Publisher Summary This chapter discusses the process of isolation, chromatography, structure, and molecular and catalytic properties of bovine pancreatic ribonuclease. At present there are three simple and widely used chromatographic procedures: (1) Hirs base their method on the carboxyl ion exchange resin IRC-50 with 0.2 M phosphate buffer pH 6.45 as the eluting medium. The principal active component of the enzyme preparation is well retarded and is universally referred to as ribonuclease-A. Several poorly resolved faster running peaks are usually seen, the area having the highest activity and running closest to A normally being called ribonuclease B. The ratio of A to B varies with the preparation but may be as high as 10 to 1. (2) Taborsky has described a system based on carboxymethyl cellulose as the exchanger operated in Tris buffer at pH 8 with a sodium chloride gradient. The excellent and adjustable resolution of this system is frequently useful. The principal peak, labeled D by Taborsky, is indistinguishable from ribonuclease-A in the IRC-50 system. (3) Crestfield found chromatography on sulfoethyl Sephadex valuable. Ribonuclease-A may develop heterogeneity during lyophilization and storage. Aggregation appears to occur. A careful study of the preparation problem has been made by Crestfield by using chromatography on Sephadex G-75, and sulfoethyl Sephadex C-25 as well as IRC-50. These authors recommended that RNase-A be stored as a solution in phosphate buffer at –20°C, that salts be exchanged by dialysis or pre-equilibrated Sephadex columns, and that concentration, if necessary, be effected by ultrafiltration. If lyophilization is necessary, it should be carried out from dilute salt-free solution to minimize aggregate formation. The aggregates can be converted to monomers by heating to 60° for a few minutes at neutral pH. The properties of the ribonuclease dimer are also discussed in the chapter.

24 Bovine Pancreatic Ribonuclease

Publisher Summary Bovine Ribonuclease has been a test protein in the study of a wide variety of chemical and physical methods of protein chemistry. It is widely employed in the course of the sequencing of RNA. This chapter discusses the usefulness of the cytoplasmic inhibitor of RNase to protect RNA in the course of the synthesis of complementary DNA by reverse transcriptase. It examines the use of the inhibitor to protect mRNA during the course of in vitro translation and during the preparation of rough microsomes and detached polysomes. The introduction of affinity chromatography has simplified the isolation of pure RNases by providing a highly efficient method for separating active enzymes from molecules that do not have an affinity for the coupled substrate analog. The technique can be used as an early step in purification or as a final step after ion exchange chromatography and gel filtration to isolate an RNase fraction of given charge and size.

12 Pancreatic Ribonuclease

Publisher Summary This chapter focuses on the different aspects of selective cleavage and modification of peptides and proteins. The selective chemical cleavages of proteins or peptides currently in use are classified into three main groups based upon the site of cleavage relative to the chemically modified residue: (1) cleavage at the α-carbon atom, (2) cleavage at the amino peptide bond, or (3) cleavage at the carboxyl peptide bond. The theme of participation of neighboring groups for peptide cleavage has stimulated numerous variations, and the search for new oxidants, alkylating reagents, and nucleophiles is still going on. Interest is almost equally divided between the dual aspects of most of these reactions; in addition to cleavage procedures, the need for selective modification of proteins continues. In addition, the knowledge of the complete tertiary structure of important enzymes, such as lysozyme, ribonuclease, α-chymotrypsin, and carboxypeptidase, has permitted a sober evaluation of the limitations of the so-called “group-specific reagents” in protein chemistry and has shown the extent of difficulty in finding an easy correlation between chemical reactivity and tertiary structure.

Selective Cleavage and Modification of Peptides and Proteins

The reversible thermal denaturation of bovine pancreatic ribonuclease A at pH 5 in 0.1 M NaCl over the range 32-70 degrees C as studied by Raman spectroscopy proceeds in a gradual manner consistent with a stepwise unfolding process rather than as a transition between two states. Conversion of residues from helical or pleated-sheet geometry to some intermediate geometry, as followed by means of the amide I and III lines, reveals that substantial amounts of the helical and pleated-sheet conformations remain at 70 degrees C. Changes in the strength of hydrogen bonding by the tyrosyl residues are indicated by the intensity ratio of the doublet at 830-850 cm(-1) and changes in the geometry of the disulfide bridges by the frequency and half-width of the Raman line near 510 cm(-1) due to the S-S vibration. Vibrations of C-S bonds in the methionines and cystines are used to monitor conformational changes in these residues. While there are small quantitative differences in temperature dependence among these probes, all agree in placing the malting temperature at or near 62 degrees C. The Raman data are quantitatively consistent with the six-stage scheme of unfolding of A.W. Burgess and H.A. Scheraga [(1975), J. Theor, Biol. 53, 403], except that no change in the environment of the tyrosines is seen until 45 degrees C.

Laser-excited Raman spectroscopy of biomolecules. 9. Laser Raman spectroscopic studies of the thermal unfolding of ribonuclease A

The chemical shift of the C-2 imidazole protons of the histidine residues of pancreatic ribo-nuclease has been studied as a function of pH in 2H2O. Upon protonation of the imidazole ring the C-2 proton magnetic resonance signal shifts about 1.0 ppm to a lower field. Thus titration curves and approximate pK values of the histidine residues are obtained. The pK values of histidine 119 and histidine 12 of the active site are unusually low and shift by 1.0 and 0.7 units to higher values when the NaCl concentration is changed from 0.1 M to 0.4 M, whereas the pK value of histidine 105 shifts only by about 0.2 units. This indicates the presence of a positively charged group in the immediate environment of histidine 119 and histidine 12. The abnormal chemical shift of the histidine 48 C-2 proton at low pH is explained by means of an interaction of histidine 48 with the carboxylate anion of aspartic acid 14. From an analysis of the titration curves of histidine 119 and histidine 12 the existence of an enzyme species in which both imidazoles are connected by a hydrogen bond is derived. The C-2 proton magnetic resonance signals of histidine 119 and histidine 12 shift to a lower field when inhibitors bind to the enzyme. Both histidines are then kept in a protonated form up to the pH range of 7–8. The apparent pK values are higher in the cytidine 2′-phosphate-ribonuclease complex than in the cytidine 3′-phosphate-ribonuclease complex. A similar shift of the histidine 119 and histidine 12 signals is observed with binding of pyrophosphate to ribonuclease indicating that only the phosphate group causes the behaviour of the histidines. Shifts of the C-6 proton magnetic resonance signal of the pyrimidine ring indicate an additional binding of the base to an aromatic side chain of the enzyme.



The mechanism for the binding process proposed in previous papers by Witzel and coworkers in which histidine 119 and lysine 41 are the binding groups of the enzyme, whereas histidine 12 takes up a proton when being released from its binding to histidine 119, is in agreement with the results reported in this communication.

NMR-studies on the structure of the active site of pancreatic ribonuclease A.

Structural studies of ribonuclease. 23. Pairing of the tyrosyl and carboxyl groups.

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Structural studies of ribonuclease. 23. Pairing of the tyrosyl and carboxyl groups.