A biochemical analysis of human cationic salivary histidine-rich polypeptides
MacDonald, Daniel Edwin
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The overall purpose of this study was the biochemical and electrophoretic characterization of a group of human salivary cationic histidine-rich proteins (HRP). The first specific aims were to examine fixation and staining procedures which would better resoIve small quantities of these components. The second aim was to determine the applicability of Bio-Gel P-2 and ammonium sulfate fractionation in the partial purification of these small macromolecules. The third aim was to elucidate the relationship between a recently characterized neutral histidine-rich component (pI 7.0) with the family of basic histidine-rich polypeptides (pI [greater than] 9.0). The final aim was to obtain HRPs from electrophoretic gels by: (a) diffusion (b) electroelution utilizing a modified disc electrophoretic collector (c) electroelution via an ISCO sample concentrator and finally (d) chemical elution. Human parotid saliva has been reported to consist of 7 major histidine-rich protein (HRP) fractions which migrate to a greater degree towards the cathode than lysozyme (pI 11.0; 14,300 MW). These proteins exhibit genetic polymorphism as well as demonstrate both antibacterial and antifungal properties. To date, not all members of this HRP family have been completely isolated or characterized. Resolution of these polypeptides was improved with a modified polyacrylamide gel electrophoresis system (PAGE) suited for the separation of small basic proteins. With this system, the seven major HRP groups were well resolved and the presence of 4 additional minor components were demonstrated. A double staining technique utilizing Coomassie blue R-25O and silver stain provided better fixing and excellent staining characteristics. It could be shown that a high concentration of trichloroacetic acid (2O%) was superior to alcoholic, acetic acid, or sulfosalicytic acid fixation procedures. Enriched fractions of basic HRPs could be obtained by gel filtration of lyophilized human parotid saliva on a Bio-Gel P-2 column. This procedure eluted five peaks, of which the second peak (peak B) contains basic HRPs. Amino acid analysis of the Bio-Gel peak B fractions revealed a three fold increase in histidine when compared with the fractions belonging to the other peaks. Furthemore, salt fractionation with ammonium sulfate of both human parotid saliva and HRP-enriched peak B resulted in partial purification of HRPs. These fracions, which migrated to a greater degree towards the cathode also remained in the supernatant at ammonium sulfate concentrations as high as 8O%. Investigators have described two groups of HRPs: the neutral-HRP characterized by a pI of 7.0 (Hay 1975; Oppenheim et al. 1985) and a group of basic-HRP with a pI in excess of 9.5 (Peters 1974; Peters and Azen 1977). A purified sample of neutral-HRP was subjected to cationic electrophoresis which demonstrated an identical mobility with the slowest member of the basic-HRP group (HRP-1). Based on the apparent similarity of charge and size, these results show for the first time that neutral-HRP is most likely a member of this group termed basic-HRP. Additional investigation into the primary and secondary structure of these components must be made before a final comparison can be drawn. Various procedures of elution from a cationic PAGE system were examined. Best results were obtained by chemical extraction with formic acid and octanol (Gibson and Gracy 1979). Chemically extracted HRP 1-7, including minor components 6a-c, revealed that basic amino acids comprised between 14-42% of each polypeptide. Moreover, between 79-89% of the amino acid residues of the basic HRPs are hydrophilic. Of interest was the amino acid analysis of HRP-7, Obtained with a modified electrophoretic collector (Hanoaka et al. 1979) and chemical elution (Gibson and Gracy 1979), which showed an arginine-rich component. The small size of HRPs (35OO-6OOO daltons) coupled with the large number of hydrophilic residues may explain their resistance to precipitation by ammonium sulfate fractionation procedures.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact email@example.com.Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1986.xiii, 140 leavesBibliography : leaves 113-140.
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