Average Surface Hydrophobicity Research Articles

Digestion-resistant whey peptides promote antioxidant effect on Caco-2 cells

Enteric endothelial cells are the first structure to come in contact with digested food and may suffer oxidative damage by innumerous exogenous factors. Although peptides derived from whey digestion have presented antioxidant potential, little is known regarding antioxidant pathways activation in Caco-2 cell line model. Hence, we evaluated the ability to form whey peptides resistant to simulated gastrointestinal digestive processes, with potential antioxidant activity on gastrointestinal cells and associated with sequence structure and activity. Using the INFOGEST method of simulated static digestion, we achieved 35.2% proteolysis, with formation of peptides of low molecular mass (<600 Da) evaluated by FPLC. The digestion-resistant peptides showed a high proportion of hydrophobic and acidic amino acids, but with average surface hydrophobicity. We identified 24 peptide sequences, mainly originated from β-lactoglobulin, that exhibit various bioactivities. Structurally, the sequenced peptides predominantly contained the amino acids lysine and valine in the N-terminal region, and tyrosine in the C-terminal region, which are known to exhibit antioxidant properties. The antioxidant activity of the peptide digests was on average twice as potent as that of the protein isolates for the same concentration, as evaluated by ABTS, DPPH and ORAC. Evaluation of biological activity in Caco-2 intestinal cells, stimulated with hydrogen peroxide, showed that they attenuated the production of reactive oxygen species and prevented GSH reduction and SOD activity increase. Caco-2 cells were not responsive to nitric oxide secretion. This study suggests that whey peptides formed during gastric digestion exhibit biological antioxidant activity, without the need for previously hydrolysis with exogenous enzymes for supplement application. The study’s primary contribution was demonstrating the antioxidant activity of whey peptides in maintaining the gastrointestinal epithelial cells, potentially preventing oxidative stress that affects the digestive system.

Changes in myofibrillar structure of silver carp (Hypophthalmichthys molitrix) as affected by endogenous proteolysis under acidic condition

SummaryEffect of endogenous proteolysis on myofibrillar conformational changes under acidic condition was investigated by spectroscopic techniques. The result of intrinsic tryptophan fluorescence showed that tryptophan residues were exposed to polar environment by acid denaturation of protein and participation of endogenous proteolysis. Extrinsic fluorescence indicated that average surface hydrophobicity (ShANS) decreased by reduction of pH but increased by endogenous proteolysis. Second‐derivative UV‐spectroscopy implied that the decrease of ShANS under acidic condition may be due to burying of some aromatic amino acid residues into hydrophobic cores induced by acid‐induced aggregation, such as tyrosine, while ShANS increased by exposure of some hydrophobic amino acids due to the effect of endogenous proteases. Raman spectra verified the changes in microenvironment of tryptophan and tyrosine residues, and showed that aliphatic amino acids were mainly exposed to polar solvent by both acid denaturation and endogenous proteolysis. In addition, new disulphide bonds were less likely to form by acid‐induced aggregation and proteolytic cleavage, while random coils increased and smaller water domains were found.

Selection of downstream steps by analysis of protein surface property: A case study for recombinant human lactoferrin purification from milk of transgenic cow

Protein purification has long been regarded as an art rather than a science. Trial and error is the widely used practice for a protein to be purified from the crude extract. In this study, we carried out a rational approach by analysis of the protein surface properties before setting out to do experiments. The target protein was recombinant human lactoferrin (rHLF), to be purified from milk of transgenic cow. We need to overcome two major problems. One is its possible co-precipitation with casein during initial separation; the other is the difficulty in separating its homologous counterpart, bovine lactoferrin (BLF). By calculation of the surface hydrophobicity, an initial separation step was decided by calcium precipitation, which removed casein but left rHLF in the supernatant. Then the average surface hydrophobicity and electric potential of rHLF were compared with those of BLF. There was a more significant difference in the electric potentials than the average surface hydrophobicity (ASH) between the homologous pairs. Therefore, the purification step should be favored by ion exchange chromatography (IEC) instead of hydrophobic interaction chromatography (HIC). Experiments were performed to verify the prediction. After removing casein, one step cationic ion exchange chromatography realized complete separation of rHLF from BLF with rHLF recovery up to 83%, while the resolution of HIC process was very limited due to the small difference in ASH between them. The laboratory process was then successfully scaled up to pilot-plant scale of 500l milk of transgenic cow per batch. Average rHLF recovery of 79%and purity of 98.9% were attained for five batches. The purified rHLF displayed bioactivities as good as the natural human-resource lactoferrin standard.

