Chemical analysis of the composition of stress induced mucus from Helix aspersa.

Astrid Helmer Svendsen, Simon Kaare Larsen, Kasper Ranum & Jorge Frederico Miranda Vélez

Studenteropgave: Semesterprojekt

Abstrakt

Slime from the land snail Helix aspersa was investigated in this project using various analytical techniques after partial solubilization in NaOH aqueous solution and separation through Size Exclusion Chromatography (SEC) on a Superose HR 6 column. The main batch of slime (FS) used here was obtained from live snails and dissolved in 10 mM NaOH in the laboratory. A second batch of slime (PS) from the same species was obtained from a farm in Poland already dissolved in 0.1 M NaOH. The solubility of freeze-dried PS was tested in a variety of solvents. Highest solubility was observed for 0.1 M NaOH and 10 mM NaOH, while lowest for 1-octanol and methanol. Water content determination performed on FS showed a composition of 93-95 % water, and 87 % of the FS was NaOH-soluble, while 13 % remained almost insoluble even in strong denaturants. Using elemental analysis the C/N ratio of FS was found to be ~7.4. Both batches of slime possessed the same SEC profile, producing a high molecular weight peak at a relative volume of approximately 0.4 (~500-700 kDa, dextran std.), a broad medium weight peak at ca. 0.6 (~80 kDa, dextran std.), and two distinct peaks above 0.9 (below the column’s linear range). One last peak eluted after the total retention volume, at ca. 1.1, due to interaction with the column. Six fractions, splitting the first peak in two, were collected for further analysis. Protein detection was performed through the Bradford assay and by measureing UV absorbance at 280 nm. FS, and fractions 1 and 2 showed consistently to contain protein. Fraction 5 showed high UV absorbance while remaining unreactive to the Bradford assay. Carbohydrate detection was performed using the periodic acid-Schiff’s reagent (PAS) assay. FS, PS and SEC fractions showed very little reaction to the PAS assay, as did a hyaluronic acid control. SDS-PAGE with Coomassie Brilliant Blue G-250 stain was performed, showing a number of distinct protein bands, some of which had lower mobility than the highest molecular weight protein in the ladder (250 kDa). Based on these results, a general structural model is discussed. Additionally, spontaneous acidification in the NaOH-soluble portions of the slime was observed, related to sample preparation, handling and storage.

UddannelserBasis - Naturvidenskabelig Bacheloruddannelse, (Bachelor uddannelse) BasisBasis - International Naturvidenskabelig Bacheloruddannelse, (Bachelor uddannelse) Basis
SprogEngelsk
Udgivelsesdato12 jan. 2016
VejlederePoul Erik Hansen & Søren Hvidt

Emneord

  • SDS-PAGE
  • #twerking
  • #latexmasterrace
  • snail slime
  • helix aspersa
  • HPLC