The inventory and chemical characterization of dissolved proteins in oceanic waters

Abstract

One-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and high resolution two-dimensional electrophoresis (2-DE) were applied to separate protein molecules in dissolved organic matter (DOM) from oceanic waters. Results were: (1) The 2-DE distinguished a total of 412 protein spots in 10 samples from five water columns over the Pacific, although fewer than 30 proteins were resolved as bands from the identical samples by SDS–PAGE. (2) Major and ubiquitous protein bands (34 and 39 kDa proteins) on the SDS–PAGE gel were resolved into horizontally spread arrays (trains) of spots on the 2-DE gels, indicating that these bands were a mixture of protein species that have the same molecular weight (MW) but different isoelectric points (p Is). (3) Proteins that exhibited such electrophoretic patterns on the 2-DE gels were glycosylated with variable linkages between the sugar and polypeptide chains. (4) N-terminal amino-acid sequencing demonstrated that individual spots within each train of spots had identical N-terminal amino-acid sequences. The N-terminal amino-acid sequences of the 39 and 34 kDa glycoprotein spots in samples collected at different sites were also identical. Protein isoforms with the same amino-acid sequence but different glycosylation profiles, termed glycoforms, were often observed on the 2-DE gel. Thirty-one and 24 spots on the 2-DE gels were glycoforms of two glycoproteins with MWs of 39 and 34 kDa, respectively; they were one protein species. The glycoforms of the 39 kDa protein were identified as a low molecular weight alkaline phosphatase (L-AP) of Pseudomonas aeruginosa PAO1 by a homology search through five amino-acid sequence databases. The present and earlier work indicates that all identified source organisms of dissolved proteins belong to the Pseudomonas group. We propose the hypothesis that proteins associated with membrane vesicles liberated from a minor member of the bacterioplankton assemblage, the marine Pseudomonas group, are one of the important sources of dissolved proteins in oceanic waters. This hypothesis may apply to the source pathway and survival not only of proteins and also to the universally occurring bacterial peptidoglycan and lipopolysaccharide components in DOM.