scholarly journals Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

Symmetry ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 153 ◽  
Author(s):  
M.M. Vorob’ev
Keyword(s):  
Tryptophan Fluorescence ◽  
Degree Of Hydrolysis ◽  
Peptide Bonds ◽  
Protein Substrates ◽  
Intrinsic Tryptophan Fluorescence ◽  
Step Model ◽  
Fluorescence Change ◽  
Β Lactoglobulin ◽  
Kinetics Of ◽  
Hydrolysis Of

To distinguish differences in enzymatic hydrolysis of various proteins, we propose an algorithm using a dataset of fluorescence spectra obtained at different moments of hydrolysis t. This algorithm was demonstrated in the example of β-lactoglobulin (β-LG) proteolysis by trypsin. The procedure involved processing the spectra to obtain the wavelength of the maximum fluorescence λmax, which was found to be proportional to the fraction of tryptophanes in hydrated proteolysis products (demasked tryptophanes). Then, the dependence λmax(t) was fitted by biexponential function with two exponential terms, one of which was responsible for the fast part of the fluorescence change during proteolysis. The contribution of this term was quite different for various protein substrates—it was positive for β-LG and negative for β-casein. The observed differences in proteolysis of different substrates were explained by different demasking processes. Combining the fluorescence data with the degrees of hydrolysis of peptide bonds allowed us to analyze the hydrolysis of β-LG in the framework of the two-step proteolysis model and estimate the ratio of rate constants of demasking and hydrolysis and the percentages of initially masked and resistant peptide bonds. This model predicted the existence of a bimodal demasking process with a fast part at the beginning of proteolysis and lag-type kinetics of release for some peptides. Compared with monitoring proteolysis in terms of the degree of hydrolysis only, the fluorescence data are helpful for the recognition of proteolysis patterns.

scholarly journals Tryptophan Fluorescence and Time-Lag Hydrolysis of Peptide Bonds during Degradation of β-Lactoglobulin by Trypsin

Catalysts ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1368
Author(s):  
Mikhail M. Vorob’ev
Keyword(s):  
Rate Constants ◽  
Time Lag ◽  
Tryptophan Fluorescence ◽  
Hydrolysis Rate ◽  
Lag Phase ◽  
Peptide Bonds ◽  
Proteolytic Action ◽  
Lag Times ◽  
Β Lactoglobulin ◽  
Hydrolysis Of

The opening of protein globules and corresponding exposure of their internal peptide bonds, the so-called demasking effect, is required for successful hydrolysis of peptide bonds by proteases. Under the proteolytic action of trypsin on β-lactoglobulin (β-LG), the evolution of tryptophan fluorescence spectra showed that the demasking process consists of two stages with different demasking rate constants for each stage. It was found that the ratio of these constants depends on the concentration of trypsin and changes are approximately threefold when the concentration of trypsin changes in the range of 0.3–15 mg/L. Simulation of hydrolysis taking into account the demasking effect demonstrated how the apparent first-order rate constants obtained experimentally are related to the true hydrolysis rate constants and demasking parameters. The lag phase in the kinetic curves corresponding to the hydrolysis of various peptide bonds in β-LG was also analyzed. The increased lag times indicated sites that are hydrolyzed by a two-stage demasking mechanism.

scholarly journals Spectrophotometric measurement of the liberation of the α-amino group of cysteine residues in polypeptides

1966 ◽  
Vol 100 (2) ◽  
pp. 309-314 ◽  
Author(s):  
S Streichman ◽  
Y Avi-Dor
Keyword(s):  
Spectral Characteristics ◽  
Cysteine Residues ◽  
Amino Group ◽  
Peptide Bonds ◽  
Spectral Changes ◽  
Sh Group ◽  
Induced Changes ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Specific Peptide

