Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

Latitudinal differentiation in the effects of the toxic dinoflagellate Alexandrium spp. on the feeding and reproduction of populations of the copepod Acartia hudsonica

Blooms of the dinoflagellate Alexandrium spp. increase in their frequency, toxicity and historical presence with increasing latitude from New Jersey (USA) to the Gaspé peninsula (Canada). Biogeographic variation in these blooms results in differential exposure of geographically separate copepod populations to toxic Alexandrium. We hypothesize that the ability of copepods to feed and reproduce on toxic Alexandrium should be higher in copepods from regions that are frequently exposed to toxic Alexandrium blooms. We tested this hypothesis with factorial common environment experiments in which female adults of the copepod Acartia hudsonica from five separate populations ranging from New Jersey to New Brunswick were fed toxic and non-toxic strains of Alexandrium, and the non-toxic flagellate Tetraselmis sp. Consistent with the hypothesis, when fed toxic Alexandrium we observed significantly higher ingestion and egg production rates in the copepods historically exposed to toxic Alexandrium blooms relative to copepods from regions in which Alexandrium is rare or absent. Such differences among copepod populations were not observed when copepods were fed non-toxic Alexandrium or Tetraselmis sp. These results were also supported by assays in which copepods from populations both historically exposed and naïve to toxic Alexandrium blooms were fed mixtures of toxic Alexandrium and Tetraselmis sp. Two-week long experiments demonstrated that when copepods from populations naïve to toxic Alexandrium were fed a toxic strain of Alexandrium they failed to acclimate, such that their ingestion rates remained low throughout the entire two-week period. The differences observed among populations suggest that local adaptation of populations of A. hudsonica from Massachusetts (USA) to New Brunswick (Canada) has occurred, such that some populations are resistant to toxic Alexandrium.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Intra-population clonal variability in allelochemical potency of the toxigenic dinoflagellate Alexandrium tamarense

Clonal variability in exponential growth rate and production of secondary metabolites was determined from clonal isolates of Alexandrium tamarense originating from a single geographical population from the east coast of Scotland. To assess variability in the selected phenotypic characteristics over a wide spectrum, 10 clones were chosen for experimentation from 67 clonal isolates pre-screened for their lytic capacity in a standardized bioassay with the cryptophyte Rhodomonas salina. Specific growth rates (μ) of the 10 clonal isolates ranged from 0.28 to 0.46 d⁻¹ and were significantly different among clones. Cell content (fmol cell⁻¹) and composition (mol%) of paralytic shellfish toxins (PSTs), analyzed by liquid chromatography with fluorescence detection (LC–FD), varied widely among these isolates, with total PST quotas ranging from 20 to 89 fmol cell⁻¹. Except for strain 3, the toxins C1/C2, neosaxitoxin (NEO), saxitoxin (STX), and gonyautoxins-1 and -4 (GTX1/GTX4), were consistently the most relatively abundant, with lesser amounts of GTX2/GTX3 evident among all isolates. Only clone 3 contained >20 mol% of toxin B1, with C1/C2, GTX2/GTX3 and NEO in almost equimolar ratios.Eight of the 10 clones caused cell lysis of both R. salina and the heterotrophic dinoflagellate Oxyrrhis marina, as quantified from the dose–response curves from short-term (24 h) co-incubation bioassays. For two clones, no significant mortality even at high Alexandrium cell concentrations (ca. 10⁴ mL⁻¹) was observed. Allelochemical activity expressed as EC₅₀ values, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, varied by about an order of magnitude and was significantly different among clones. No correlation was observed between growth rate und allelochemical potency (as EC₅₀) indicating that at least under non-limiting growth conditions no obvious growth reducing costs are associated with the production of allelochemically active secondary metabolites.     

Morphogenetic diversity and biotoxin composition of Alexandrium (Dinophyceae) in Irish coastal waters

The diversity of Alexandrium spp. in Irish coastal waters was investigated through the morphological examination of resting cysts and vegetative cells, the determination of PSP toxin and spirolide profiles and the sequence analysis of rDNA genes. Six morphospecies were characterised: A. tamarense, A. minutum, A. ostenfeldii, A. peruvianum, A. tamutum and A. andersoni. Both PSP toxin producing and non-toxic strains of A. tamarense and A. minutum were observed. The average toxicities of toxic strains for both cultured species were respectively 11.3 (8.6 S.D.) and 2.3 (0.5 S.D.) pg STX equiv. cell⁻¹. Alexandrium ostenfeldii and A. peruvianum did not synthesise PSP toxins but HPLC–MS analysis of two strains showed distinct spirolide profiles. A cyst-derived culture of A. peruvianum from Lough Swilly mainly produced spirolides 13 desmethyl-C and 13 desmethyl-D whereas one of A. ostenfeldii, from Bantry Bay, produced spirolides C and D. Species identification was confirmed through the analyses of SSU, ITS1-5.8S-ITS2 and LSU rDNA genes. Some nucleotide variability was observed among clones of toxic strains of A. tamarense, which all clustered within the North American clade. However, rDNA sequencing did not allow discrimination between the toxic and non-toxic forms of A. minutum. Phylogenetic analysis also permitted the differentiation of A. ostenfeldii from A. peruvianum. Resting cysts of PSP toxin producing Alexandrium species were found in Cork Harbour and Belfast Lough, locations where shellfish contamination events have occurred in the past, highlighting the potential for the initiation of harmful blooms from cyst beds. The finding of supposedly non-toxic and biotoxin-producing Alexandrium species near aquaculture production sites will necessitate the use of reliable discriminative methods in phytoplankton monitoring.     

Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach

Dinoflagellates of the genus Alexandrium are known producers of paralytic shellfish toxins. Species within the genus have similar phenotypes making morphological identification problematical. The use of Alexandrium rDNA sequence data is therefore increasing, resulting in the improved resolution of evolutionary relationships by phylogenetic inferences. However, the true branching pattern within Alexandrium remains unresolved, with minimal support shown for the main phylogentic branch. The aim of this study is to improve phylogenetic resolution via a concatenated rDNA approach with a broad sample of taxa, allowing inference of the evolutionary pattern between species and toxins. 27 Alexandrium strains from 10 species were tested with HPLC for PSP toxin presence and additionally sequenced for 18S, ITS1, 5.8S, ITS2 and 28S rDNA before being phylogenetically inferred together with all available orthologous sequences from NCBI. The resulting alignment is the largest to date for the genus, in terms of both inferred characters and taxa, thus allowing for the improved phylogenetic resolution of evolutionary patterns there in. No phylogenetic pattern between PSP producing and non-producing strains could be established, however the terminal tamarense complex was shown to produce more PSP analogues than basal clades. Additionally, we distinguish a high number of polymorphic regions between the two copies of A. fundyense rDNA, thus allowing us to demonstrate the presence of chimeric sequences within GenBank, as well as a possible over estimation of diversification within the tamarense complex.     

Detection of bacteria originally isolated from Alexandrium spp. in the midgut diverticula of Mytilus edulis after water-borne exposure

Bacteria associated with toxic dinoflagellates have been implicated in the production of paralytic shellfish poisoning (PSP) toxins, but it has not been substantiated that bacteria are truly capable of autonomous PSP toxin synthesis or what role bacteria may play in shellfish toxification. In this study, different putatively PSP toxin producing bacteria originally isolated from toxic Alexandrium spp. were exposed to the blue mussel Mytilus edulis. To document that these bacteria accumulated in the digestive tract of the mussels, hybridization techniques that use rRNA targeted oligonuceotides for in situ identification of these bacteria were applied. The mussel hepatopancreas was dissected and paraffin and frozen sections were made. The dissected glands were hybridized with digoxigenin-labelled 16S rRNA oligonucleotide probes. Results demonstrate that mussels will readily uptake and accumulate these bacteria in the hepatopancreas. However, the mussels were not rendered toxic by the ingestion of the bacteria as determined by HPLC with UV detection for PSP toxins and determination of sodium channel blocking activity using the mouse neuroblastoma assay. Thus, although the role that bacteria play in mussel toxification remains unclear, methods are now available which will aid in further investigation of this relatively unexplored area.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

Toxin production in migrating dinoflagellates: a modelling study of PSP producing Alexandrium

A mechanistic model of dinoflagellate physiology, previously developed and parameterised to simulate paralytic shellfish poison (PSP) content and cell growth for Alexandrium fundyense in response to N and P nutrition, was operated within a vertical water structure in which the organism migrated. Simulations showed the expected development of vertical migration behaviour in response to light and mineral nutrient interactions. Growth in a N-limited water column resulted in a continual, though low level, PSP production with a large population biomass. A sequence of P-stress and nutrient re-feeding during vertical migration stimulated an enhancement of PSP content even with only moderately elevated supply of N:P ratios. This was exacerbated by low absolute P concentrations below the nutricline as well as by the N:P ratio. Although the final biomass was lower in these P-limited simulations, the total toxin production was much higher. The simulations suggest that vertical migration in stratified waters in even moderately high N:P waters could result in the formation of highly toxic populations of Alexandrium. One may expect a similar enhancement of toxicity in other harmful algal species that are engaged in vertical migration, where nutrient supply ratios affect toxin production.     

The toxigenic marine dinoflagellate Alexandrium tamarense as the probable cause of mortality of caged salmon in Nova Scotia

