The Rate of Air-Sea CO2 Exchange: Chemical Enhancement and Catalysis by Marine Microalgae.

Chapter 2: Introduction: Carbonic Anhydrase in Marine Algae and the Sea-surface Microlayer

Carbonic anhydrase (hereafter referred to as CA) is an enzyme which catalyses the hydration and dehydration of CO2. CA plays a critical role in the physiology, both of respiration in animals, and of photosynthesis in plants, including marine microalgae. These physiological roles, the properties and kinetics of the enzyme and the practical means of investigating them, will be introduced later in this chapter. Firstly, however, we should consider the origin of the hypothesis, that carbonic anhydrase may be significant in catalysing the chemical enhancement of air-sea CO2 exchange.

2.1 Carbonic anhydrase and air-sea CO2 exchange

The first link between carbonic anhydrase and the air-sea CO2 exchange rate was made by Kanwisher (1963), who deliberately added Bovine CA to his small wind-wave tank. With a "stagnant film thickness" of 50-70μm (see section 1.2.4), he found that doubling the reaction rate by addition of enzyme had little effect, and it had to be increased tenfold to significantly increase the measured transfer velocity. This experiment demonstrated that the chemical enhancement effect was real, even if not important in average conditions. Quinn and Otto (1971) used their iterative model to predict that about 3mg l-1 (≈10-7 Molar) CA would be needed to significantly increase the transfer velocity in typical conditions at sea. However, Kanwisher (1963) also observed that CA was rapidly denatured in seawater, and did not suggest that it would be present naturally.

Berger and Libby (1969) first proposed that CA produced by marine organisms might naturally catalyse the rate of air-sea CO2 exchange. Samples of seawater were kept in barrels and aerated with air, which at the time was enriched by bomb 14C (see section 1.2.11). They found that the 14C entered seawater samples taken from 60m depth, about 20 times faster than seawater samples taken from the surface off Santa Monica beach. However, the rate of penetration into surface water samples taken from the same location a few months later, to which 0.5 mg l-1 CA had been added, was similar to that of the deep water samples. On this basis, they suggested that CA might have been present in the deep samples, whereas it was denatured by oxidation in surface waters.

To examine this hypothesis, Goldman and Dennet (1983) investigated the transfer of CO2 into samples of natural and artificial seawater in a small stirred tank, measuring the CO2 in the headspace using an infra-red gas analyser. They found that addition of bovine CA at either 0.5 or 20 mg l-1 increased the exchange rate by a maximum factor of 2 or 3, at medium stirring speeds (corresponding to a stagnant film thickness of about 450μm). Addition of the enzyme inhibitor ethoxyzolamide (see section 2.7.3) reduced the exchange rates back to their original values, suggesting that there was no significant amount of CA originally present in the natural seawater samples, which were collected from various locations, both at the surface and at depth, and also included samples from an aquarium. Goldman and Dennet (1983) therefore refuted the hypothesis of Berger and Libby (1969), and proposed that the very low exchange rate which they found using their first surface water samples might have been caused by an organic film inhibiting gas exchange (see section 1.4.5), which was not present a few months later.

The hypothesis was also investigated by Williams (1983), who compared the rate of the CO2 hydration reaction in distilled water, with that in natural lake-water samples and one seawater sample, measuring the reaction rate using the stopped-flow pH indicator technique (see also section 2.6.1). Generally, they found no significant difference, and the measured reaction rates supported earlier literature values (see section 1.5.2). In the case of the alkaline "Walker Lake", however, the hydration rate measured in lake water was about 25% faster than in the control, which might possibly be attributed to catalysis by CA. It should be noted that Peng and Broecker (1980) found that 14C invasion into this lake was 5 times higher than predicted.

A general problem with all these experiments is that CA has a short lifetime in seawater (Kanwisher 1963, and experimental results in section 3.5 and section 7.6), and we are not told how much time elapsed between collecting the samples, and measuring the gas exchange. Moreover, a general conclusion regarding the whole ocean cannot be drawn from so few samples. On the other hand, it was hardly plausible that the fish, shellfish and other large marine organisms then known to produce CA, as referred to by Berger and Libby (1969) and subsequent authors, might release enough enzyme to significantly catalyse the global atmosphere-ocean CO2 flux. It was not until the more recent discovery of the importance of CA in marine microalgae (see section 2.3), together with the continuing discrepancy between the global average transfer velocities calculated from the 14C data, compared to those measured using inert trace gases (see section 1.2.7), that interest revived in this hypothesis.

