Studies on Some Biological Reactions of Agricultural Interest

Edmond, John (1967) Studies on Some Biological Reactions of Agricultural Interest. PhD thesis, University of Glasgow.

Abstract

The general introduction out lines briefly, with particular reference to ribonucleases, the character of the enzymes which depolymerise nucleic acids. The publication by H. S. Kaplan and L. A. Heppel in J. Biol. Chem. 222 907 (1956) that a heat stable ribonuclease M. W. 2,000 - 5,000 could be isolated from calf spleen led to the research work presented in this thesis. These workers reported the purification of a ribonuclease similar to pancreatic ribonuclease in heat stability and specificity. Publications by other workers revealed that several ribonuclease activities could be extracted from calf spleen. The work reported here describes the procedures taken to purify the low molecular weight ribonuclease. Since several ribonucleases were reported, the original purification scheme of Kaplan and Heppel was adhered to initially. Section I outlines this procedure and the salient points are highlighted. In addition to a heat treatment there were four fractionations by conventional precipitation techniques, a lengthy dialysis and an Amberlite resin treatment. Section II, in addition to drafting the criteria for enzyme isolation and purification, reports the investigation of the techniques used originally by these workers. The unfavourable results obtained are presented in detail. Although an equivalent purification was achieved the yield of heat stable ribonuclease activity was poor. Each of the steps rejected ~50% of the activity. With the exception of the heat treatment which was considered essential, this original system was abandoned in favour of preliminary fractionation end concentration by precipitation with ammonium sulphate before and after the heat treatment. In this way ~75% of the heat stable activity was concentrated ready for molecular sieve and ion exchange chromatography. Section III reports the chromatography procedures undertaken to develop a purification scheme for the heat stable spleen ribonuclease. Some early experiments on the gel filtration behaviour of the active sample, particularly with respect to pancreatic ribonueleaee, are described. Gel filtration on a Sephadex G-75 column, 5cm x 75cm, developed to desalt the crude spleen sample. This measure eliminated the lengthy dialysis and achieved a complete recovery of activity with some purification. The desalting technique was followed by ion exchange chromatography. Chromatography on a Carboxymethyl-cellulose column fractionated the heat stable sample into two ribonuclease active peaks "A" and "B". A satisfactory adsorption of the crude sample on the C. M. cellulose was difficult to effect initially thus the sample was reduced by passing it through Diethylaminoethyl-cellulose as a pretreatment. All the ribonuclease activity passed through the column leaving ~37% of the contaminants adsorbed. The preliminary column work necessary to achieve the fractionation on C. M. cellulose using a gradient elution system is described. This column method was scaled up tenfold to cope with the large quantity of crude spleen preparation and prepare sufficient amounts of the active peaks "A" and "B" for rachromatography. Rechromatography on Carboxymethyl Sephadex revealed an elution irregularity at the chromatography on C.M. cellulose. Although ribonuclease active peak "B" was eluted as a single peak, the active peak "A" on rechromatography split into two peaks, one of which was eluted at a similar position to active peak "B". This indicated a distribution of activity for peak "A" similar to the chromatography of the crude preparation. On subsequent chromatography the rechromatographed peak "A" did not split again. These results indicated that calf spleen contains two heat stable ribonucleases. The activity "A" amounted to 16% of the total heat stable ribonuclease as determined by the general assay method. This activity was shown to be as heat stable as the bulk of the reparation. Attempts are made to explain the irregular chromatography effect. Section IV outlines the merit of disc electrophoresis on polyacrylamide gels as a technique for estimating the purity of protein samples. The spleen ribenuclease fractions "A" and "B" were examined by this technique. It was demonstrated that active fraction "B" had been extensively purified and had an electrophoretic mobility similar to pancreatic ribonuclease. Active fraction "A" though considerably purified contained at least three contaminants. An estimate of the molecular weights of the two ribonucleases is presented in Section V. A linear relationship exists between elution volume and log. (molecular weight) for globular proteins at gel filtration. To carry out the estimation, a Sephadex G-75 column was calibrated by protein standards of known molecular weight and gel filtration behaviour. After determining the elution volumes for the heat stable spleen ribonucleases, molecular weights of ~24,000 and 10,000 were attributed to activity "A" and activity "B" respectively. Ho evidence could be found to support the previous report that a heat stable ribonuclease M.W. 2,000-5,000 was present in calf spleen.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry, Organic chemistry, Agriculture
• Date of Award: 1967
• Depositing User: Enlighten Team
• Unique ID: glathesis:1967-78467
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 28 Feb 2020 12:09
• Last Modified: 28 Feb 2020 12:09
• URI: https://theses.gla.ac.uk/id/eprint/78467