Theoretical and Experimental Evaluation of Hydrophobicity of Proteins to Predict their Partitioning Behavior in Aqueous Two Phase Systems: A Review

Hydrophobicity is the main determinant in the partitioning of proteins in Aqueous Two-Phase Systems (ATPSs). There are a range of different methods available to evaluate hydrophobicity, including hydrophobic interaction chromatography, reverse phase chromatography, precipitation with ammonium sulphate, average surface hydrophobicity (3-D structure), aminoacid (AA) composition, hydrophobic imbalance, and a statistical description of the AA distribution. This paper reviews the present state of the art in the practical and theoretical evaluation and measurement of a protein's hydrophobicity and evaluates which are the best methodologies to estimate hydrophobicity to predict behaviour in ATPSs. The two methods that are more attractive, depending on the ATPS used, are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one that uses the parameter 1/m*, which is very simple to determine experimentally.

Efficient separation of homologous α-lactalbumin from transgenic bovine milk using optimized hydrophobic interaction chromatography

Transgenic bovine milk could be a rich source of recombinant human proteins. However, the co-presence of bovine and human homologous proteins can be a challenge for product purification. In this study, the average surface hydrophobicity and electric potential of human α-lactalbumin (HLA) and bovine α-lactalbumin (BLA) were analyzed and compared through the exposure area calculation of different amino acids. Based on the analysis, calcium independent hydrophobic interaction chromatography was selected for separation of recombinant human α-lactalbumin (rHLA) from BLA in transgenic bovine milk. The operating conditions for the best separation of two proteins were predicted by fluorescence data. Three commercially available HIC resins (Butyl Sepharose 4 FF, Octyl Sepharose 4 FF, Phenyl Sepharose 6 FF) were compared. The transgenic milk was skimmed and treated by pH adjustment to remove a large quantity of casein protein. The supernatant was loaded on the hydrophobic interaction chromatographic matrix. The correct elution fraction was further treated with gel filtration chromatography. The overall recovery of rHLA was up to 67.1% with the purity greater than 95%. Circular dichroism spectroscopy (CD) and mass spectrogram (MS) confirmed the native state and glycosylated form of the purified rHLA.

Prediction of the partitioning behaviour of proteins in aqueous two-phase systems using only their amino acid composition

The prediction of the partition behaviour of proteins in aqueous two-phase systems (ATPS) using mathematical models based on their amino acid composition was investigated. The predictive models are based on the average surface hydrophobicity (ASH). The ASH was estimated by means of models that use the three-dimensional structure of proteins and by models that use only the amino acid composition of proteins. These models were evaluated for a set of 11 proteins with known experimental partition coefficient in four-phase systems: polyethylene glycol (PEG) 4000/phospate, sulfate, citrate and dextran and considering three levels of NaCl concentration (0.0% w/w, 0.6% w/w and 8.8% w/w). The results indicate that such prediction is feasible even though the quality of the prediction depends strongly on the ATPS and its operational conditions such as the NaCl concentration. The ATPS 0 model which use the three-dimensional structure obtains similar results to those given by previous models based on variables measured in the laboratory. In addition it maintains the main characteristics of the hydrophobic resolution and intrinsic hydrophobicity reported before. Three mathematical models, ATPS I–III, based only on the amino acid composition were evaluated. The best results were obtained by the ATPS I model which assumes that all of the amino acids are completely exposed. The performance of the ATPS I model follows the behaviour reported previously, i.e. its correlation coefficients improve as the NaCl concentration increases in the system and, therefore, the effect of the protein hydrophobicity prevails over other effects such as charge or size. Its best predictive performance was obtained for the PEG/dextran system at high NaCl concentration. An increase in the predictive capacity of at least 54.4% with respect to the models which use the three-dimensional structure of the protein was obtained for that system. In addition, the ATPS I model exhibits high correlation coefficients in that system being higher than 0.88 on average. The ATPS I model exhibited correlation coefficients higher than 0.67 for the rest of the ATPS at high NaCl concentration. Finally, we tested our best model, the ATPS I model, on the prediction of the partition coefficient of the protein invertase. We found that the predictive capacities of the ATPS I model are better in PEG/dextran systems, where the relative error of the prediction with respect to the experimental value is 15.6%.