The toxins associated with paralytic shellfish poisoning (PSP) are potent neurotoxins produced by natural populations of the marine dinoflagellate Alexandrium tamarense. In early June 2000, a massive bloom (>7×10⁵ cells l⁻¹) of this dinoflagellate coincided with an unusually high mortality of farmed salmon in sea cages in southeastern Nova Scotia. Conditions in the water column in the harbour were characterised by the establishment of a sharp pycnocline after salinity stratification due to abundant freshwater runoff. In situ fluorescence revealed a high sub-surface (2–4 m depth) chlorophyll peak related to the plankton bloom. The intense bloom was virtually monospecific and toxicity was clearly related to the concentration of Alexandrium cells in plankton size fractions. Cultured clonal isolates of A. tamarense from the aquaculture sites were very toxic on a per cell basis and yielded a diversity of PSP toxin profiles, some of which were similar to those from plankton concentrates from the natural bloom population. The toxin profile of plankton concentrates from the 21–56 μm size fraction was complex, dominated by the N-sulfocarbamoyl derivative C2, with levels of other PSP toxins GTX4, NEO, GTX5 (=B1), GTX3, GTX1, STX, C1, and GTX2, in decreasing order of relative abundance. Although no PSP toxin was found systemically in the fish tissues (liver, digestive tract) from this salmon kill event, the detection of Alexandrium cells and low levels of PSP toxins in salmon gills provide evidence that the enhanced mortalities were caused by direct exposure to toxic Alexandrium cells and/or to soluble toxins released during the bloom.     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K⁺ ion channels (2–6 pA) using the patch-clamp technique when the seals are less than 1 GΩ (about 100 MΩ). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K⁺ flux is due solely to concentration differences. Although conductances are in the range expected for K⁺ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.     

Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species

A series of experiments was conducted to examine effects of four strains of the estuarine dinoflagellate, Pfiesteria shumwayae, on the behavior and survival of larval and adult shellfish (bay scallop, Argopecten irradians; eastern oyster, Crassostrea virginica; northern quahogs, Mercenaria mercenaria; green mussels, Perna viridis [adults only]). In separate trials with larvae of A. irradians, C. virginica, and M. mercenaria, an aggressive predatory response of three strains of algal- and fish-fed P. shumwayae was observed (exception, algal-fed strain 1024C). Larval mortality resulted primarily from damage inflicted by physical attack of the flagellated cells, and secondarily from Pfiesteria toxin, as demonstrated in larval C. virginica exposed to P. shumwayae with versus without direct physical contact. Survival of adult shellfish and grazing activity depended upon the species and the cell density, strain, and nutritional history of P. shumwayae. No mortality of the four shellfish species was noted after 24 h of exposure to algal- or fish-fed P. shumwayae (strains 1024C, 1048C, and CCMP2089) in separate trials at ≤5 × 10³ cells ml⁻¹, whereas higher densities of fish-fed, but not algal-fed, populations (>7–8 × 10³ cells ml⁻¹) induced low (≤15%) but significant mortality. Adults of all four shellfish species sustained >90% mortality when exposed to fish-fed strain 270A1 (8 × 10³ cells ml⁻¹). In contrast, adult M. mercenaria and P. viridis exposed to a similar density of fish-fed strain 2172C sustained <15% mortality, and there was no mortality of A. irradians and C. virginica exposed to that strain. In mouse bioassays with tissue homogenates (adductor muscle, mantle, and whole animals) of A. irradians and M. mercenaria that had been exposed to P. shumwayae (three strains, separate trials), mice experienced several minutes of disorientation followed by recovery. Mice injected with tissue extracts from control animals fed cryptomonads showed no response. Grazing rates of adult shellfish on P. shumwayae (mean cell length ±1 standard error [S.E.], 9 ± 1 μm) generally were significantly lower when fed fish-fed (toxic) populations than when fed populations that previously had been maintained on algal prey, and grazing rates were highest with the nontoxic cryptomonad, Storeatula major (cell length 7 ± 1 μm). Abundant cysts of P. shumwayae were found in fecal strands of all shellfish species tested, and ≤45% of the feces produced viable flagellated cells when placed into favorable culture conditions. These findings were supported by a field study wherein fecal strands collected from field-collected adult shellfish (C. virginica, M. mercenaria, and ribbed mussels, Geukensia demissa) were confirmed to contain cysts of P. shumwayae, and these cysts produced fish-killing flagellated populations in standardized fish bioassays. Thus, predatory feeding by flagellated cells of P. shumwayae can adversely affect survival of larval bivalve molluscs, and grazing can be depressed when adult shellfish are fed P. shumwayae. The data suggest that P. shumwayae could affect recruitment of larval shellfish in estuaries and aquaculture facilities; shellfish can be adversely affected via reduced filtration rates; and adult shellfish may be vectors of toxic P. shumwayae when shellfish are transported from one geographic location to another.     

Histopathology in Pacific oyster (Crassostrea gigas) spat caused by the dinoflagellate Prorocentrum rhathymum

The recognition of an apparent association between seasonal oyster spat mortalities (up to 40%) and high Prorocentrum rhathymum density in the Little Swanport Estuary, Tasmania, prompted further experimental investigation into the toxicity by this dinoflagellate. Standard brine shrimp, haemolysis assays and intraperitoneal mouse bioassays revealed fast acting toxins in methanol but not aqueous extracts of P. rhathymum, with mice dying in less than 20 min. Oyster bioassays involved feeding spat (4 mm shell width) for 21 consecutive days on a diet of cultured P. rhathymum at simulated bloom densities (10⁴ cells ml⁻¹). No oyster mortality was observed, however, histopathological signs of thin, dilated gut tubules and sloughing of gut cells resembled those seen in affected field samples. In contrast to field samples, gill pathology was also observed in experimental exposure oysters.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.