An investigation has been made of the role of the four disulfide bonds of bovine pancreatic ribonuclease in maintaining the protein in a biologically active form. Studies were carried out to determine the effect of reductive cleavage of these bonds on the ability of the enzyme to catalyze the hydrolysis of ribonucleic acid. The appearance of sulfhydryl groups was taken as evidence that reduction of the protein had occurred.
<P>
No significant reduction or loss of enzymic activity of ribonuclease could be demonstrated when the protein was treated with the reducing agents sodium or potassium borohydride in aqueous solution at room temperature,pH 8.
<P>
Thioglycolic acid was found to be an effective agent for reducing ribonuclease disulfide bonds. At room temperature, in an aqueous solution containing a Large excess of thioglycolate over protein, reduction proceeded slowly at pH 8. At the end of five hours, approximately one disulfide bond was broken, with the loss of 20 per cent of the original enzyme activity. The addition of urea greatly facilitated reduction.
The rate of reduction was especially rapid in solutions of urea concentration greater than 4 molar. In 8 M urea at pH 8, treatment of ribonuclease with thioglycolate resulted in the complete loss of enzymic activity in one half hour, with the simultaneous rupture of two or three disulfide bonds. Under these conditions, maximum reduction was achieved in approximately two hours, with cleavage of between three and four
disulfide bonds per molecule. In the pH range from 3 to 10, rate of activity loss was most rapid at pH 10, slightly less rapid at pH 3, and
reached a minimum near pH 5. The effects of pH and urea were additive in that the maximum rate of inactivation occurred at pH 10 in 8 M urea (97 per cent activity loss in 10 minutes), and the minimum rate st pH 5 in the absence of urea (20 per cent loss in 28 hours).
<P>
Inactivation was markedly inhibited by phosphate ions. A solution of protein which was O.36 M in phosphate at pH 8 lost activity very slowly when treated with thioglycolic acid, even in the presence of 4 M urea. These findings, together with the observation of other workers that polyvalent ions such as phosphate reverse the denaturetion of ribonuclease in urea, suggest that phosphate inhibits reduction by stabilizing the protein in its native configuration, whereas urea facilitates reduction by denaturing the protein.
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Air oxidation of fully or partially inactivated protein resulted, in some cases, in the recovery of up to 4O per cent of the enzyme activity which had been lost as a result of reduction.
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The relation between loss of activity and reduction was analyzed by correlating the data obtained in those experiments in which both the
sulfhydryl concentration and the enzymic activity of samples of modified protein were determined. The experiments were carried out under a variety
of conditions of pH and urea concentration. On the basis of these data, it is concluded that the inactivation of thioglycolate-treated ribonuclease
is probably not a unique function of extent of reduction, but depends in part on the method by which the reduction is achieved.
| Identifer | oai:union.ndltd.org:YALE_med/oai:ymtdl.med.yale.edu:etd-06062011-103356 |
| Date | 06 June 2011 |
| Creators | Steiner, Lisa Amelia |
| Contributors | Frederic M.Richards MD, Werner A Klee MD |
| Publisher | Yale University |
| Source Sets | Yale Medical student MD Thesis |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://ymtdl.med.yale.edu/theses/available/etd-06062011-103356/ |
| Rights | unrestricted, I hereby grant to the Yale School of Medicine the non-exclusive license to photocopy, archive and make accessible, under the conditions specified below, my print and electronic thesis, in whole or in part, in all forms of media. <p> I agree that the Yale School of Medicine may electronically store, copy or translate my thesis to any medium or format for the purpose of preservation and accessibility. The Yale School of Medicine is not under obligation to reproduce or display my thesis in the same format in which it was originally deposited. <p> I retain all ownership rights to the thesis, including but not limited to the right to use in future works (such as articles and books) all or part of this thesis. |
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