Fractionation of Hagfish Slime Gland Secretions: Partial Characterization of the Mucous Vesicle Fraction

Abstract

This paper deals with the collection, fractionation and partial characterization of the slime gland secretion of the Pacific hagfish (Eptatretusstouti) with emphasis on the mucous fraction. Secretions were collected by electrical stimulation of the glands of anesthetized hagfish and, using three different methods, separated into three fractions: 1) the thread cells, 2) the mucous vesicles of the mucous cells, and 3) the soluble fraction. The methods take advantage of the stabilization of the thread cells and mucous vesicles by ammonium sulfate and sodium citrate. In the first method, the secretions were stirred into 1 M ammonium sulfate containing 1.75 M sucrose. The thread cells were collected by centrifugation, and the mucous vesicles were recovered from the resulting supertatant by centrifugation after dilution with an equal volume of 1 M ammonium sulfate. The remaining supernatant represented the soluble fraction. In the second and third methods, the secretions were stirred into 1 M ammonium sulfate or 1 M sodium citrate, respectively, and the resulting suspension filtered successively through 60 μm, 41 μmand 20 μm nylon meshes. The bulk of the thread cells were retained by the 60 μrn mesh, while the mucous vesicles passed through all of the meshes. The vesicles were subsequently removed by centrifugation. The supernatant, after removal of the mucous vesicles, represented the soluble fraction. The hagfish mucus was characterized as containing, on a dry weight basis, about 77% protein, 12% carbohydrate, 6% sulfate and 5%lipid. Electrophor-etically, the mucus was found to contain two major components and several minor components, as revealed by Coomassie blue staining or photochemical silver staining. The two major components include a very high molecular weight component (does not enter a 3% stacking gel) and a 52k dalton component. The amino acid and sugar compositions somewhat resemble mucin glycoproteins, but differ from other mucins in that the proline content is high, the carbohydrate content is low, the ratio of neutral sugars to amino sugars is low, while the sulfate content is high.