1. The reaction between beta-bromopyruvic acid and SH groups of cysteine residues in reduced ribonuclease and in some other polypeptides was investigated. 2. One molecule of the acid was found to be necessary to block one SH group in reduced ribonuclease. The stoicheiometry of the interaction and the spectral characteristics of the compound formed suggested that the product is and S-oxalomethyl (R.S.CH(2).CO.CO(2)H) derivative of reduced ribonuclease. 3. Digestion of reduced S-oxalomethylated ribonuclease by trypsin or chymotrypsin induced changes in the spectrum that could be attributed to the liberation of the alpha-amino group of S-oxalomethylated cysteine residues from peptide bonds. The spectral changes that accompanied the hydrolysis of specific peptide bonds in reduced S-oxalomethylated ribonuclease and S-oxalomethylated co-poly(l-Lys,l-CySH) allowed the kinetics of the digestion to be followed. 4. Possible applications of the spectrophotometric method in the study of protein structure are discussed.

Kinetics of changes in plasmin activity and proteolysis on heating milk

2005 ◽  
Vol 72 (4) ◽  
pp. 493-504 ◽  
Author(s):  
Anthony Crudden ◽  
Jorge C Oliveira ◽  
Alan L Kelly
Keyword(s):  
Rate Constant ◽  
Thermal Inactivation ◽  
Plasmin Activity ◽  
Casein Micelles ◽  
Complete Inactivation ◽  
System A ◽  
Β Lactoglobulin ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Thermal Stability Of

Heat-induced inactivation of bovine plasmin, denaturation of β-lactoglobulin (β-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of β-casein was studied in a model system consisting of phosphocasein and β-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of β-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of β-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65–80 °C was its interaction with β-lg (kr at 60 °C, 0·0526; Ea, 176 KJ/mol). At the point of complete inactivation of plasmin ~45% of the β-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of β-lg with the casein micelles at 60 °C had a rate constant of 3·71×10−5 min−1 and an Ea of 259 KJ/mol; thermal denaturation of β-lg was of much less importance, with a rate constant at 60 °C of the order of 1×10−10 min−1 and an Ea of 250 KJ/mol. On denaturation of all β-lg in the system, a maximum of ~55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of β-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of β-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with β-lg.

Correlation of base consumption with the degree of hydrolysis in enzymic protein hydrolysis

2001 ◽  
Vol 68 (2) ◽  
pp. 251-265 ◽  
Author(s):  
FERNANDO CAMACHO ◽  
PEDRO GONZÁLEZ-TELLO ◽  
MARÍA-PURIFICACIÓN PÁEZ-DUEÑAS ◽  
EMILIA-MARÍA GUADIX ◽  
ANTONIO GUADIX
Keyword(s):  
Working Conditions ◽  
Whey Proteins ◽  
Degree Of Hydrolysis ◽  
Bacterial Protease ◽  
Alkaline Conditions ◽  
Neutralizing Agent ◽  
Hydrolytic Process ◽  
The Relationship ◽  
Kinetics Of ◽  
Hydrolysis Of

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the α-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein–protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 °C, pH 8·0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

scholarly journals The V30M Amyloidogenic Mutation Decreases the Rate of Refolding Kinetics of the Tetrameric Protein Transthyretin

2012 ◽  
Vol 27 ◽  
pp. 343-348 ◽  
Author(s):  
Catarina S. H. Jesus ◽  
Daniela C. Vaz ◽  
Maria J. M. Saraiva ◽  
Rui M. M. Brito
Keyword(s):  
Tryptophan Fluorescence ◽  
Amyloid Formation ◽  
Monomeric Species ◽  
Intrinsic Tryptophan Fluorescence ◽  
Tetrameric Structure ◽  
Partially Unfolded ◽  
Kinetics Of ◽  
Refolding Kinetics

