Bovine pancreatic ribonuclease,

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 5.6 Bovine pancreatic ribonuclease A contains 124 amino acid residues, none of which are tryptophan. Four intrachain disulfide bridges (S—S) form cross-links in this... 

Thannhauser, T. W., McWherter, C. A., and Scheraga, H. A., Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. II. A two-dimensional technique for determination of disulfide pairings using a continuous-flow disulfide detection system, Anal. Biochem., 149, 322, 1985. 

Rait VK, O Leary TJ, Mason JT. Modeling formalin fixatin and antigen retrieval with bovine pancreatic ribonuclease A I—structural and functional alterations. Lab. Invest. 2004 84 292-299. 

Navon A, Ittah V, Laity JH, et al. Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A. Biochemistry 2001 40 93-104. 

Crook EM, Mathias AP, Rabin BR. Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2, 3 -phosphate. Biochem. J. 1960 74 234-238. 

Yan YB, Zhang J, He HW, et al. Oligomerization and aggregation of bovine pancreatic ribonuclease A characteristic events observed by FTIR spectroscopy. Biophys. J. 2006 90 2525-2533. 

Hartman, F.C., and Wold, F. (1967) Cross-linking of bovine pancreatic ribonuclease A with dimethyl adipimidate. Biochemistry 6, 2439-2448. 

Goldberg, R. F., Epstein, C.)., Anfinsen, C. B., Acceleration of reactivation of reduced bovine pancreatic ribonuclease by a microsomal system from rat liver, /. Biol. Chem. 238 (1963), p. 628-635... 

N Fujii, H Yajima. Total synthesis of bovine pancreatic ribonuclease A. Parts 1 to 6. J Chem Soc Perkin Trans 1 789, 1981. 

S. Capasso, A. Di Donato, L. Esposito, F. Sica, G. Sorrentino, L. Vitagliano, A. Zagari, L. Mazzarella, Deamidation in Proteins The Crystal Structure of Bovine Pancreatic Ribonuclease with an Isoaspartyl Residue at Position 67 , J. Mol. Biol. 1996, 257, 492 -496. 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Fig. 1. Schematic drawing of the polypeptide backbone of ribonuclease S (bovine pancreatic ribonuclease A cleaved by subtilisin between residues 20 and 21). Spiral ribbons represent a-helices and arrows represent strands of /3 sheet. The S peptide (residues 1-20) runs down across the back of the structure.

Sokolovsky, M., and A. Patchornik Nonenzymatic cleavage of peptide bonds The hall cystine residues in bovine pancreatic ribonuclease. J. Amer. chem. Soc. 86, 1859—1860 (1964). 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

An example of enzyme depletion is the ribonuclease inhibitor isolated from human placenta by Blackburn, Wilson Moore. This protein forms a 1 1 complex with bovine pancreatic RNase A and is a noncompetitive... 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

The S-ribonuclease is the complex formed between an eicosapeptide and the S-RNAse. While replacement of various amino acids by fluorinated analogues does not modify the activity of the native complex, replacement of His-12 by 4-F-His has a strong influence. Indeed, the S-ribonuclease, formed between the bovine pancreatic S-RNAse and the fluoro peptide that contains 4-F-His, has no more catalytic activity, but it is stable. This loss of enzymatic activity is probably due to the significant lowering of the pAia of the catalytic His (2.5 units), which results from the presence of the fluorine atom. It is known that histidine plays an important role in nucleophilic and acid-base processes, which are connected to the catalytic activity of numerous enzymes. 

Fig. 1. The two-step hydrolysis of ribonucleic acid as catalyzed by bovine pancreatic ribonuclease A.

A series of carboxyl derivatized polyglucoses were studied as inhibitors of ribonuclease activity, in an attempt to relate charge density to inhibitory activity.202 In comparison with other factors, it was concluded that coulombic forces probably play a major role in complex-formation between enzyme and substrate, and between enzyme and inhibitor. However, other specific, nonelectrostatic forces were shown to participate in the binding of bovine pancreatic ribonuclease to ribonucleic acid.204... 

These reactions are the easiest to tackle, since they require only one phosphoryl oxygen to be substituted in both the substrate and the product. The classic example of this experiment is the first step in the hydrolysis of RNA catalyzed by bovine pancreatic ribonuclease. As discussed in detail in Chapter 16, ribonucle-ase catalyzes the hydrolysis of RNA by a two-step reaction in which a cyclic intermediate is formed. The stereochemistry of the first step (cyclization) (equation 8.35),... 

Bovine pancreatic RNase A is a member of a homologous superfamily. In addition, there is a separate family of guanine-specific microbial RNases that have evolved to have a similar active site.192,193 Ribonuclease T1 from Aspergillus oryzae and the 110-residue bamase from Bacillus amyloliquefaciens of Mr 12 392 (see Chapter 19) are the best known examples. One of the histidine residues is replaced by a glutamate in these enzymes. The microbial enzymes are much more amenable to study by protein engineering. 

Bovine pancreatic ribonuclease catalyzes the hydrolysis of RNA by a two-step process in which a cyclic phosphate intermediate is formed (equation 16.35). The cyclization step is usually much faster than the subsequent hydrolysis, so the intermediate may be readily isolated. DNA is not hydrolyzed, as it lacks the 2 -hydroxyl group that is essential for this reaction. There is a strong specificity for the base B on the 3 side of the substrate to be a pyrimidine—uracil or cytosine. 

What is the driving force for protein adsorption Is the adsorption driven by overall energetic (enthalpic) interactions or does the entropic contribution prevail Do both entropic and enthalpic contributions play a major part in the adsorption process, the extent of each depending on the particular protein and surface in question An illuminating thermodynamic analysis given by Norde and Lyklema 62,66) for the adsorption of two different globular proteins (human serum albumin, HSA, and bovine pancreatic ribonuclease, RNase) on polystyrene latices will be presented. We believe this analysis has general validity. 

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