Keller (1994) and Emerson (1995) suggested that it might be possible to constrain the maximum concentration of CA in seawater, by considering the concentration of zinc, since each molecule of CA requires at least one zinc (or possibly cadmium) atom at the active site of catalysis (see section 2.2.2). The concentration of zinc in ocean waters is low, typically less than one nanomolar (nM), but it may be enriched by an order of magnitude or more in the surface microlayer (see section 2.4.4) where any catalysis of air-sea CO2 transfer will take place. Keller (1994) calculated that an enzyme concentration of 2x10-8 M in the microlayer would result in a global average enhancement factor (see section 3.3.4) of 1.24 for an average film thickness of 65μm. Emerson (1995) also discussed how much CA might significantly enhance the air-sea CO2 flux, but calculated very high rates of catalysis inconsistent with experimental results. I believe this was due to a misinterpretation of the enzyme kinetic constants, and so it is important to consider how these are derived, and how they should be applied to the conditions of air-sea CO2 transfer.

Before delving further into such calculations, however, it is necessary to introduce the properties of the enzyme and its physiological role in marine microalgae. This may be particularly important, because recent theoretical work and culture studies (see section 2.3.4) have shown that many species of marine microalgae only produce CA in low pCO2 conditions. Although Keller (1994) demonstrated that the small bias in the reaction rate via the OH- reaction pathway towards lower pCO2 conditions could make a big difference to the net global air-sea CO2 flux (see section 1.5.6 and section 3.3), he did not consider that a bias in the distribution in carbonic anhydrase might have a similar effect. The use of a "physiological distribution" of CA, in calculating the possible effect of catalysis of the net global air-sea CO2 flux, will be demonstrated by the calculations in the chapter 3 and chapter 9.

2.2 Carbonic Anhydrase in mammalian respiration

2.2.1 Discovery of carbonic anhydrase

It was the study of CO2 transport in the blood and across the surface of the human lungs which led to the discovery of carbonic anhydrase. At the pH of blood, which is similar to that of seawater, most dissolved CO2 is in the form of bicarbonate ions, but the dehydration reaction was known to be slow. As early as the 1850s, Lavoisier was investigating the transport of CO2 out of the human lung, and suggested that an "acide pneumique" might be produced in the alveoli to ensure rapid dehydration of carbonic acid, but this theory was later dismissed. In 1928 Henriques calculated the flux of CO2 out of the lung based on the uncatalysed CO2 dehydration rate, and found it was much smaller than actually measured in expired air. He then showed experimentally that blood could release CO2 much faster than serum. This led to the search for an enzyme, which was first isolated by Meldrum and Roughton (1933), although its structure and mechanism were not known until much later. The story of this discovery, which was compared earlier with the current problem of the global air-ocean carbon flux (see overview), is told well by Roughton (1935).

2.2.2 Structure and Mechanism

Since then, there has been much research on the structure, mechanism and physiology of carbonic anhydrases in mammals. It was found that there are several distinct types, with different physiological functions. For instance, carbonic anhydrases catalyse the hydration /dehydration of esters as well as of carbonic acid. Maren et al (1976) showed that CO2 hydration /dehydration by human CA types "A" and "B" (or "I") was strongly inhibited by chloride ions in the blood (see also section 2.7.3), and these must therefore have some other function than that of a carbonic anhydrase. It is thus the human CA type "C" (or "II") which performs the CO2 hydration /dehydration function, and this also has similar properties to the commercially available bovine CA and to CA found in some microalgae.

The structure of human carbonic anhydrase C was described by Liljas et al (1972). It is a protein composed of 260 amino acids wrapped around a central zinc atom bound to 3 histidine ligands which forms the active site, as shown in Figure 2-1(a). The zinc atom can sometimes be substitued by cobalt or cadmium in marine microalgae (Yee et al 1996, Lee et al 1995). The mass of one molecule (or one active unit of a macromolecule -see section 2.6.3) is about 35,000 Da, and the diameter 5 nm. Recent analysis and quantum-mechanical simulations (e.g. Aqvist et al 1992, Liang and Lipscomb 1989, Silverman 1991) have now led to a consensus over the catalytic mechanism, which is shown in Figure 2-1(b).

