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

Effect of enzymatic cross-linking of β-casein on proteolysis by pepsin

Structural studies of ribonuclease. XXI. The reaction between ribonuclease and a water-soluble carbodiimide.

Peptic digestion of beta-casein. Time course and fate of possible bioactive peptides.

Structural Studies of Ribonuclease. XVIII. An Investigation of the Peptic Digestion Products of Ribonuclease

Structural Studies of Ribonuclease. XIX. Location of the Buried Tyrosyl Residue in Pepsin-Inactivated Ribonuclease*

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Structural Studies of Ribonuclease. XVIII. An Investigation of the Peptic Digestion Products of Ribonuclease

Structural Studies of Ribonuclease. XIX. Location of the Buried Tyrosyl Residue in Pepsin-Inactivated Ribonuclease*