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#25a: Enzymatic Hydrolysis of Recalcitrant Steroids with Engineered Arylsulfatases
ABSTRACT:
INTRODUCTION
Over the last decade, detecting sulfated anabolic androgenic steroids (AAS) has been described as preferred analytical methods. However, its direct detection can be limited due to poor cleavage of the sulfate, especially in testosterone and its derivative, boldenone. Poor recovery of dehydroepiandrosterone sulfate (DHEAS) has also been a major challenge in steroid testing community.
OBJECTIVE
There are no reported sulfatases that can cleave sulfated metabolites of testosterone, boldenone or dehydroepiandrosterone. These are the first reported, genetically engineered variants of sulfatases completely hydrolyze testosterone- boldenone- 17beta sulfates and DHEA-3beta sulfates.
METHODS
Steroid standards, internal standards, and sulfated metabolites were from Cerilliant, Steraloids, TRC and Fisher. Chemicals were purchased from MilliporeSigma and Fisher Scientific. Drug free human urine controls were from UTAK. Urine controls were fortified with sulfated metabolites and hydrolyzed up to 4 hours at 37°C. After hydrolysis, samples were diluted to 50% methanol and eluted through a β-Gone Plus plate from Phenomenex. 10 uL of diluted sample was injected on a Thermo Scientific® Vanquish® UHPLC system coupled to Thermo Scientific® Endura® Triple Quadrupole Mass Spectrometer using a Phenomenex Kinetex® 2.6 um Biphenyl 100 Å, 50 x 4.6 mm column. Mobile phases A and B were 0.1% formic acid in water and methanol, respectively. Calibration curves had r2 ≥ 0.99 and quality controls were within ± 20%.
RESULTS
Engineered arylsulfatases from P. aeruginosa can hydrolyze difficult substrates such as boldenone sulfate and dehydroepiandrosterone sulfate within 2 hours at 37°C, whereas all prior purified sulfatases have little or no observed activity towards these analytes.
CONCLUSION
Difference in reactivities were observed for sulfatases towards 3beta and 17beta sulfates. Milder reaction conditions were established for liberating sulfated steroids in urine. Larger, comparative studies need to be performed in a broader range of biological matrices before enzymes are recommended for routine use in steroid testing.
We systematically purified pDNA from bacterial lysates containing plasmids of different sizes (3262 – 8484 bps) using our automated method employing loosely-packed silica tips. The effectiveness of our method was benchmarked against commercially available spin plates and magnetic bead kits. Our method demonstrated higher overall recoveries when compared to magbeads and comparable yields when compared to the manual spin plate, yielding pDNA amounts between 8.92±0.62 µg and 12.28±0.24 µg. Moreover, the resulting purity compared well to alternative methods, with 260/280 and 260/230 ratios of 1.85 and 2.03 across all samples.
Overall, yields and purity were comparable to the spin-plate method. Time from sample isolation to purification was under one hour for up to 96 samples, comparable to the spin-plate method and faster than the magbead workflow (75 minutes). This work represents an adaptation of a traditional plasmid purification workflow for automated liquid handlers. Challenges such as carryover and buffer selection when the purification is automated in pipette tips are identified and how these challenges were overcome are discussed. The workflow can process 96 samples quickly (<60 minutes), while maintaining high yields (>10 µg) and excellent purity.
