Isolation and characterization of canine submaxillary mucin

The isolation and properties of mucin from submaxillary glands of a number of species of animals have been reported (reviewed by Pigman & Gottschalk, 1966).The present communication describes a method for preparation of purified canine submaxillary mucin involving gel filtration on Sephadex G-200 with an additional fractionation step using Bio-Gel CM-100.This method yields a native and highly purified material from submaxillary glands and from submaxillary secretions.The composition of the purified mucin indicates that it is a sulphated glycoprotein containing 35 % protein, 61 % carbohydrate and 3.8% sulphate.The findings extend previous work on canine submaxillary mucin reported by Hashimoto & Pigman (1962).Canine submaxillary glands either were obtained from dogs under anaesthesia and immediately frozen in liquid N2, or were obtained frozen from Pel-Freeze Inc. (Rogers, Ark., U.S.A.).The glands were decapsulated, freed from connective tissue and fat, cut into small pieces and extracted with 2vol. of ice-cold distilled water containing NaN3 (5mM) for 24h at 4°C (extract 1).Extracts 2 and 3 were obtained by repeating the extraction twice with 1 vol. of water for 48h.The extracts were combined and dialysed overnight in the cold against 0.1 M-sodium acetate buffer, pH 3.9, and a small precipitate was removed by centrifugation.Then 80ml of the combined dialysed supernatant, containing 610mg of protein, was applied to a Sephadex G-200 column (5cm x 81cm) previously equilibrated with 0.1 M-sodium acetate buffer, pH3.9.Elution was carried out with the same buffer at a flow rate of 36ml/h.Fractions of volume 20ml were collected, and samples were analysed for protein by measuring the absorbance at 280nm, for hexoses by the orcinol method (Winzler, 1955) and for sialic acid by the direct Ehrlich method (Werner & Odin, 1952).Virtually all of the sialic acid, 93 % of the hexose and 43 % of the protein were eluted in the void volume.The remaining protein was eluted in two retarded peaks, one of which contained some hexose.Re- coveries of sialic acid, hexose and protein were greater than 95 %.For large-scale preparations 300ml (2.29g of protein) was applied to a column of larger size (9.7cmx 105 cm).The fractions constituting the Vol.128