Molecular ecological responses of the dinoflagellate Karenia mikimotoi to phosphate stress

Karenia mikimotoi is a toxic, widespread dinoflagellate which could produce hemolytic toxins and ichthyotoxins affecting fisheries within the area of its bloom. Previous ecophysiological studies indicated that the enhance of environmental phosphate concentration could promote the growth of K. mikimotoi. Intrinsic mechanisms regarding the effects of external phosphate on its photosynthesis, cell cycle succession and differential proteins’ expressions are still unknown. K. mikimotoi was cultured in phosphate-deprived medium, while the culture in f/2 medium (Guillard, 1975) was introduced as phosphate-sufficient control experiment. Cell counts and phosphate concentration detection were performed every other day. Flowcytometry was applied to measure cell cycle succession and chlorophyll fluorescence intensity fluctuation. Differential proteomics expression was examined by SDS-PAGE tandem LTQ Orbitrap MS/MS spectrometry. Functions of each differential protein were searched within NCBInr protein database and Swissprot database. Our study demonstrated that phosphate stress inhibited growth and cell cycle succession of K. mikimotoi remarkably (p < 0.01). Algal chlorophyll fluorescence intensity was significantly affected by phosphate deprivation (p < 0.05). 11 species of differential proteins were detected only in phosphate-limited culture sample which related to stress signal transduction, vacuolar phosphate release, phospholipid degradation, organic acid synthesis and phagotrophy. 4 kinds of differential proteins were identified only in f/2 medium culture sample which referred to cell proliferation, glycolysis, SAM cycle and polyamine production. Based on analysis of differential proteomic functional annotation, we hypothesized proteomic response mechanism of K. mikimotoi to phosphate stress. Molecular biological responses of dinoflagellate K. mikimotoi to phosphate stress was explored.     

Detection of Norspermidine and Norspermine in Medicago sativa L. (Alfalfa)

Shoot meristem tissues of alfalfa, Medicago sativa L., were found by high performance liquid chromatography analyses to contain the uncommon polyamines, norspermidine and norspermine. The chemical structures of norspermidine and norspermine, purified from alfalfa, were confirmed by comparison of mass spectra with those from authentic standards. The discovery of norspermidine and norspermine in alfalfa implicates the presence of at least two biosynthetic enzymes, a polyamine oxidase and a previously uncharacterized aminopropyltransferase.     

Occurrence of uncommon polyamines in cultured tissues of maize

Summary The uncommon polyamines, norspermidine and norspermine, were detected in maizein vitro cultures of three different genotypes. The common polyamines, spermidine and spermine, along with the diamine, putrescine, were also observed. The total amounts of the uncommon polyamines, norspermidine and norspermine, were comparable to the total amounts of the common polyamines, spermidine and spermine, in the maize tissues. The titer for norspermidine was 6- to 15-fold greater than that of its common counterpart (spermidine) in the three genotypes. Norspermidine was the predominant polyamine among all triamines and tetramines detected in cell cultures of two of the three genotypes of maize examined and was predominant along with spermine in the third genotype. Enzyme assays performed with extracts from callus of one of the genotypes suggested a likely mechanism to account for the biosynthesis of the uncommon polyamines in cultured maize cells, through the actions of putrescine aminopropyltransferase, polyamine oxidase, and Schiff-base reductase/decarboxylase enzyme activities. This is the first report of the detection of uncommon polyamines in maize tissues, as well as the first report of these uncommon polyamines in a monocotyledonous plant.     

Comparative role of polyamines division and plastid differentiation of Euglena gracilis

Regulation of polyamine biosynthesis during growth and differentation of Euglena gracilis was investigated. Increased activity of l-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotropically or photoautotrophically grown cells. In photoatotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition from quiescence of active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decaboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena, i.e., a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. α-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed only by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.     

Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.     

Polyamines of the hyperthermophilic archaebacteria belonging to the genera Thermococcus and Methanothermus and two new genera Caldivirga and Palaeococcus

Cellular polyamines of eight new thermophilic archaebacteria were investigated to determine the chemotaxonomic significance of polyamine distribution profiles. Hyperthermoacidophilic Caldivirga maquilingensis belonging to the family Thermoproteaceae of the Crenarchaeota have a unique polyamine profile comprising spermidine, norspermidine and norspermine as the major polyamines. Within the order Thermococcales of the Euryarchaeota, the major polyamines of an extremely thermophilic terrestrial species of Thermococcus, T. zilligii, were spermidine and agmatine, whereas hyperthermophilic submarine species of Thermococcus and hyperthermophilic submarine Palaeococcus ferrophilus contained a quaternary branched penta-amine, N4-bis(aminopropyl)spermidine, as a major polyamine. A hyperthermophilic methanogen, Methanothermus sociabilis, belonging to Euryarchaeota, contained spermidine and spermine as the major polyamine.     