New approaches for predicting protein retention time in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is an important technique for the purification of proteins. In this paper, we review three different approaches for predicting protein retention time in HIC, based either on a protein's structure or on its amino-acidic composition, and we have extended one of these approaches. The first approach correlates the protein retention time in HIC with the protein average surface hydrophobicity. This methodology is based on the protein three-dimensional structure data and considers the hydrophobic contribution of the exposed amino acid residues as a weighted average. The second approach, which we have extended, is based on the high correlation level between the average surface hydrophobicity of a protein's hydrophobic interacting zone and its retention time in HIC. Finally, a third approach carries out a prediction of the average surface hydrophobicity of a protein, using only its amino-acidic composition, without knowing its three-dimensional structure. These models would make it possible to test different operating conditions for the purification of a target protein by computer simulations, and thus make it easier to select the optimal conditions, contributing to the rational design and optimization of the process.

Predicting the behaviour of proteins in hydrophobic interaction chromatography: 1: Using the hydrophobic imbalance (HI) to describe their surface amino acid distribution

This paper focuses on the prediction of the dimensionless retention time of proteins (DRT) in hydrophobic interaction chromatography (HIC) by means of mathematical models based on characteristics of the surface hydrophobicity distribution. We introduce a new parameter, called hydrophobic imbalance (HI), obtained from the three-dimensional structure of proteins. This parameter quantifies the displacement of the superficial geometric centre of the protein when the effect of the hydrophobicity of each amino acid is considered. This parameter is simpler and less expensive than those reported previously. We use HI as a way to incorporate information about the surface hydrophobicity distribution in order to improve the prediction of DRT. We tested the performance of our DRT predictive models in a set of 15 proteins. This set includes four proteins whose DRTs are known as very difficult to predict. By means of the variable HI, it was possible to improve the predictive characteristics obtained by models based on the average surface hydrophobicity (ASH) by 9.1%. Also, we studied linear multivariable models based on characteristics determined from the HI. By using this multivariable model, a correlation coefficient of 0.899 was obtained. With this model, we managed to improve the predictive characteristics shown by previous models based on ASH by 31.8%.

Is it possible to predict the average surface hydrophobicity of a protein using only its amino acid composition?

Hydrophobicity is one of the most important physicochemical properties of proteins. Moreover, it plays a fundamental role in hydrophobic interaction chromatography, a separation technique that, at present time, is used in most industrial processes for protein purification as well as in laboratory scale applications. Although there are many ways of assessing the hydrophobicity value of a protein, recently, it has been shown that the average surface hydrophobicity (ASH) is an important tool in the area of protein separation and purification particularly in protein chromatography. The ASH is calculated based on the hydrophobic characteristics of each class of amino acid present on the protein surface. The hydrophobic characteristics of the amino acids are determined by a scale of aminoacidic hydrophobicity. In this work, the scales of Cowan–Whittaker and Berggren were studied. However, to calculate the ASH, it is necessary to have the three-dimensional protein structure. Frequently this data does not exist, and the only information available is the amino acid sequence. In these cases it would be desirable to estimate the ASH based only on properties extracted from the protein sequence. It was found that it is possible to predict the ASH from a protein to an acceptable level for many practical applications (correlation coefficient > 0.8) using only the aminoacidic composition. Two predictive tools were built: one based on a simple linear model and the other on a neural network. Both tools were constructed starting from the analysis of a set of 1982 non-redundant proteins. The linear model was able to predict the ASH for an independent subset with a correlation coefficient of 0.769 for the case of Cowan–Whittaker and 0.803 for the case of Berggren. On the other hand, the neural model improved the results shown by the linear model obtaining correlation coefficients of 0.831 and 0.836, respectively. The neural model was somewhat more robust than the linear model particularly as it gave similar correlation coefficients for both hydrophobicity scales tested, moreover, the observed variabilities did not overcome 6.1% of the mean square error. Finally, we tested our models in a set of nine proteins with known retention time in hydrophobic interaction chromatography. We found that both models can predict this retention time with correlation coefficients only slightly inferior (11.5% and 5.5% for the linear and the neural network models, respectively) than models that use the information about the three-dimensional structure of proteins.

