SCHOOL OF ENVIRONMENTAL AND LIFE SCIENCES
Conjoint Professor Tim Roberts

University Drive, Callaghan
NSW 2308 Australia
Phone: +61 2 49215702
Fax:+61 2 49217282
Email: tim.roberts@newcastle.edu.au

28 July 2015
Digestion of Exorphins (Caseomorphin, Dermorphin, Gluteomorphin) with ZYMAX sample provided by:
Direct HealthPty Ltd
39 Evelyn St North,
Sylvania NSW2224

EXORPHINS.
Beta-Casomorphin-7 H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH
• final concentration of 1mg/ml in Phosphate Buffered Saline
• AusPep#2246 BatchM41480
Dermorphin H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2
• final concentration of 1mg/ml in PBS
• AusPep#2029 BatchM41555
Gluten Exorphin-C H-Try-Pro-Ile-Ser-Leu-NH2
• final concentration of 1mg/ml in PBS
• Sigma # E-2019 Lot64H5846
METHOD:
Digestion reaction
A mixture of three opioid peptides (at final concentration of 1mg ml-1 for each peptide) was used as the substrate of the digestion reaction.
One capsule of Zymax was suspended in 1ml of PBS and the supernatant obtained after centrifugation at 13000rpm for 1 minute was used in the digestion reaction.
The digestion reaction tubes contained 1.8mL of substrate and either 0.2mL of Zymax or O.2mL of PBS. The mixture was incubated at 37oC for 4 hours. Samples were taken from the tubes at time 0 min, 10min, 20min, 30min, 60 min 120min, 150min, 180min, 210 min & 240min. Enzyme activity was inactivated by adding 2microlitres of 3M HCl to the samples immediately on collection.
Peptide analysis by High Performance Liquid Chromatography
The digested samples were applied to the HPLC using an Agilent 1100 Autosampler. HPLC was performed using Agilent 1100 Series HPLC-DAD (Agilent Technologies, Waldbronn, Germany) on Phenomenex Jupiter 300 C18 columns part number 00F-4053-E0 with dimensions 4.6 mm  150mm, 5 m particle size and 300 Å pore size at 30oC based on the method by Solaas, Skjeldal, Gardner, Kase and Reichelt {, 2002 #2104} with modifications. Gradient elution was as follows: eluant A was 0.1% trifluoroacetic acid (TFA) in distilled water and eluant B was 0.1% TFA in acetonitrile (ACN). Gradient step 1 was 1 percent eluant B and 99 percent eluant A for 15 minutes to extract most of the amino acids and salts. Gradient step 2 from 15 to 75 minutes was solvent B increasing linearly from 1 to 40 percent by volume. Gradient step 3 from 75 to 80 minutes was solvent B increasing linearly from 40 to 60 percent. Gradient step 4 from 80 to 89 minutes was at 60 percent solvent B. Gradient step 5 from 89 to 94 minutes was solvent B decreasing back to 1 percent. Re-equilibration was run isocratically with 1 percent solvent B from 94 to 115 minutes. Flow rate was 1 ml min-1. The injection volume was 5 microlitres and a needle wash in 100% Milli-Q water was employed immediately prior to sample drawing to minimise sample carryover. Peptides extracted by HPLC were detected by an Agilent 1100 Diode Array Detector at 215 nm and at 280 nm (reference 390 nm). All chromatographic and spectral data was obtained using Agilent Technologies’ G2170BA & G2180BA 3D LC Chemstation (Rev. B.01.01 SR1) running under Microsoft Window XP. The peak areas at 215 and 280 nm were integrated. Analyses were monitored at a retention time from 47 to 54 min.

RESULTS:
The Zymax supernatant was able to digest the three exorphin peptides.
Dermorphin was digested at a marginally slower rate than the exorphins derived from wheat and milk.
Tim Roberts
Conjoint Professor of Biology