Spermidine promotes human hair growth and is a novel modulator of human epithelial stem cell functions

Background

Rapidly regenerating tissues need sufficient polyamine synthesis. Since the hair follicle (HF) is a highly proliferative mini-organ, polyamines may also be important for normal hair growth. However, the role of polyamines in human HF biology and their effect on HF epithelial stem cells in situ remains largely unknown.

Methods and Findings

We have studied the effects of the prototypic polyamine, spermidine (0.1–1 µM), on human scalp HFs and human HF epithelial stem cells in serum-free organ culture. Under these conditions, spermidine promoted hair shaft elongation and prolonged hair growth (anagen). Spermidine also upregulated expression of the epithelial stem cell-associated keratins K15 and K19, and dose-dependently modulated K15 promoter activity in situ and the colony forming efficiency, proliferation and K15 expression of isolated human K15-GFP+ cells in vitro. Inhibiting the rate-limiting enzyme of polyamine synthesis, ornithine decarboyxlase (ODC), downregulated intrafollicular K15 expression. In primary human epidermal keratinocytes, spermidine slightly promoted entry into the S/G2-M phases of the cell cycle. By microarray analysis of human HF mRNA extracts, spermidine upregulated several key target genes implicated e.g. in the control of cell adherence and migration (POP3), or endoplasmic reticulum and mitochondrial functions (SYVN1, NACA and SLC25A3). Excess spermidine may restrict further intrafollicular polyamine synthesis by inhibiting ODC gene and protein expression in the HF''s companion layer in situ.

Conclusions

These physiologically and clinically relevant data provide the first direct evidence that spermidine is a potent stimulator of human hair growth and a previously unknown modulator of human epithelial stem cell biology.     

Cadaverine: a common polyamine in zygotic embryos and somatic embryos of the species Capsicum chinense Jacq.

The behavior of endogenous polyamines was studied in somatic embryos and zygotic embryos of Habanero pepper (Capsicum chinense). In the first part of the work, the polyamine content was evaluated in both types of embryos (somatic and zygotic). As a result, in addition to the common polyamines (putrescine, spermidine and spermine), it was also possible to detect cadaverine, a polyamine rarely found in plants. In general, all the polyamines were found to be more abundant in somatic embryos than in zygotic embryos, with significantly higher contents of putrescine and cadaverine. Subsequently, the content of putrescine, spermidine, spermine and cadaverine, in their different forms (free, bound and conjugated) was determined in somatic embryos which were cultured in non-ventilated and ventilated containers. Detection of polyamines was carried out at 28 and 42 days of culture by the HPLC method. The ethylene content was monitored during the process in both culture conditions (ventilated and non-ventilated). As a result of the analysis, cadaverine was always found present, indicating that it is a common polyamine in the species. Ethylene was detected in containers without ventilation throughout the culture, except during replenishment of the culture medium (R1, R2 and R3). The behavior pattern of each polyamine, analyzed under different culture conditions (ventilated and non-ventilated) and at different moments of culture (28 and 42 days of culture), show that the polyamines are not only involved in morphogenic processes in plants; polyamines are also significantly affected by the surrounding environment. However, the most novel result, presented for the first time in this paper, is that cadaverine is found to be a common polyamine in C. chinense since it is present in both zygotic embryos and somatic embryos.

     

Ribosome Modulation Factor,an Important Protein for Cell Viability Encoded by the Polyamine Modulon

We searched for proteins whose synthesis is enhanced by polyamines at the stationary phase of cell growth using an Escherichia coli polyamine-requiring mutant in which cell viability is greatly decreased by polyamine deficiency. The synthesis of ribosome modulation factor (RMF) was strongly enhanced by polyamines at the level of translation at the stationary phase of cell growth. In rmf mRNA, a Shine-Dalgarno (SD) sequence is located 11 nucleotides upstream of the initiation codon AUG. When the SD sequence was moved to the more common position 8 nucleotides upstream of the initiation codon, the degree of polyamine stimulation was reduced, although the level of RMF synthesis was markedly increased. Polyamine stimulation of RMF synthesis was found to be caused by a selective structural change of the bulged-out region of the initiation site of rmf mRNA. The decrease in cell viability caused by polyamine deficiency was prevented by the addition of a modified rmf gene whose synthesis is not influenced by polyamines. The results indicate that polyamines enhance cell viability of E. coli at least in part by enhancing RMF synthesis.     

The recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism

Rooting of wild-type tobacco (Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation.     

Polyamines, both common and uncommon, under heat stress in rice (Oryza sativa) callus

Enhanced production and accumulation of free and conjugated polyamines as well as increased activities of their biosynthetic enzymes in plants have been associated with heat stress. Perchloric acid-soluble free, as well as conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated polyamines, as well as arginine decarboxylase (EC 4.1.1.19) and polyamine oxidase (EC 1.4.34) activities were higher in tolerant than in sensitive callus under non-stressed conditions. Heat stress caused greater accumulation of free and conjugated polyamines in callus of the heat-tolerant cultivar N22 than in that of the heat-sensitive cultivar IR8. In particular, the uncommon polyamines norspermidine and norspermine were detected in cv. N22, which increased appreciably during stress, but they were not detected in callus of cv. IR8. Arginine decarboxylase and polyamine oxidase activities increased to a larger extent in N22 than in IR8 callus during stress, activities that were well correlated with the increased levels of common and uncommon polyamines. Increased levels of transglutaminase activity indicated the high titre of conjugated polyamines.     

Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi

Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms.     

Individual-based modelling of the development and transport of a Karenia mikimotoi bloom on the North-west European continental shelf

In 2006, a large and prolonged bloom of the dinoflagellate Karenia mikimotoi occurred in Scottish coastal waters, causing extensive mortalities of benthic organisms including annelids and molluscs and some species of fish (Davidson et al., 2009). A coupled hydrodynamic-algal transport model was developed to track the progression of the bloom around the Scottish coast during June–September 2006 and hence investigate the processes controlling the bloom dynamics. Within this individual-based model, cells were capable of growth, mortality and phototaxis and were transported by physical processes of advection and turbulent diffusion, using current velocities extracted from operational simulations of the MRCS ocean circulation model of the North-west European continental shelf. Vertical and horizontal turbulent diffusion of cells are treated using a random walk approach. Comparison of model output with remotely sensed chlorophyll concentrations and cell counts from coastal monitoring stations indicated that it was necessary to include multiple spatially distinct seed populations of K. mikimotoi at separate locations on the shelf edge to capture the qualitative pattern of bloom transport and development. We interpret this as indicating that the source population was being transported northwards by the Hebridean slope current from where colonies of K. mikimotoi were injected onto the continental shelf by eddies or other transient exchange processes. The model was used to investigate the effects on simulated K. mikimotoi transport and dispersal of: (1) the distribution of the initial seed population; (2) algal growth and mortality; (3) water temperature; (4) the vertical movement of particles by diurnal migration and eddy diffusion; (5) the relative role of the shelf edge and coastal currents; (6) the role of wind forcing. The numerical experiments emphasized the requirement for a physiologically based biological model and indicated that improved modelling of future blooms will potentially benefit from better parameterisation of temperature dependence of both growth and mortality and finer spatial and temporal hydrodynamic resolution.     

Pigment compositions and toxic effects of three harmful Karenia species,Karenia concordia,Karenia brevisulcata and Karenia mikimotoi (Gymnodiniales,Dinophyceae), on rotifers and brine shrimps

