Protein concentrates native

Before protein molecules attain their native folded state they may expose hydrophobic patches to the solvent. Isolated purified proteins will aggregate during folding even at relatively low protein concentrations. Inside cells, where there are high concentrations of many different proteins, aggregation could therefore occur during the folding process. This is prevented by... 

Fig. 40. Far-UV CD spectra of /Flactamase from Bacillus cereus (A), horse apomyo-globin (B), and horse ferricytochrome c (C) as a function of HC1 concentration. Protein concentrations were 10 fiM. The numbers refer to the HC1 concentration (mM). The spectra of the native state (A), the A state induced by KC1, pH ss 2), (O) and GdmCl-unfolded state (4-5 M GdmCl, 25 mM phosphate buffer, pH 7.0) ( ) are shown for comparison. From Goto et al (1990a). 1990, with permission of the authors.

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

The extreme stability of amyloid and amyloid-like fibrils is difficult to understand in terms of the three classes of fibril models. For the Refolding models, it has been suggested that the amyloid conformation is a default conformation for a polypeptide chain (Dobson, 1999). However, these models do not give a clear indication of what types of interactions differ in the amyloid conformation versus the native conformation, and so it is unclear why the amyloid conformation should be more stable. Also, it seems that the elevated protein concentrations associated with fibril formation might disproportionately favor nonspecific aggregation of the destabilized intermediate over amyloid fibril formation. 

For immunoprecipitations from native tissues, one requires antibodies directed against both the fish and the bait proteins. Further, these antibodies should not bind to epitopes within the putative protein-protein BDs. It is technically difficult to determine low affinity or transient association among proteins by immunoprecipitation because low-affinity interactions may be lost by washing immune pellets to remove nonspecifically bound proteins. Also, one cannot manipulate protein concentrations to favor protein association as one can in a pull-down assay. Under these circumstances, probably the best method to use would be FRFT. 

Figure 3. DSC thermograms of native CBH I and of fragments produced by limited proteolysis. For native CBH I and the core fragment, protein concentrations were in the range from 0.91 to 1.31 mg/ml. The concentration of the tail fragment was 0.41 mg/mL. Scan rate was 30 C/hr. Each division on the ordinate represents 20,000 cal/mol/deg.

Enzymic Activity. Lysozyme activity was determined by following the rate of lysis of dried Micrococcus lysodeikticus cells according to the method of Shugar ( ). Assays were run at room temperature in O.IM phosphate buffer pH 7.0, with an enzyme concentration of about 0.05 mg/ml. A solution of native lysoz3mie at the same protein concentration was always assayed as standard, along with ozonized lysozymes. 

Protein concentrations were approximately 0.4%. Circular dichroism data were presented as mean residue ellipticity [0], in degrees X cm x decimole . The same mean residue weight 112.4 was employed for native lysozyme and its ozonized products, since the deviation caused by the ozonolysis of few amino acid residues of lysozyme is far less than the experimental error. 

Claussen, . C., Str0mmen,l.,Egelandsdal,B., Straetkvern, K. O. (2007). Effects ofdrying methods on functionality of a native potato protein concentrate. Drying Technology, 25, 1091-1098. 

Formation of protein gel structures can occur under conditions which disrupt the native protein structure provided that the protein concentration, thermodynamic conditions and other conditions are optimal for the formation of the tertiary matrix. The most Important food processing techniques relative to protein gelation Involve divalent cations (calcium) and/or heat treatment. 

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

Figure B3.5.5 Near-UV CD spectra. (A) Bovine a -casein peptide under a variety of conditions (data from Alaimo et al., 1999). Peptide concentration 0.631 mg/ml in 2 mM PIPES, 4 mM KCI, pH 6.75 scan rate 40 sec/nm path length 10 mm bandwidth 1.5 nm. The loss of aromatic dichroism with increasing temperature indicates denaturation, which is, however, not complete at 70°C or in 6 M guanidine hydrochloride. The shift in maximum wavelength indicates loss of tryptophan asymmetry, but less so of tyrosine. (B) Seed coat soybean peroxidase under native and denaturing conditions (data from Kamal and Behere, 2002). Protein concentration 15 pM and path length 10 mm. The negative aromatic band centered around 280 nm and the Soret band around 410 nm both disappear at 90°C, indicating the loss of net conformational asymmetry of the aromatic and heme chromophores.

Figure B3.6.11 The binding of retinol to p-lactoglobutin that has been denatured by exposure to high pressure. The sample contaiining 270 pM p-lactoglobulin was pressurized to 400 MPa for 15 min. After release of pressure, retinol in ethanol was added and fluorescence was measured as a function of time. Parameters final protein concentration 8.5 pM in 20 mM phosphate buffer . ex = 330 nm k9m = 470 nm. Circles, native p-lactoglobulin triangles, pressurized p-lactoglobulin. Reprinted from Ikeuchi et al. (2001) with permission from the American Chemical Society.

Figure 10.16 Effect of temperature on fluorescence intensity of native (A) and guanidine unfolded ( ) AEDANS-RNase. Intensities are expressed relative to that measured at 10°C for the same sample (native or unfolded). The buffer is 50 mM cacodylate, pH 6.5, in the absence or presence of 6 M guanidine hydrochloride. The protein concentration is 10 5 M. Excitation wavelength 350 nm emission wavelength 476 nm. Source Jullien, M., Garel, J-R., Merola, F. and Brochon J.-C. (1986). European Biophysical Journal, 13, 131-137, Figure No. 1. With kind permission of Springer Science and Business Media (1,2, and 3).

Fig. 39. Optical absorption spectra (room temperature) of native, type-2-depleted (T2D), T2D + 30-fold excess H202 (TT2D) and TT2D + 100-fold excess NJ laccase (pH = 6.0 protein concentration 1.5 mM) (from Ref. 104)...

The mitogenic activities of native (tetravalent) and succinylated or acetylated (bivalent) con A were compared.336 Whereas the protein concentration-dependent, DNA synthetic response of mouse spleno-cytes to native con A exhibited a sharp peak, succinyl con A stimulated DNA synthesis over a broad range of protein concentration.336 Mixing of native con A with its dimeric, succinylated derivative at pH 4.5 resulted in the formation of hybrid molecules. A dimer species consisting of equimolar amounts of native con A protomer and its succinyl derivative was isolated, and shown3481 to have a molecular weight of50,000 at both pH 5 and pH 7. 

Tolkach, A., and Kulozik, U. (2005). Optimization of thermal pretreatment conditions for the separation of native a-lactalbumin from whey protein concentrates by means of selective denaturation of p-lactoglobulin. J. Food Set., 70(9), 557-566. 

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