Isolation of subpollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules
2021
Abstract. Within the last years pollen grains have gained
increasing attention due to their cloud-forming potential. Especially the
discovery that ice nucleating macromolecules (INMs) or subpollen particles
(SPPs) obtained from pollen grains are able to initiate freezing has stirred
up interest in pollen. INMs and SPPs are much smaller and potentially more
numerous than pollen grains and could significantly affect cloud formation
in the atmosphere. However, INMs and SPPs are not clearly distinguished. This
has motivated the present study, which focuses on birch pollen and
investigates the relationship between pollen grains, INMs, and SPPs. According
to the usage of the term SPP in the medical fields, we define SPPs as the
starch granules contained in pollen grains. We show that these insoluble SPPs
are only obtained when fresh pollen grains are used to generate aqueous
extracts from pollen. Due to the limited seasonal availability of fresh
pollen grains, almost all studies have been conducted with commercial pollen
grains. To enable the investigation of the SPPs we develop an alternative
extraction method to generate large quantities of SPPs from commercial pollen
grains. We show that INMs are not bonded to SPPs (i.e. can be washed off with water). Further, we find that purified SPPs are not ice nucleation active:
after several times of washing SPPs with ultrapure water the ice nucleation
activity completely disappears. To our knowledge, this is the first study to
investigate the ice nucleation activity of isolated SPPs. To study the
chemical nature of the INMs, we use fluorescence spectroscopy. Fluorescence
excitation–emission maps indicate a strong signal in the protein range
(maximum around λex = 280 nm and λem = 330 nm) with all ice nucleation active samples. In contrast, with purified SPPs
the protein signal is lost. We also quantify the protein concentration with
the Bradford assay. The protein concentration ranges from 77.4 µ g mL −1 (highly concentrated INMs) to below 2.5 µ g mL −1 (purified
SPPs). Moreover, we investigate the connection between proteins and ice
nucleation activity by treating the ice nucleation active samples with
subtilisin A and urea to unfold and digest the proteins. After this
treatment the ice nucleation activity clearly diminished. The results
indicate a linkage between ice nucleation activity and protein
concentration. The missing piece of the puzzle could be a glycoprotein
which exhibits carboxylate functionalities, can bind water in tertiary
structures, and displays degeneration and unfolding of its secondary
structure due to heat treatment or reaction with enzymes. Even though
purified SPPs are not ice nucleation active they could act as carriers of INMs
and distribute those in the atmosphere.
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