A theory of protein–resin interaction in hydrophobic interaction chromatography

Docking simulations were performed in order to investigate surface area of interaction between several ribonucleases and a reduced model for the hydrophobic moiety used in Phenyl Sepharose™ using the program AutoDock 3.0. For each ribonucelase, 80 independent simulations with populations consisting of 100 random structures were performed and from these the most probable docked protein–ligand conformations were obtained. A new methodology was used to select the most probable conformations, based on qualitative and quantitative considerations. The interacting amino acids in each protein were identified. The average surface hydrophobicity of the interfacial zone (local hydrophobicity, LH) was determined. The LH showed a high correlation level ( r 2 = 0.99) with the “hydrophobic contact area” (HCA) experimentally determined for the different ribonucleases as well as with the dimensionless retention time ( r 2 = 0.90). This study allowed us to identify the zones on the protein surface most probably involved in protein retention in HIC, without tedious experimental work. Given the good correlation level obtained, this new methodology may constitute a novel approach that could be used to predict protein behavior in HIC.

Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography

The effect of surface hydrophobicity distribution of proteins on retention in hydrophobic interaction chromatography (HIC) was investigated. Average surface hydrophobicity as well as hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface hydrophobicity but different surface hydrophobicity distribution. It was shown experimentally that surface hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter “hydrophobic contact area,” which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the hydrophobic patches can therefore have an important effect on protein retention in HIC, and the hydrophobic contact area adequately describes this.

Characterization of a Truncated Form of Recombinant Porcine Growth Hormone Generated in Vitro During Solubilization of Inclusion Bodies

During the development of a novel solubilization procedure (1) for bacterial inclusion bodies (IB′s) using the cationic surfactant cetyltrimethylammonium chloride (CTAC; (CH 3) 3N +C 16H 33Cl) significant proportions of an apparently truncated, lower molecular weight (MW) variant form of recombinant pig growth hormone (rPGH) were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis relative to pig pituitary derived GH. The formation of this rPGH-like species, designated P-band, was found to occur in vitro during solubilization of IB′s by CTAC and was dependent on pH and temperature of solubilization, but was not due directly to the use of CTAC, as purified soluble rPGH of the correct MW could not be converted to P-band by exposure to CTAC alone. The bacterial proteolysis suspected as being responsible for the in vitro formation of P-band could not be inhibited by the use of a "cocktail" of defined antiproteolytic agents but was inhibited by pH and temperature, and by solubilization of IB′s in 5% SDS, 6 M gnHCl or 7.5 M urea. Detailed characterization of the structure of P-band by N-terminal amino acid sequencing, electrospray mass spectrometry, radio-receptor binding assay, peptide mapping, and C-terminal peptide sequencing confirmed that P-band was approximately 950 mass units smaller than normal rPGH and lacked eight C-terminal amino acids. A significant finding was that P-band is unable to bind to the pig liver-membrane GH receptor in a competitive radioreceptor assay. Analysis of the relative secondary and tertiary structure of P-band by circular dichroism spectra, intrinsic tryptophan-dependent fluorescence, and average surface hydrophobicity (2) suggested small but measurable changes to the overall structure of P-band relative to normal rPGH. Consequently, our results also suggest that the C-terminal portion of rPGH, including in particular the last eight amino acids, is of major importance in the binding of rPGH to the pig liver membrane GH receptor.