Measurement of Protein Hydrophobicity

36 mM cis-parinaric acid (CPA) in absolute ethanol containing 3.6 mM butylated hydroxytoluene (BHT) 

Specific buffer conditions (pH and salt) are selectedfor the environment of the food sample to be simulated. Generally, pH 7.0 and 0 NaCl are appropriate. 

Prepare duplicate 2-ml aliquots of each protein concentration. 

Add 10 pi ANS or CPA fluorescent probe to one tube at each concentration, and add 10 pi of the solvent used to prepare the probe solution to the other tube as a control (blank). 

Standardize the spectrofluorometer by adjusting the reading for equivalent concentrations of ANS in methanol or CPA in decane to an arbitrary value, such as 70% as the full scale. 

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B5.2 Measurement of Protein Hydrophobicity B5.3 Water Retention Properties of Solid Foods... 

The hydrophobic character of proteins and peptides depends primarily on their amino acid composition, i.e., the relative amount and lipophilicity of nonpolar residues in the molecule. A measurement of protein hydrophobicity (average hydrophobicity HOave) can be derived theoretically as the average of the individual A/t values based on the molar amino acid composition, where A/, is the change in free energy for the transfer of one mole of amino acid from ethanol to water (63-65). The average hydrophobicity values of the principal source proteins of cosmetic interest are listed in Table 3. A different way to express protein hydrophobicity is the so-called surface hydrophobicity, an entity measured experimentally on proteins by the adsorption of a chemical compound (cis-... 

Nakai, S Li-Chan, E and Arteaga. G.E. 1996. Measurement of surface hydrophobicity. In Methods of Testing Protein Functionality (G.M. Hall, ed.) pp. 226-259. Blackie Academic and Professional, London. 

Comparison of protein hydrophobicity measured using different methods is tabulated in Nakai et al. (1996). 

Protein content, particularly in urine or cerebrospinal fluid, may also be estimated by methods based on precipitation using sulfosalicylic acid (an anionic protein precipitant) or heat. The turbidity, which is a measure of protein concentration, can be quantitated by spectropho-tometric absorbance methods or light scattering analysis. Absorbance of a hydrophobic indicator dye that binds to protein and changes color is also used. 

A second analytical measurement of protein purity, which should be conducted, is HPLC analysis. Various chromatography columns can be utilized to verify the purity of the protein. The most commonly employed methods are ion exchange chromatography, molecular sieve chromatography (also known as gel permeation chromatography), and hydrophobic interaction chromatography (HlC). Each of these techniques probe a different chemical aspect of the protein and provide excellent independent check of purity and homogeneity. 

Abstract We discuss recent experiments and theories concerning protein collapse and folding. Experiments using multicomponent solutions have revealed much about the mechanism of folding. Simulation and theory have been used to interpret thermodynamic and fluorescence correlation spectroscopy experimental results. We consider the theoretical arguments using variations of the free energy with respect to fluctuations in number and composition to consider recent experiments. We find new measures of protein stability tendencies offer a different view than the often poorly defined hydrophobic effect. 

In an extensive SFA study of protein receptor-ligand interactions, Leckband and co-workers [114] showed the importance of electrostatic, dispersion, steric, and hydrophobic forces in mediating the strong streptavidin-biotin interaction. Israelachvili and co-workers [66, 115] have measured the Hamaker constant for the dispersion interaction between phospholipid bilayers and find A = 7.5 1.5 X 10 erg in water. 

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

To conclude, a strong correlation was found to exist between the net charge of the proteins in solution, the net charge of the SUM surface, and the extent of protein adsorption, which was expressed in terms of flux losses measured after filtration of the different protein solutions. Moreover, in the case of charge-neutral SUMs, flux losses increased with the hydrophobicity of the nucleophiles bound to the S-layer lattice. All proteins caused higher flux losses on SUMs modified with HDA than on those modified with GME or... 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

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