Whole-saliva proteolysis and its impact on salivary diagnostics.
Author(s): Thomadaki K, Helmerhorst EJ, Tian N, Sun X, Siqueira WL, Walt DR, Oppenheim FG
Publication: J Dent Res, 2011, Vol. 90, Page 1325-30
PubMed ID: 21917601 PubMed Review Paper? No
Suggested by: Eva J. Helmerhorst, Boston University
Purpose of Paper
The purpose of this paper was to determine the effects of 19 different protease inhibitors, pH, storage duration and temperature and boiling on the saliva proteome and the stability of exogenous histatin 5.
Conclusion of Paper
Only 5 of the tested protease inhibitors prevented hydrolysis, but even when these 5 were combined with EDTA, saliva proteome degradation was only partially attenuated. Reducing the pH of saliva specimens to 3.0 or increasing the pH to 10.0 stabilized the native proteins, but only reducing the pH to 3.0 stabilized exogenous histatin 5. As expected, histatin 5 degradation increased with increasing saliva storage temperature. Boiling the specimens (10 min at 100°C) increased the 1/2 life of histatin 5 at 37°C from 15 min to 9 h.
Studies
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Study Purpose
The purpose of this study was to determine the effects of 19 different protease inhibitors, pH, storage duration and temperature, and boiling on the saliva proteome and the stability of exogenous histatin 5. Saliva was obtained from 5 individuals after 10 min mastication of paraffin wax. All specimens were pooled, placed on ice and aliquoted. Some aliquots were centrifuged for 20 min at 14,000 g at 4°C. Hydrolytic activity was assayed by monitoring the fluorescence after addition of the substrates benzyloxycarbonyl-Phe-His-Glu-Lys-7-amino-4-methylcoumarin (Z-FHEK-AMC) and benzyloxycarbonyl-Arg-Gly-Tyr-Arg-7-amino-4-methylcoumarin (Z-RGYR-AMC). Saliva pH was adjusted by the addition of hydrochloric acid or sodium hydroxide.
Summary of Findings:
Of the 19 protease inhibitors tested, only 4-benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, pancreatic trypsin inhibitor, leupeptin, and antipain showed >85% inhibition of the hydrolysis of exogenous Z-FHEK-AMC and Z-RGYR-AMC. A cocktail of the 5 effective protease inhibitors and EDTA attenuated changes in the proteome peak patterns and delayed degradation of exogenous histatin 5, statherin, and proline-rich protein 1 (PRP1) during 8 h of storage at 37°C in uncentrifuged saliva and even more so in centrifuged saliva. While decreasing the pH to 4.0 had little effect on endogenous protein stability at 37°C, it stabilized histatin-5 for 5 h (as opposed to <1 h), and further decreasing the pH to 3.0 stabilized the endogenous proteome and exogenous histatin-5 for 24 h. Increasing the pH to 10.0 also stabilized the endogenous proteome but only had a small effect on histatin-5 stability. The 1/2 life of histatin 5 decreased with increasing storage temperature (4 h 20 min, 3 h 10 min, 35 min and 22 min at 0°C, 4°C, 22°C, and 37°C, respectively). Boiling the specimens (10 min in a 100°C water bath) increased the 1/2 life of histatin 5 at 37°C from 15 min to 9 h.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide 1D/2D gels Peptide HPLC Protein Fluorometry Protein HPLC Protein 1D/2D gels Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components pH 3
4
7.2
10
Biospecimen Aliquots and Components Centrifugation Not centrifuged
Centrifuged
Biospecimen Aliquots and Components Biospecimen components Histatin 5 added
Statherin added
PRP1 added
Storage Storage temperature 0°C
4°C
22°C
37°C
Storage Storage duration 0 min
5 min
15 min
30 min
1 h
1.5 h
3 h
5 h
7 h
8 h
24 h
Analyte Extraction and Purification Protease inhibitor Alpha-1 antitrypsin
Soybean trypsin inhibitor
Chymostatin
6-Aminohexanoic acid
E-64
N-ethylmalemide
Phosphoramidon
Bestatin
Pepstatin
Aprotinin
Pancreatic trypsin inhibitor
Leupeptin
Antipain
EDTA
2-PDS
4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF)
PMSF
Benzenecarboximidamide (benzamidine)
TLCK
Storage Storage conditions Boiled
Not boiled