Trypsin activity enhancement

The presence of SDS significantly reduces the efficiency of tryptic digestion. In a comparison between SDS and ALS, it was found that SDS resirlts in a 80% reduction of the trypsin activity, while with ALS only 1% reduction is observed [37]. During in-solution digestion, it acts as a mild protein denaturanf thereby enhancing the digestiom It is compatible with ESI-MS as well. 

Peptides are not as commonly analyzed by CIEF as are proteins one reason is their lower resolution, another their lower (or lack of) detectability at 280 nm (the wavelength mostly used). The separation of tryptic digests (peptide mapping) of proteins have been performed by using absorption detection at 280 nm and refractive index gradient imaging detection no exact correlations were observed between measured and calculated p7 values [1,5]. Refractive index detection is a universal detection method (i.e., independent of chromophores like tyrosine and tryptophan) but suffers from low sensitivity. Assays of trypsin activity have also been performed with laser-induced fluorescence detection for enhanced sensitivity, with detectability down to picomolal concentrations [5,6]. 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

The presence of crown ethers in aqueous medium before lyophilization can greatly enhance the activity of proteases (e.g., chymotrypsin, subtilisin, trypsin) for peptide synthesis in organic solvents. - The crown ether must also be rinsed away from the lyophilized enzyme before use. The activity enhancement is solvent dependent. It is speculated that crown ethers accelerate enzymatic rates either by preventing a salt bridge from forming in the enzyme s secondary structure or inducing microscopic changes in the structure of the solid phase. ... 

Eugenol, aqueous extracts of allspice, and allspice oil, along with numerous other spices and their volatile oils, have been demonstrated to enhance trypsin activity they also exhibit larvicidal properties. ... 

It is generally believed that proteases enhance cancer spread primarily by catalyzing degradation of the extracellular matrix. Since multiple substrates are encountered in this matrix, a number of different proteases are likely to be required to complete the metastatic process. Multiple proteases may also be required to activate different inactive precursor forms. Thus, in vitro, plasmin (D7), cathepsin B (D7), and a trypsin-like protease (K12) can all activate pro-uPA, while plasmin, which results from the action of uPA on plasminogen, can activate certain metalloproteases (M4). As mentioned earlier, completion of the metastatic process may require a cascade of different proteases operating, as shown in Fig. 2. 

Pancreatic effect. Ginger (50 mg%), administered orally to rats, significantly enhanced pancreatic lipase activity and significantly stimulated trypsin and chymot-rypsin. The stimulatory influence was not observed when the treatment was restricted to a single dose . 

Fig. 9 demonstrates the proportionality between the amount of immobilized trypsin and the lytic activity of the trypsin-containing material. The higher lytic efficiency of the binary trypsin-heparin systems seems unexpected (cf. curves 2 and 4, and 5 and 6 of Fig. 9), however. Thus, immobilized trypsin and heparin of the described polymeric materials mutually affected each other. This is manifested by the enhanced anticoagulant action of heparin promoted by trypsin and by greater lytic efficiency of trypsin provoked by heparin. 

Immobilization in a sol-gel matrix accelerated the propanolysis of N-acetyl-i-phenylalanine ethyl ester in cyclohexane for several serine proteases compared to the non-immobilized lyophilized enzymes 31-fold for Subtilisin Carlsberg, 43-fold for a-chymotrypsin, and 437-fold for trypsin (van Unen, 2001). The activity yield upon immobilization was 90% (a-chymotrypsin). The rate enhancement effect of immobilization on the enzyme activity is highest in hydrophobic solvents. 

Neoglycosylation of proteins was also applied to the modification of the psychrophilic Atlantic cod trypsin the applicability of psychrophilic enzymes is limited because of their lower thermodynamic stability, despite their higher catalytic rate. It has been shown that the thermodynamic stability could be enhanced appreciably by covalent chemical modification with an oxidized sucrose polymer without affecting the enzymatic activity. The acquired stability of cod trypsin was found to be on par with the mesophilic porcine trypsin.40... 

Trypsin-encapsulated sol-gel (alkoxysilane-based) was fabricated in situ onto the sample reservoir of a PMMA chip. This was employed for enzymatic conversion of NBD-labeled ArgOEt and bradykinin, followed by CE separation of the products. The enzymatic activity of the encapsulated trypsin as given by the Km value was found to be 19 times higher than that of the free trypsin. The stability of trypsin was 1 week at 4°C. This enhanced enzyme stability was possibly caused by the prevention of enzyme autolysis by the sol-gel matrix [1062],... 

In the SI pockets of /92, no such large structural changes are noted upon the introduction of IFNy-induced subunits (/92i), as was the case with /91i. Ser-32 and Asp-53 of pi are replaced by glutamine in the pl subunit. It is conceivable that the trypsin-like activity of the pit subunit is enhanced by the acidic Glu residues, which would stabilize the basic substrate residue in the pocket. 

Solutions containing active enzymatic proteins (protease, lipase, trypsin, pepsin, prophase, or cellulase) or their mixtures, adjusted to the nature of the matrix of the solid material of biological origin [79, 83, 84]. The aim of the procedure is to break up proteins, polysaccharides, or fat chains and release the constituent amino acids, sugars, or short aliphatic chains. Enzymatic decomposition of the matrix can be considerably enhanced by application of ultrasound the process can, for example, increase the efficiency of disintegration of cell walls in yeast and thus improve the recovery of selenium by as much as 20 % [85]. 

FIGURE 1. (A) Catalytic triad in the serine protease trypsin (PDB ID lAKS), wherein the nucle-ophilicity of the nucleophilic Serl95 is dramatically enhanced by H-bonding (dotted lines) with H57. (B) The metaUotriad active site in serralysin (PDB ID ISRP), showing the sandwiched nucleophihc water (in the form of hydroxide) by the active-site Zn(II) and Glul77 via a coordination bond and a H-bond... 

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