University of the Pacific Theses and Dissertations

Campus Access Only

All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Title

Binding study of inorganic phosphate to chicken erythrocyte histone H4 by ³²PO₄ micropartition and by ³¹P-NMR. ¹H-NMR assignment of akrhrkv and its binding to PO₄ : a dissertation

1993

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Micahel Minch

Abstract

New methods were developed to purify histone H4 from chicken erythrocyte nuclei. Hydroxylapatite gel sedimentation required approximately one hour to isolate whole histones from chromatin as opposed to days when the traditional HTP chromatography was employed. In addition, a selective membrane ultrafiltration technique was proven to be as effective as the HTP gel in separating histones from DNA. A reversed-phase high performance liquid chromatography elution profile was developed to isolate histone H4 from other histones. A linear gradient from 95% H$\sb2$O:5% CH$\sb3$CN (both solvents contained 0.1% v/v trifluoroacetic acid) to 42% H$\sb2$O:58% CH$\sb3$CN through a C18 column (Axxiom, 3$\mu$m, spherical particles, 60 A pore size, 4.6 mm x 100 mm) at a flow rate of 1 ml/min for 1 hour yielded electrophoretically pure histone H4. The binding of $\sp{32}$PO$\sb4$ to purified histone H4 was studied by equilibrium partitioning across a 5 kD MWCO semipermeable membrane in an Amicon micro-partition system. A linear Scatchard plot was obtained indicating a stoichiometric ratio of 5 molecules of ligand to every molecule of histone H4 (n = 5.4) and a dissociation constant in the millimolar range $(\rm K\sb{d} = 2.8\times10\sp{-3} M).$ The $\sp{31}$P-NMR linewidths increased markedly and progressively as a function of histone H4 concentration. However, the $\sp{31}$P peak became narrower, although never back to the original value for PO$\sb4$ alone, when the (PO$\sb4$):(H4) ratio was greater than 5. At ratios higher than 5:1, free phosphate contributes significantly to the observed relaxation rate. Hence, both types of studies suggest the same 5 to 1 stoichiometry for the inorganic phosphate-histone H4 interaction. Two-dimensional high-resolution NMR was used to assign all resolvable peaks from a 500 MHz $\sp1$H-NMR spectrum of N-acetyl-AKRHRKV, an oligopeptide that resembles an important part of the N-terminal end of histone H4. Binding of this peptide segment with inorganic phosphate was studied by $\sp{31}$P- and $\sp1$H-NMR. Analysis of $\sp{31}$P-NMR linewidths with added peptide revealed a dissociation constant of $7.04\times10\sp{-2}$M. The apparent spin-spin relaxation times for free, $\rm T\sbsp{2}{F},$ and histone-bound phosphate, $\rm T\sbsp{2}{B},$ were calculated to be 28.7 mseconds and 6.3 mseconds respectively. $\sp1$H-NMR TOCSY and NOESY spectra indicated that this peptide adopts a loop conformation with the C-terminal valine H-bonded to the histidine carbonyl. The involvement of the histidine imidazole ring in phosphate binding is also indicated by the shifts of the H2 and H4 protons in the presence of PO$\sb4$.

Pages

224

To access this thesis/dissertation you must have a valid pacific.edu email address and log-in to Scholarly Commons.

Share

COinS

If you are the author and would like to grant permission to make your work openly accessible, please email