Trypsin solubility

All samples were digested with trypsin and analyzed by cIEF in the first dimension followed by LC-MS/MS as described above. Samples were analyzed in duplicate. Sequence searching was performed using OMSSA. Analysis of the soluble fraction yielded a total of 2856 identified proteins, while the insoluble fraction yielded 3227 proteins. Combined, the fresh-frozen sample yielded 3902 protein identifications. The FFPE portion yielded 2845 protein identifications from 14,178 distinct tryptic peptide sequences, on a par with the fresh-frozen soluble fraction. Combining all identifications gave 4145 proteins. While, the soluble fraction and the FFPE extraction yielded similar numbers of protein identification, both found 25% of their respective protein set uniquely (Fig. 20.4). 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

In the past, dissociation of the nucleoprotein complex has been brought about by salt solutions or by heat denaturation,129 but, more recently, decomposition has been effected by hydrolysis with trypsin,126 or by the use of dodecyl sodium sulfate130 or strontium nitrate.131 Some virus nucleoproteins are decomposed by ethyl alcohol.132 This effect may be similar to that of alcohol on the ribonucleoproteins of mammalian tissues. If minced liver is denatured with alcohol, and the dried tissue powder is extracted with 10% sodium chloride, the ribonucleoproteins are decomposed to give a soluble sodium ribonucleate while the deoxyribonucleoproteins are unaffected.133 On the other hand, extraction with 10 % sodium chloride is not satisfactory unless the proteins have first been denatured with alcohol. Denaturation also serves to inactivate enzymes of the tissues which might otherwise bring about degradation of the nucleic acid during extraction. 

The majority of the studies about the benefits of Allium components have been done with nonaqueous extracts. In our study, only water-soluble components are responsible for the biological effects observed. On the other hand, garlic extracts are considered the most efficient for biomedical effects. In our experiments with 0.5 g/mL of cmde aqueous extracts of different parts of Allium plants (bulb, green leaf, white stalk), the red onion bulb extract showed the highest inhibition of acrosin and especially trypsin activity. The acrosin inhibition was reported regarding with spermicidal effect of garlic [8], but the protease inhibition has not been reported before in relation to the clinical effects of these plants. 

HPMA copolymers are water-soluble biocompatible polymers, widely used in anticancer drug delivery (reviewed in Reference [22]). HPMA copolymers containing reactive groups at side-chain termini were previously used for the modification of trypsin [23], chymotrypsin [23,24], and acetylcholinesterase [25]. The modification dramatically increased the acetylcholinesterase survival in the blood stream of mice and the thermostability of modified enzymes when compared to the native proteins. However, the modification involved multipoint attachment of the copolymers to the substrates, which may cause crosslinking. To modify proteins or biomedical surfaces by one point attachment, semitelechelic polymers should be used. 

Supplementing the fermentation of gutted herring with equivalent amount of pure bovine and Greenland cod trypsins resulted in increased solubilization of protein. While the cod enzyme was effective in solubilizing protein, the percentage of solubilized protein which was soluble in 10% trichloroacetic... 

Plasmin [EC 3.4.21.7], also known as fibrinase and fibri-nolysin, is a peptidase (a member of the peptidase family SI) that exhibits preferential cleavage at Lys—Xaa > Arg-Xaa (there is actually greater selectivity than displayed by trypsin). Plasmin converts fibrin into soluble products. It is formed from plasminogen by proteolysis, resulting in multiple forms of the active plasmin. 

Native RNase is quite resistant to digestion with trypsin, even at a w/w ratio of 1 20, but small or unfolded fragments would be expected to be digested. When the synthetic enzyme was treated with trypsin, a 70% recovery of protein with a specific activity of 61% was obtained. Treatment of this material with saturated ammonium sulfate (diluted 16 26), pH 4.6, gave 33% of amorphous precipitate and 66% of soluble RNase A. The overall yield from the first Val residue was only 3%, but the specific activity was quite high at 78%. This is as far as the purification was carried out at that time. 

Figure 9.12 The uptake of water-soluble proteins (p) from an aqueous solution to an AOT micellar solution as a function of the salt concentration in the water phase. Note the remarkable difference between cytochrome-C (Cyt-c) and a-chymo-trypsin (a-Chym), so that in principle it is possible to separate one from the other.

In order to overcome the main limitations of the impregnation processes, connected to the limited solubility of the compounds in the supercritical fluids, Perman [68] proposed an alternative method. A supercritical impregnation process was coupled with a liquid solvent (preferentially water) to enhance the drug solubilization. The system composed of a liquid drug solution and the polymeric support was pressurized with the supercritical fluid. Consequently, the swelled polymer allows rapid diffusional transport of the solute into the polymeric substrate. In different examples, bovine serum albumin microspheres were impregnated with insulin, trypsin and gentamicin (see Table 9.9-5). 

PROTEASE. A proteolytic enzyme that weakens or breaks the peptide linkages in proteins, They include some of the more widely known enzymes such as pepsin, trypsin, ficin, bromelm, papain, and rennin. Being water soluble they solubilize proteins and are commercially used for meat tendenzers, bread baking, and digestive aids. 

At present, the binary water-soluble preparation of heparin and proteolytic enzymes is being applied for the treatment of thromboses. For instance, injection into the bloodstream of heparin-plasmin complex or a heparin-plasmin-streptokinase preparation leads to the total dissolution of the thrombus, while if introduced separately, heparin and streptokinase do not display the lytic action at all, and plasmin, alone or together with streptokinase, dissolves the thrombus only partially 132>. The treatment of acute thrombophlebitis with trypsin resulted in a full dissolution of the thrombus and in an increase of antithrombin III in the blood 133). Administration of trypsin together with heparin has an effect similar in efficiency to the action of the heparin-plasmin complex 134>. The use of a mix of heparin and urokinase for improving tbrom-boresistance of polymeric materials was also described 13S). These substances were immobilized by preliminary coating of the surface of a polymer with a graphite layer and subsequent adsorption of heparin and the enzyme. 

---