Preparation and properties of poly-L-tyrosyl acetamidinated ribonuclease Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/8049g806n

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • Polypeptidyl derivatives of bovine pancreatic ribonuclease (RNase) containing two or three tyrosine chains with an average of two to three residues per chain have been prepared. The effects of various perturbants such as neutral salts, ethanol, urea and guanidine hydrochloride on the thermal transition of the derivatives have been studied. The forces responsible for maintaining the native conformation of RNase appear to be unaffected by chemical modification. In the association-dissociation reaction of the polypeptidyl derivatives of RNase, hydrophobic interactions are strongly implicated. Acetamidinated-RNase I was prepared by treating a RNase solution with excess acetimidate hydrochloride at pH 7.8 in 0.1 N NaHCO₃ buffer containing Na₂SO₄. After removal of reagents by dialysis, polypeptidylation was carried out under the same conditions by reacting the acetamidinated-RNase derivative with excess N-carboxy-L-tyrosine anhydride. There were then obtained a soluble fraction (IA) and an insoluble fraction (IB) of polytyrosyl-acetamidinated RNase (PT-Ac-RNase). IB could be dissolved in 0.0025 N HCl (IC). Fractions IA and IC were purified by gel filtration prior to experimental investigation. Another preparation (PT-Ac-RNase II) was carried out in phosphate buffer under comparable conditions as the preparation of PT-Ac-RNase I. Analysis for added amino acid residues and for the number of sites amidinated or peptidylated indicated that IA contained three peptide chains of average chain length slightly less than three tyrosines. The fraction IC had 17 moles of tyrosine per mole of protein, giving an average chain length of about six tyrosines. PT-Ac-RNase IIA had 4 moles of tyrosine per mole of protein attached to two sites. The enzymatic activities of the PT-Ac-RNase derivatives were shown to be about 5 percent of the unmodified enzyme by two methods of assay. The melting temperatures (Tm) as determined by difference spectra at 287 mμ and 237 mμ for RNase, acetamidinated RNase and PT-Ac-RNase in 0.05 M salt at pH 2.1 were very similar and varied between 33° and 35°. But calculations for Δε₂₈₇ mμ and Δε₂₃₇ revealed respective values of 1000 and 3000 for the PT-Ac-RNase derivatives. The Tm of PT-Ac-RNase IA was shown to be dependent on PH, urea, ethanol and guanidine trydrochloride. Lowering pH or increasing the concentration of perturbant would shift the Tm to lower temperature. However, the same observation has also been reported for native RNase. PT-Ac-RNase IC became insoluble between pH 5 and pH 9, but IA was soluble throughout this pH range. Although turbidity formation was enhanced by increasing concentration of KCI, addition of guanidine hydrochloride could prevent turbidity formation. Increasing concentration of KCI in the PT-Ac-RNase II solution at pH 2.1 shifted the Tm to higher temperatures and could induce turbidity formation at 1.2 M concentration. A similar effect of increasing the Tm of PT-Ac-RNase II was observed with CaCl₂, at pH 2.1. With 0.5 M KSCN precipitation of the protein derivative occurred. A higher enrichment of tyrosine as a result of peptidylation must account for these experimental observations. Spectrophotometric titration of PT-Ac-RNase II revealed seven normal and three abnormal tyrosines, indicating that the tyrosine residues covalently attached to the surface of the protein molecule are titrated normally. A thermodynamic treatment of the melting profiles of native RNase, Ac-RNase and PT-Ac-RNase derivatives in the presence of various concentrations of CaCl₂, was carried out. The approximation of two-state transition was employed to estimate the various thermodynamic parameters. The increase in heat capacity due to addition of extra tyrosine residues in the PT-Ac-RNase derivatives could probably account for the striking curvature at both low and high temperatures in the van't Hoff plots. However, the theory of gradual "unwinding" of the molecule cannot be excluded.
Resource Type
Date Available
Date Copyright
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Academic Affiliation
Non-Academic Affiliation
Keyword
Subject
Rights Statement
Peer Reviewed
Language
Digitization Specifications
  • File scanned at 300 ppi using Capture Perfect 3.0 on a Canon DR-9050C in PDF format. CVista PdfCompressor 5.0 was used for pdf compression and textual OCR.
Replaces
Additional Information
  • description.provenance : Approved for entry into archive by Katy Davis(kdscannerosu@gmail.com) on 2014-05-20T16:40:20Z (GMT) No. of bitstreams: 1 LynnJackson1966.pdf: 3324439 bytes, checksum: 99269cdb9ced5c8b206bf6b7ae332583 (MD5)
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2014-05-20T13:13:01Z (GMT) No. of bitstreams: 1 LynnJackson1966.pdf: 3324439 bytes, checksum: 99269cdb9ced5c8b206bf6b7ae332583 (MD5)
  • description.provenance : Made available in DSpace on 2014-05-20T16:40:20Z (GMT). No. of bitstreams: 1 LynnJackson1966.pdf: 3324439 bytes, checksum: 99269cdb9ced5c8b206bf6b7ae332583 (MD5) Previous issue date: 1966-05-12
  • description.provenance : Submitted by Georgeann Booth (gbscannerosu@gmail.com) on 2014-05-20T00:11:43Z No. of bitstreams: 1 LynnJackson1966.pdf: 3324439 bytes, checksum: 99269cdb9ced5c8b206bf6b7ae332583 (MD5)

Relationships

Parents:

This work has no parents.

Last modified

Downloadable Content

Download PDF

Items