Trypsin ribonuclease

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. 

MALTASE PEPSIN PROTEASE RENNIN TRYPSIN RIBONUCLEASE UREASE ZYMASE... 

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Levitt, M. Sander, C. Stem, P.S., Protein normal-mode dynamics - trypsin-inhibitor, crambin, ribonuclease and lysozyme, 7. Mol. Biol. 1985,181, 423 47... 

One simple case of disordered structure involves many of the long charged side chains exposed to solvent, particularly lysines. For example, 16 of the 19 lysines in myoglobin are listed as uncertain past C8 and 5 of them for all atoms past C/J (Watson, 1969) for ribonuclease S Wyckoff et al. (1970) report 6 of the 10 lysine side chains in zero electron density in trypsin the ends of 9 of the 13 lysines refined to the maximum allowed temperature factor of 40 (R. Stroud and J. Chambers, personal communication) and in rubredoxin refined at 1.2 A resolution the average temperature factor for the last 4 atoms in the side chain is 9.2 for one of the four lysines versus 43.6, 74.4, and 79.3 for the others. Figure 57 shows the refined electron density for the well-ordered lysine and for the best of the disordered ones in ru-... 

Honeychurch and Ridd [97] used chronopotentiometry to study reduction of five disulfide-containing proteins (bovine serum albumin, insulin, ribonuclease A, transferrin, and trypsin) adsorbed on HMDE. All studied proteins exhibited a reduction step at —0.6 V (versus SCE), due to reduction of disulfide groups. 

Ribonuclease A, soybean trypsin inhibitor, thaumatin, a-lactalbumin AOT/isooctane Solubilization [71]... 

Cellulose albumin y-globulin ribonuclease trypsin antibodies concana valin... 

The five N-terminal residues and the six or seven C-terminal residues cannot be seen in the high resolution electron density map, and the loop referred to above, formed by residues 44 to 53, appears at only one-third to one-half the amplitude of the well-resolved parts of the map. The lack of clarity in these three regions might possibly result from poor phasing or some other crystallographic factor, but we consider it more likely that these predominantly hydrophilic sections of the peptide project in a disordered way into the solvent. In this connection, it is interesting that in the presence of Ca2+ and pdTp trypsin cleaves inhibited nuclease at only two points between residues 5 and 6 and between residues 48 and 49 (36-38) which are at the very extremity of the loop. It also seems relevant that ribonuclease S also shows lack of clarity at the ends of the peptide chains and in the region of a relatively exposed loop (56). 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

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).

Figure 6.7 Comparative peptide map of enzymatic digests of bovine ribonuclease B. (A) TIC trace of digested RCM-glycoprotein with trypsin. (B) TIC trace of digested RCM-glycoprotein with trypsin and PNGase F. (Reprinted with permission from Liu et al., 1993. Copyright 1993 Elsevier.)...

Enzymes which catalyze the hydrolysis of the unit linkage of sequential residues of oligomers or polymers determine their substrate specificity by recognizing the particular unit residue in the sequential chain as well as the direction of the chain. For example, ribonuclease cleaves the 3 -phosphate of a pyrimidine nucleotide residue but not the 5 -phosphate, and trypsin hydrolyzes peptide bonds which involve the arginine or lysine residue at the carbonyl end but not at the amino end. This is also the case for the hydrolysis of a variety of synthetic substrates and quasi-substrates (Sect. 4.1). Synthetic trypsin substrates are ester or amide derivatives in which the site-specific group (positive charge) is contained in their carbonyl portion. 

FIGURE 13 Plot of the logarithm of the retention volume (In VR) versus the concentration of the displacing salt, ammonium sulphate, in the HP-HIC mode with the proteins I, insulin B-chain 2, bovine trypsin inhibitor 3, bovine trypsinogen 4, insulin A-chain 5, ribonuclease 6, sperm whale myoglobin 7, horse heart cytochrome c. Data from Ref. 42. 

During last decades the domains C-2 symmetry (the dyad rotation symmetry) of low-B palindrome was established in many enzymes (chymotrypsin, trypsin, aspartyl proteinases, HIV-1 protease, carboxypeptidase A, phospholipase A-2 ribonuclease, etc.) (Lumry, 2002 and references therein). It is proposed that the pair domain closure causes constrain of pretransition state complex that activates cleavage or formation of chemical bonds. Thus control of strong bonds by the cooperation of many matrix or knots bonds takes place. As an example, in the active site of carboxypeptidase A the zinc ion is attached to one of the catalytic domains by histidine 69 and glutamine 72 and connected by hystidine 196 to the second domain. Similar structures were found in the chymotrypsin and pepsin active sites where protons are driven under compression of the domains closure. 

Carbonic anhydrase, a-lactalbumin, trypsin inhibitor, oval-bumn, conalbumin, hemoglobin variants Ribonuclease, insulin, a-lactalbumin Lysozyme, a-chymotrypsinogen, ribonuclease A, cytochrome c... 

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