International Journal of Biological Macromolecules 26 (1999) 353–356 AFM imaging of surface adsorbed polymeric 3-alkylpyridinium salts from the marine sponge Reniera sarai Kristina Sepc ˇic ´ a, *, Igor Mus ˇevic ˇ b,c , Gojmir Lahajnar a,c , Tom Turk a , Peter Mac ˇek a a Department of Biology, Biotechnical Faculty, Uniersity of Ljubljana, Vec ˇna pot 111, 1111 Ljubljana, Sloenia b Faculty of Mathematics and Physics, Uniersity of Ljubljana, Jadranska 19, 1000 Ljubljana, Sloenia c Joz ˇef Stefan Institute, Jamoa 39, 1000 Ljubljana, Sloenia Accepted 26 July 1999 Abstract Bioactive 3-alkylpyridinium polymers (poly-APS) have recently been isolated from the marine sponge Reniera sarai. Previous results have shown that these molecules in aqueous solutions form supramolecular aggregates with an average hydrodynamic radius of 23 2 nm. To obtain additional evidences about the shape and the dimensions of poly-APS aggregates, we used atomic force microscopy (AFM) operating in tapping mode. The images clearly showed adsorbed aggregates with a lateral dimension of 40 nm and a thickness of the order of 1 nm. The distribution of volumes of the adsorbed aggregates is very similar to the distribution of hydrodynamic radii as obtained from the dynamic light scattering experiments. The volume distribution of these aggregates shows a maximum at 1750 nm 3 , which corresponds to a sphere with a radius of 7.5 nm. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Atomic force microscopy; Polymeric 3-alkylpyridinium salts; Reniera sarai www.elsevier.com/locate/ijbiomac 1. Introduction Recently, high molecular weight polymeric alkylpyri- dinium salts (poly-APS) were isolated from the crude extract of the marine sponge Reniera sarai [1]. Poly- APS are 3-octylpyridinium polymers, which are ‘head to tail’ connected between the nitrogen of one ring and the terminus of the 3-octyl chain of the next. According to MALDI-TOF spectrometry, poly-APS are a mixture of two polymers with the same basic structure but with different molecular weights, i.e. 5520 and 18 900 Da. These correspond to polymers built of 29 and 99-100 3-octylpyridinium units, respectively. Poly-APS exert a broad spectrum of different biolog- ical activities, especially a strong inhibition of several cholinesterases [2,3], hemolysis and cytotoxicity [4]. In contrast to other 3-alkylpyridinium polymers that have been isolated from sponges so far and that are soluble exclusively in organic solvents [5–8], poly-APS are soluble in water only. This difference is very likely a consequence of the higher polymerisation degree, as also observed by Gil et al. [9] for synthetic 3-alkylpyri- dinium oligomers and polymers mimicking the poly- APS structure. In contrast to MALDI-TOF spectrometry which re- mains ambiguous in the estimation of the m.w. of non-covalent aggregates of organic polymers [10], size- exclusion chromatography of poly-APS on Sephadex G-100 or Sephadex G-200 beds always revealed only one peak that eluted with the void volume. The elution profile was not dependent on poly-APS concentration. It was concluded that the eluted species should be aggregates of 5520 and 18 900 Da poly-APS molecules. In order to obtain more information about poly-APS aggregates in aqueous solutions, dynamic light scatter- ing (DLS) and static light scattering (SLS) were applied to poly-APS water solutions [1]. DLS measurements demonstrated that poly-APS form particles with a di- ameter ranging from 15 to 80 nm and an average Abbreiations: AFM, atomic force microscopy; DLS, dynamic light scattering; MALDI-TOF, matrix assisted laser desorbtion/ionization time-of-flight; poly-APS, polymeric alkylpyridinium salts; SLS, static light scattering. * Corresponding author. Tel.: +386-61-123-3388; fax. +386-61- 273-390. E-mail address: kristina.sepcic@uni-lj.si (K. Sepc ˇic ´) 0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S0141-8130(99)00106-3