Transthyretin (TTR) is a homotetrameric protein implicated in several amyloid diseases. The mechanism by which TTR is converted into elongated fibrillar assemblies has been extensively investigated, and numerous studies showed that dissociation of the native tetrameric structure into partially unfolded monomeric species precedes amyloid formation. The small differences observed in the crystal structures of different TTR variants, as well as the thermodynamics and kinetics of tetramer dissociation, do not seem to completely justify the amyloidogenic potential of different TTR variants. With this in mind, we have studied the refolding kinetics of WT-TTR and its most common amyloidogenic variant V30M-TTR, monitoring changes in intrinsic tryptophan fluorescence at different urea and protein concentrations. Our results demonstrate that thein vitrorefolding mechanisms of WT- and V30M-TTR are similar, involving a dimeric intermediate. However, there are large differences in the refolding rate constants for the two variants, specially close to physiological conditions. Interestingly, tetramer formation occurs at a much slower rate in the amyloidogenic variant V30M-TTR than in WT-TTR, which in thein vivosetting may promote the accumulation of monomeric species in the extracellular environment, resulting in higher susceptibility for aggregation and amyloid formation instead of spontaneous refolding.

Study on the Kinetics of Salmon Skin Proteolysis

2019 ◽  
Vol 11 (12) ◽  
pp. 1706-1710
Author(s):  
Yiping Xia ◽  
Hao Yu ◽  
Yaoguang Zhong
Keyword(s):  
Enzymatic Hydrolysis ◽  
Kinetic Model ◽  
Kinetic Mechanism ◽  
Reaction Rate Constant ◽  
Hydrolysis Rate ◽  
Degree Of Hydrolysis ◽  
Hydrolysis Process ◽  
Skin Protein ◽  
Kinetics Of ◽  
Hydrolysis Of

In this study, in order to study the kinetic mechanism of enzymatic hydrolysis of salmon protein, the kinetic model of enzymatic hydrolysis of salmon skin protein by papain was established. The skin protein of salmon was hydrolyzed by papain under the following conditions: the mass concentration of salmon skin protein is 55 g/L, the initial papain concentration is 2.0 g/L, the pH of enzymatic solution is 7.2 and the temperature of enzymatic hydrolysis is 55 °C. Finally, the kinetic model of hydrolysis was established as follows: Hydrolysis rate R =(27.217E0–0 0357S0exp[–0.2587(DH)]; Degree of hydrolysis DH = 3.879 ln[1 + 7.0165E0/S0 –0.0092t]. The reaction rate constant k3 = 27.217 min–1 and the enzyme deactivation constant kd = 7.0752 min–1 were deduced to control the enzymatic hydrolysis process. Further verification tests showed that the theoretical value of the degree of hydrolysis of the model was basically consistent with the actual value, and the kinetic model had certain practical value, indicating that the established salmon skin protease kinetic model could be used to guide and optimize the enzymatic hydrolysis process.

scholarly journals Methodology for Determining Degree of Hydrolysis of Proteins in Hydrolysates: A Review

2010 ◽  
Vol 93 (5) ◽  
pp. 1515-1522 ◽  
Author(s):  
Shane M Rutherfurd
Keyword(s):  
Protein Hydrolysate ◽  
Hydrolytic Enzymes ◽  
Degree Of Hydrolysis ◽  
Soluble Nitrogen ◽  
Trinitrobenzenesulfonic Acid ◽  
Peptide Bonds ◽  
Ph Stat ◽  
Terminal Amino ◽  
Stat Method ◽  
Hydrolysis Of

Abstract Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Several methods exist for determining DH; the most commonly used of these include the pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis; the TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. The SN-TCA method measures the amount of TCA-soluble nitrogen, rather than DH. The pH-stat is the simplest and most commonly used method, but does not determine peptide bonds directly. In addition, the accuracy of the method depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. Generally, the TNBS and OPA methods compare well and do directly determine DH. However, the assumption that the response factor for all derivatized N-terminal amino acids is similar may lead to inaccuracies. In conclusion, there is no consensus as to the best method for determining the DH of protein hydrolysates; consequently, there is a need for a standardized approach if interstudy comparisons are to be made.

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