Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

• Published in: Journal of chromatography. B Vol. 861; no. 2; pp. 227 - 235
• Main Authors: Satheesh Babu, A.K., Vijayalakshmi, M.A., Smith, Gary J., Chadha, Kailash C.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 15.01.2008; Elsevier Science
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blotting, Western; Catalysis - drug effects; Chromatography, Affinity; Chymotrypsin - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Male; Medical sciences; Pharmacology. Drug treatments; Prostate cancer; Prostate-specific antigen; Prostate-Specific Antigen - antagonists & inhibitors; Prostate-Specific Antigen - isolation & purification; Prostate-Specific Antigen - metabolism; Prostatic Neoplasms - metabolism; Prostatic Neoplasms - pathology; Substrate Specificity; T-PSA; Thiophilic-interaction chromatography; Zinc; Zinc - pharmacology; T-gel; f-PSA; Prostate-specific antigen; Thiophilic-interaction chromatography; Zinc; TIC; PSA-A 2M; T-PSA; PSA-AT; PSA-PCI; SDS/PAGE; PSA-ACT; Prostate cancer; PSA; ELISA; Urinary system disease; Prostate disease; Interaction; Transition metal; Malignant tumor; Chromatography; Prostate specific antigen; Tissue; Male genital diseases; Cancer
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2007.11.039

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored “thiophilic-interaction chromatography” (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the “free” form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn 2+ ions (10 nM to 50 μM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn 2+ (10 μM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn 2+ (5 μM and lower), the enzymatic activity was enhanced.