Trypsin denaturation

A second set of data are temperature jump studies of trypsin denaturation near pH 2 made by Pohl (59), Fig. 20. Temperature changes near the transition temperature can, of o>urse, change the degree of folding markedly in Pohl s setup rather large jumps can be m e, but these are relatively slow, the maximum observable rate is only 0.5sec ... 

Figure 19.2 A generalized proteomics work flow for the extraction and identification of proteins in FFPE tissue. Formalin-fixed tissues acquired by sectioning, needle dissection, or laser capture are deparaffinized in xylenes and are rehydrated in graded alcohols. The material is resuspended in buffer which generally contains a detergent/ protein denaturant and the sample is heated to complete the extraction process. The protein extract is reduced, alkylated, and digested with trypsin before protein profiling.

For the MSn measurement of proteins, that is, the measurement of peptides, one has to denature and reduce proteins in the tissue samples, followed by enzyme digestion. Therefore, protein samples should be treated with trypsin after membrane transfer. Trypsin can be attached to the membrane and can be performed in the same steps as in the matrix coating method. 

The protocol for using isobaric tags differs from that described previously for the ICAT or ECAT type reagents. In the following method, the proteins are denatured and the disulfides reduced and then alkylated to block them permanently. This eliminates disulfide re-association and also prevents the isobaric tags from forming thioester modification with cysteine thiols. Next, the proteins are digested with trypsin and then modified with an isobaric tag. Each sample is labeled with a different isobaric compound so that the samples can be differentiated upon MS/MS analysis. 

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. 

Evidence demonstrating that the degradation observed is catalyzed by enzyme(s) was obtained by typical denaturing treatments. Late extracellular protein fractions from both strains present the same characteristics resistance to heat up to 100°C, partial resistance to acidity as low as pH 1.0 (samples being returned to pH 7.8 prior to assaying for activity), but are completely inactivated by proteolysis with a mixture of trypsin and chy-motrypsin (Table II). 

Gabel, D. (1973) The denaturation by urea and guanidinum chloride of trypsin and N-acetylated-trypsin derivatives bound to sephadex and agarose. Eur. J. Biochem., 33, 348-356. 

NADH or MgATP or ATP plus AMP protect against proteolysis by pronase or trypsin and against heat denaturation [45]... 

The inactive precursors are called trypsinogen, pepsinogen, chymotrypsino-gen, and procarboxypeptidase. These precursors are converted to the active enzymes by hydrolytic cleavage of a few specific peptide bonds under the influence of other enzymes (trypsin, for example, converts chymotrypsinogen to chymotrypsin). The digestive enzymes do not appear to self-destruct, probably because they are so constructed that it is sterically impossible to fit a part of one enzyme molecule into the active site of another. In this connection, it is significant that chymotrypsin attacks denatured proteins more rapidly than natural proteins with their compact structures of precisely folded chains. 

RNase can be completely denatured by boiling or by treatment with chaotropic agents (e.g., urea), yet can refold to its fully active form on cooling or removal of the denaturant. By contrast, when enzymes of the trypsin family and carboxypeptidase A are denatured, they do not regain full activity on renaturation. What aspects of trypsin and carboxypeptidase A structure preclude their renaturation to the fully active form ... 

Renaturation of denatured protein is dictated by the primary structure of the protein. The trypsin family of enzymes and carboxypeptidase A are synthesized as proenzymes that are proteolytically activated. The proteolyzed, active enzymes have primary structures different from the gene product and are not active upon renaturation. In addition, zinc is a cofactor required for carboxypeptidase A activity. 

Figure 16.13 The free energy of denaturation AfjG as a function of temperature for a number of proteins Lys = lysozyme Rna = ribonuclease A Ct = a-chymotrypsin Cyt = cytochrome c Mb = metmyoglobin Tr = Trypsin and PTI2 = the dimer of pancreatic trypsin inhibitor. Reprinted with permission from P. L. Privalov, Stability of Proteins — Small Globular Proteins, Adv. Prot. Chem., 33, 167 (1979).

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