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Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection

Section of Biochemistry and Molecular Biology of the Department of Sciences Applied to Biosystems, Cagliari University, Strada Provinciale Monserrato-Sestu Km 0.700, 09042 Monserrato, Cagliari, Italy; Center for the Chemistry of Receptor and Biologically Active Molecules, C.N.R., Rome, Italy; Department of Odontostomatology, Cagliari University, Cagliari, Italy; Institute of Chemistry and Clinical Chemistry, Faculty of Medicine, Catholic University, Rome, Italy
Journal of Chromatography B: Biomedical Sciences and Applications DOI:10.1016/S0378-4347(00)00466-7 pp.153-160

ABSTRACT A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, α-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.

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    Article: Analysis of the Human Salivary Peptidome by Differential Peptide Display and LC-MS/MS Overview Sequencing
    N Le Yondre, H Tammen, R Hess, P Budde, M Jürgens
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    ABSTRACT: In recent years interest in the characterization of the human salivary proteome has increased in order to explore its diagnostic potential. Major constituents of human saliva are highly polymorphic proteins that may have biological roles in oral lubrication and protection, e.g. proline-rich proteins (PRPs), statherins, histatins and cystatins. Interestingly, many of theses proteins are rapidly degraded in the oral cavity by host-and bacteria-or viral-derived proteases. Thus, compre-hensive analysis of peptides up to 15 kDa (peptidomics) of whole saliva may yield basic information on proteolytic pat-terns. By employing a LC-ESI-MS/MS approach a total of 107 native peptides from 17 distinct protein precursors was identified from whole saliva. Subsequently the catalog of peptides was used to analyze inter-individual differences in sa-liva samples from four donors by differential peptide display technology. Genetic polymorphisms were found in peptides from the PRB4M_HUMAN and PROL4_HUMAN precursors. Analysis of the proposed N-and C-termini of the peptides revealed frequent cleavage after Lys or Arg which is characteristic for salivary kallikrein enzymes. Furthermore, we high-light the cleavage motif Gln/Gly in the PRP-C precursor, which suggests a new proteolytic pattern in saliva.
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Keywords

diode-array detection
 
Direct HPLC analysis
 
high-molecular-mass salivary proteins
 
histatin 1
 
histatin 5
 
human salivary peptides
 
intrinsic protease activities
 
mucins
 
normal subjects
 
oral
 
peaks pertaining
 
phosphate buffer
 
resulting acidic sample
 
reversed-phase high-performance liquid chromatography
 
salivary peptides
 
Sample pretreatment
 
sample viscosity
 
simultaneous inhibition
 
uric acid