Karenia concordia, Karenia brevisulcata and Karenia mikimotoi are anomalously-pigmented gymnodinoids characterised by having fucoxanthin and two acyl-oxyfucoxanthin derivatives, instead of having peridinin as in the majority of photosynthetic dinoflagellates. HPLC pigment analyses of all three species, cultured in identical conditions, revealed two different patterns of pigmentation: (1) 19′-hexanoyl-oxyfucoxanthin-rich with very little fucoxanthin, as represented by K. concordia and K. brevisulcata, and (2) fucoxanthin-rich with much less acyl-oxyfucoxanthins, as represented by K. mikimotoi. Moreover, zeaxanthin was detected in both K. concordia and K. brevisulcata, but only trace amount in K. mikimotoi (generally not detected in this species). Both K. concordia and K. brevisulcata lacked carotene-β,ɛ and carotene-ɛ,ɛ. These differed from K. mikimotoi which generally produced all three carotenes (β,β; β,ɛ and ɛ,ɛ). At exponential growth phase, chlorophyll a content on a per cell basis of K. mikimotoi was more than double that of K. concordia and six times greater than that of K. brevisulcata. Toxicological tests conducted on rotifers elicited distinct responses – single-strength lipophilic cell extracts of both K. concordia and K. brevisulcata killed rotifers in tens of minutes (fast-acting), while that of double-strength K. mikimotoi, in tens of hours (slow-acting). Additionally, mature and nauplii forms of brine shrimps exposed to lipophilic cell extract of K. concordia appeared to be temporarily ‘anaesthetised’, but recovered in c. 45 min and 3 h respectively. No such ‘anaesthetic’ effect was observed on both forms of brine shrimps exposed to lipophilic extracts of either K. brevisulcata or K. mikimotoi. The former, however, killed both forms of brine shrimps in a matter of tens of hours, while those of the latter, did not cause any harm to either form tested.     

Biosynthesis of long-chain polyamines by crenarchaeal polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.     

Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms

Blooms of the toxic dinoflagellate Karenia mikimotoi (K. mikimotoi) have occurred frequently in the East China Sea in recent decades and were responsible for massive mortalities of abalones in Fujian coastal areas in 2012, however, little is known about the effects of these blooms on other marine organisms. In this study, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Calanus sinicus, and Neomysis awatschensis) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated. At a bloom density of 3 × 10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Moreover, the intact cell suspension was toxic to all tested species, whereas cell-free culture and the ruptured cell suspension had no significant effects on the tested organisms. Possible mechanisms for this toxic effect, including reactive oxygen species (ROS) and hemolytic toxins, were evaluated. For K. mikimotoi, 0.014 ± 0.004 OD/(10⁴ cells) superoxide (O₂⁻) and 3.00 ± 0.00 nmol/(10⁴ cells) hydrogen peroxide (H₂O₂) were measured, but hydrogen peroxide did not affect rotifers at that concentration, and rotifers were not protected from the lethal effects of K. mikimotoi when the enzymes superoxide dismutase and catalase were added to counteract the ROS. The lipophilic extract of K. mikimotoi had a hemolytic effect on rabbit erythrocytes but exhibited no significant toxicity. These results suggest that this strain of K. mikimotoi can have detrimental effects on several typical marine organisms and that its toxicity may be associated with intact cells but is not related to ROS or hemolytic toxins.     

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo ( Raphidophyceae ), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua . These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.     

Arrest of cell division blocks the utilization of polyamines in synchronized cultures of photoautotrophically grown Euglena

A trimodal change in the cellular levels of three major polyamines: spermidine, N,N′-bis(3-aminopropyl)-1, 3-propanediamine (BAP) and 3,3′-diaminodipropylamine (DAD) was observed during two successive cell cycles in synchronously dividing cultures of the algal flagellate, Euglena gracilis Z photoautotrophically grown in a 24-h light-dark cycle. The intracellular levels of these three polyamines decreased as cells divided and then were enhanced as cells exited the G₁ phase and proceeded through the S and G₂ phases. Spermidine, BAP and DAD concentrations increased about 2.5-fold during the S phase. Putrescine and 1,3-diaminopropane levels did not vary significantly. One peak of polyamine synthesis occurred in the G₁ phase prior to DNA synthesis, followed by a second more important peak during the S-G₂ phases before cell division; both peaks were observed during the light period. A third minor peak was observed during the pre-G₁ (or G₀) phase in the dark period after mitosis had been completed. In contrast, when the cells attained the “stationary” phase of growth, there was no significant increase in the content of polyamines during the light period although spermidine and BAP increased slightly twice during the dark period (putrescine and 1,3-diaminopropane and DAD levels remained almost constant). To ascertain whether the synthesis of polyamines was merely a direct effect of the photoperiod, parallel experiments with synchronous cultures were carried out in the presence and absence of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, a photosynthetic inhibitor. Although a slight decrease in the concentration of polyamines was observed, the three maxima of polyamines synthesis were observed as in normal cultures. These results clearly suggest that polyamine biosynthesis is closely related to DNA replication and cell division in Euglena cells.     

Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.Abbreviations PAs polyamines - Put putrescine - Spd spermidine - Spm spermine - S free polyamine fraction - SH conjugated polyamine fraction - PH bound polyamine fraction