Date of Award

1993

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Supervisor

Lee, Lana,

Rights

info:eu-repo/semantics/openAccess

Abstract

1. Platelet factor 4 (PF4) is a platelet protein believed to be involved in hemostasis and the immune response. $\sp1$H-NMR spectroscopy was used to study histidine residues in human and bovine PF4. In bovine PF4, one histidine has a pKa of 6.51, while the second exhibits pKa values of 5.56 and 5.67 at an ionic strength of 0.75 M NaCl. The deuterium exchange half times of the two histidines were 3.2 and 3.9 days at pH 8.8. Differentially exchanged bovine PF4 was digested with V8 protease, and histidine containing peptides isolated. Analysis of the degree of exchange of these histidines allowed assignment of the lower pKa histidine to His-38, and the higher pKa histidine to His-50. The pKa values human PF4 histidines were determined at 0.75 M NaCl to be 5.09 and 5.31 for the low pKa histidine, and 6.34 for the high pKa histidine. The two histidines had similar deuterium exchange rates of 3.8 and 3.9 days at pH 8.2, which precluded assignment. The low pKa histidine was attributed to His-23, and the high pKa histidine to His-35 based upon similarity in behavior to the homologous bovine histidines. The interaction of human and bovine PF4 with a 16.5 kDa heparin was studied using histidine resonances as spectroscopic probes. The lower pKa histidine in both proteins experienced greater perturbation than the high pKa histidine. The pKa of bovine His-38 increased by 0.26 pH units to 5.87 in the presence of saturating amounts of heparin, while the pKa of His-50 increased by only 0.07 pH units to 6.58. Similarly, the pKa of human His-23 increased 0.57 pH units to 5.77, and the pKa of His-35 increased by 0.17 pH units to 6.51. The greater perturbation of His-38, which is located in a belt of positive charge, supports the heparin biding model that has heparin crossing over the $\alpha$-helices at right angles rather than binding in the groove between the helices. 2. ArsC is a 16 kDa protein believed to be involved in arsenical resistance in E. coli. It was found to be sensitive to air oxidation, which resulted in high order aggregates. This aggregation could be reversed by reduction of the protein with DTT. Titration of ArsC with either arsenate or arsenite did not cause noticeable spectral perturbation. Source: Dissertation Abstracts International, Volume: 54-09, Section: B, page: 4656. Adviser: Lana Lee. Thesis (Ph.D.)--University of Windsor (Canada), 1993.

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