Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

G. G. Kenefick


The purpose of this research was to develop a more efficient procedure that would enable purification of adequate RNase necessary to further characterize the physical, chemical, and catalytic properties-of the enzymes involved. Two cultivars of winter barley were selected because of previous observations showing differences in RNase amounts and types which was related to freeze survival potential. Plant RNases from many species and cultivars of plants have been studied. Reddi proposed a classification system for plant ribonucleases that was later extended by Wilson. All enzymes that cleave a polynucleotide can be identified as either a nuclease or a phosphodiesterase. Wilson classified plant nucleases into three main groups: (a) RNase I, (b) RNase II, (c) nuclease I. RNase is used to describe only those enzymes that are capable of hydrolyzing RNA. Both RNase I and RNase II split RNA by a transfer reaction to form 2'(3')-cyclic nucleotides. However, RNase I hydrolyzes only the purine cyclic nucleotides to produce a 3' nucleotide final product, whereas RNase II hydrolyzes both purine and pyrimidine cyclic nucleotides to 3' nucleotide final products. RNase I has an optimal pH of 5.0 to 6.0 whereas the RNase II optimum is pH 6.0 to 7.0. Enzymes that can hydrolyze DNA and RNA are classed as sugar non-specific enzymes or nucleases. Partially purified RNase preparations have been obtained from various plant tissues, including roots, seed, shoots and leaves. Efforts to obtain pure RNases from plant tissues have been generally unsuccessful; however, there are a few examples of reliable purification. Maize endosperm RNase I has been purified 4,600-fold by Wilson, barley leaf RNase I was purified 29,000-fold by Lantero and Klosterman, and mung bean nuclease I was purified 45,000-fold by Kowalski et al. Purification factors in these reports are of higher magnitude than those usually reported. It was surmised that the low specific activity obtained by Wong might be explained by the presence of large amounts of inactive enzyme which could be removed by an affinity procedure. A purification procedure was adopted in this report that used four different properties of proteins for enzyme isolation. The four properties exploited were: a) differential solubility, b) net charge, c) molecular weight and d) active site specificity. Techniques used include ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and affinity chromatography, respectively.

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South Dakota State University


No Copyright - United State