Abstract
Abstract Bovine lactoferrin has a molecular weight of 77,100 ± 1500 in dilute aqueous solution as determined by the method of sedimentation equilibrium in the ultracentrifuge. In 6 m guanidine hydrochloride in the presence or absence of mercaptoethanol, bovine lactoferrin has a molecular weight of 72,500 to 77,200 ±1300 as measured by sedimentation equilibrium in the ultracentrifuge or by gel filtration on agarose columns. The molecular weight obtained by gel electrophoresis in the presence of sodium dodecyl sulfate is 76,000 ± 2400. Tryptic peptide maps reveal a number of peptides approximately equal to the content of lysine plus arginine in the molecule. Quantitative end group analysis shows 1 residue of alanine at the amino terminus of the molecule. These data demonstrate that bovine lactoferrin is composed of a single polypeptide chain. Analysis of the carbohydrate content revealed 1 residue of terminal sialic acid, 10 to 11 residues of N-acetyl glucosamine, 5 to 6 residues of galactose, and 15 to 16 residues of mannose per molecule of lactoferrin. Another protein, isolated as a major contaminant in commercial preparations of bovine α-lactalbumin showed identical properties to bovine lactoferrin except that it appeared, based upon molecular weight measurements, to be an aggregating system.
Highlights
In the ultracentrifuge or by gel filtration on agarose columns
Quantitative end group analysis shows 1 residue of alanine at the amino terminus of the molecule. These data demonstrate that bovine lactoferrin is composed of a single polypeptide chain
An incompletely dissociated or z-linked molecule would yield a molecular weight by this technique lower than the molecular weight determined by sedimentation equilibrium since this method depends upon the hydrodynamic volume as a function of chain length
Summary
JfateriulsThe lactoferrin used in these studies was either isolated from commercial preparations of bovine cu-lactalbumin (Pentex, Inc., Kankakee, Illinois) or generously donated by Dr Philip Aisen.Both preparations were homogeneous as judged by disc gel electrophoresis at pH 8.5 [8] and pH 3.2 in the presence of 6.25 M urea [9].Trypsin (treated with tosylphenylalanylchloromethyl ketone) was obtained from Worthington.Guanidine hydrochloride was purchased from Heico, Inc. (ultra high purity) and used without further purification.Iodoacetic acid was purchased from Matheson, Coleman, and Bell and recrystallized from hexane. The lactoferrin used in these studies was either isolated from commercial preparations of bovine cu-lactalbumin (Pentex, Inc., Kankakee, Illinois) or generously donated by Dr Philip Aisen. Both preparations were homogeneous as judged by disc gel electrophoresis at pH 8.5 [8] and pH 3.2 in the presence of 6.25 M urea [9]. Trypsin (treated with tosylphenylalanylchloromethyl ketone) was obtained from Worthington. Guanidine hydrochloride was purchased from Heico, Inc. (ultra high purity) and used without further purification. Iodoacetic acid was purchased from Matheson, Coleman, and Bell and recrystallized from hexane.
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