mM EPPS, pH 7.3 T, 25°C scan rate, 10 mV/s. The cathodic (reducing branch, 7 < 0) and anodic (oxidizing branch, 7 > 0) peak potentisds Emd the resulting midpoint potential are indicated. SHE, standEU d hydrogen electrode.

Prepare the protein to be modified in a non-amine-containing buffer at a slightly basic pH (i.e., avoid Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the protein in the reaction buffer may vary from pg/ml to mg/ml, but highly dilute solutions will result in less efficient modification yields. A protein concentration from 1 to 10 mg/ml works well in this reaction. 

Reactions done with NHS-PEG -biotin compounds typically are done with the reagent in molar excess over the amount of protein being modified. The efficiency of the reaction is dependent on the concentrations of reactants and the solvent exposed area of the amine groups on the protein. Reactions done with a 10-fold molar excess of NHS-PEG -biotin usually will result in at least 2-3 biotin labels per protein, while doubling the molar excess should provide 4-6 biotinylations. The optimal number of biotin groups added to a particular protein should be determined experimentally to provide the best performance in the intended application. 

Of the large number of protein interactions that take place in cells, perhaps the vast majority may be described as transient. Most proteins that modify other molecules do so very rapidly and so interact only briefly with their substrates or binding partners (i.e., enzymes). In addition, since proteins within cells are highly compartmentalized, the affinity of most interactions doesn t have to be very great, because each potential binding partner is within short diffusion distances and the relative concentration of molecules within these small volumes is high. 

Soya Proteins. Early attempts to make albumen substitutes from soya protein also ran into problems. A bean flavour tended to appear in the finished product. A solution to these problems has been found. Whipping agents based on enzyme modified soy proteins are now available. The advantage of enzymatic modification is that by appropriate choice of enzymes the protein can be modified in a very controlled way. Chemical treatment would be far less specific. In making these materials the manufacturer has control of the substrate and the enzyme, allowing the final product to be almost made to order. The substrates used are oil-free soy flakes or flour or soy protein concentrate or isolate. The enzymes to use are chosen from a combination of pepsin, papain, ficin, trypsin or bacterial proteases. The substrate will be treated with one or more enzymes under carefully controlled conditions. The finished product is then spray dried. 

Whole oilseeds and legumes and their derivatives (defatted flours, and protein concentrates and isolates) are used in traditional foods as sources of protein and for their texture-modifying functions. This article reviews, on a comparative basis, processes for preparation of vegetable food proteins, compositions and characteristics of the resulting food ingredients, and their functionalities and uses in traditional foods. 

Using NaDOC, the protein concentration precipitable by TCA is below 1 pg/ml. The precipitation protocol is modified as follows Add 0.1 ml Soln. C per milliliter to a sample. Mix and incubate at RT for 10 min. Add 0.1 ml of Soln. A, cool to 0 °C and continue as described above. In the presence of SDS the NaDOC-TCA precipitation does not work. 

Figure 9. Effect of pH on emulsifying capacity of native and chemically modified (succinylation, acetylation) sunflower seed protein concentrates (47)...

Considerable effort has been devoted to the improvement of functional properties of fish protein concentrate. Spinelli et al. (44) conducted a study to determine the feasibility of modifying myofibrillar fish proteins by partially hydrolyzing them... 

Even though liquid whey has been successfully commercialized in the form of alcoholic and nonalcoholic beverages, these are still a rarity in most countries. Most whey is converted to whey solids as ingredients for human food or animal feeds by traditional processes such as spray drying, roller drying, concentration to semisolid feed blocks, or production of sweetened condensed whey. Jelen (1979) reported other traditionally established processes including lactose crystallization from untreated or modified whey, production of heat-denatured whey protein concentrate, or recovery of milk fat from whey cheese in whey butter. ... 

Figure B1.1.2 shows the color response curves obtained with the Modified Lowry Protein Assay Reagent using BSA and BGG. The graph shows the net absorbance at 750 nm versus the protein concentration at seven concentrations from 125 to 2000 pg/ml for each protein. Note that the response curve for BGG is higher than the response curve for BSA.

Immediately purify the maleimide-activated avidin or streptavidin away from excess cross-linker and reaction by-products by gel filtration on a desalting column (Sephadex G-25 or the equivalent). Use 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, as the chromatography buffer. Pool the fractions containing protein (the first peak eluting from the column). After elution, adjust the protein concentration to 10 mg/ml for the conjugation reaction (centrifugal concentrators work well for this step). At this point, the maleimide-activated avidin may be frozen and lyophilized to preserve its maleimide activity. The modified pro-... 

Protein concentrations were determined according to the method of Lowry et al. (30). Electrophoresis of proteins in polyacrylamide gels was carried out at 4°C, using the discontinuous buffer system No. 1 described by Maurer (31) and modified by Emert et al. (1). Protein was stained with 0.1% Coomassie Brilliant Blue R250 in a water-acetic acid-methanol (45 10 45) solution. Carbohydrates were stained with the periodic acid-Schiff (PAS) reagent using the method described by Lang (32). 

Two other findings from the MCAT experiments have relevance to achieving total biosynthesis in vitro. Investigations into the effects of varying protein concentrations on polyketide production demonstrated that the optimal composition of a minimal PKS is not stoichiometric in each of the components (KS, CLF, and ACP) [30], Increasing the proportion of the ACP relative to the KS/CLF pair caused an enhancement in turnover even at more than 100-fold excess of ACP, there appeared to be no indication of saturation. This result has important implications for the composition of efficient one-pot biosynthetic assemblies. The experiments also illustrated that domains can be usefully engineered to improve yields. In this case, the ACP was modified to prevent its inactivation by formation of an internal disulfide between its active thiol and a remote cysteine. Optimizing... 

Figure 2.8 Empirical relationship between the content of myoglobin and the content of FABP in different areas of the human heart and in various types of skeletal muscles. Both protein concentrations are expressed in terms of mg protein/g wet weight (ww) of tissue. (Modified from Van Nieuwenhoven et al., 1995.)...

An important issue is whether the structure of the adsorbed protein is modified by the addition of electrolyte. The electrolyte is added only at the beginning of the light scattering experiment,1 and one might expect that during the experiment (about 2 min), and at low electrolyte concentrations, not much modification in the structure of the adsorbed layer can occur. This expectation, however, is probably not accurate. The strong specific ionic effects... 

This modified Scatchard Plot is used if the protein concentration is unknown (Rosenthal 1967). 

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