The zinc ion aids the electron transfer around the six-membered ring, but it is the transfer of protons to or from this active site which is actually the rate limiting process. Khalifah (1971) pointed out that carbonic anhydrase was such an effective catalyst that the hydration / dehydration appeared to occur faster than diffusion could carry away or supply protons. To explain this paradox he proposed that buffer ions, which are abundant in blood and other physiological media, must carry the protons to the surface of the enzyme. Later, Liang and Lipscomb (1989) explained how protons are transferred from the surface to the active site by a neat relay mechanism involving three partially ordered water molecules trapped inside the protein, a theory recently expanded by Lu and Voth (1998). High concentrations of buffer are used in most enzyme kinetic experiments, so proton supply should not be a problem and catalysis was generally reported to be buffer independent. However, using a novel 18O isotope method, Silverman and Tu (1975) showed that buffer concentrations below 10mM can be rate limiting. This may therefore be a problem in seawater, as discussed later (section 2.3.2).

2.3 Carbonic anhydrase in photosynthesis

2.3.1 Evolution of plant carbonic anhydrase

In the 1950s carbonic anhydrase was found to be equally important for the reverse process in plants, i.e. the supply of CO2 for photosynthesis (see for example Graham et al 1984). CA was isolated from agricultural plants such as spinach and peas, although these molecules have a much greater mass possibly consisting of several similar subunits, and also differ from mammalian CA in their response to enzyme inhibitors. CA was also extracted from freshwater algae, particularly Chlamydomonas Reinhardii which has been intensively investigated (see references in Table 2-1 ), and the activity (Bundy 1986, see Table 2-2) and protein sequence of Chlamydomonas CA were determined. Hewettemmett and Tashian (1996) have recently analysed the amino acid sequences of many different carbonic anhydrases from both animals and plants, and from this have derived an evolutionary tree showing that the "eukaryotic" CA of freshwater algae is much closer to that of mammals than it is to the "prokaryotic" CA of the higher plants, which seems to be closer to the CA extracted from bacteria and cyanobacteria. Henry (1996), comparing physiological mechanisms, concluded that CA evolved as an enzyme for trans-membrane facilitated CO2 transport, and any secondary metabolic roles developed later. However, Roberts et al (1997) found that the protein sequence of CA from a marine diatom was quite different from earlier studies of CA from freshwater green algae, and suggested they either had different physiological roles, or their evolution was convergent.

2.3.2 Functions and location of carbonic anhydrase in marine microalgae

Such facilitated CO2 transport is required in land plants and some freshwater plants and microalgae, because the dissolved CO2 concentration in equilibrium with the air is low. However, for many years it was thought that marine algae had an abundant supply of CO2 as the concentration of total inorganic carbon (mostly bicarbonate) in seawater is about 100 times that of dissolved CO2 (see section 1.1.2). Moreover, until recently, CA was not found in most extracts from marine algal cultures. It was only when investigators started growing cultures in equilbrium with atmospheric CO2, rather than the 5% CO2 atmosphere often used to encourage rapid growth, that they found evidence of carbonic anhydrase (using a variety of measurement techniques discussed in section 2.6). During the last 10 years, CA has been found in many species of marine microalgae (see Table 2-1 ).

The various physiological roles of CA in microalgae are discussed in several recent reviews, for instance those by Badger and Price (1992, 1994), and Raven (1995), who discusses the link between the production of CA and the efficient use of various resources, such as carbon, nitrogen, energy and trace metals. He also points out that there are various non-photosynthetic biochemical roles of CA in cyanobacteria and microalgae, as it can catalyse many other carboxylase reactions.

There are two distinct types of CA found in microalgae, corresponding to two different locations: "internal" CA is used to facilitate CO2 transport within the cell and is concentrated in the chloroplasts, whereas "external" CA is used to transfer CO2 into the cell from the surrounding water, and is located outside the cell membrane. Internal CA is soluble, whereas external CA is surface active, part of it soluble in water and part of it soluble in the lipid bilayer of the membrane. They can be distinguished experimentally in vivo by the use of membrane impermeable enzyme inhibitors such as dextran bound sulphanamide (e.g. Nimer et al 1997) which only affect external CA, and also by isotopic methods (e.g. Aizawa and Miyachi 1986).

The main function of internal CA is well understood. In the dark reaction of photosynthesis, CO2 is fixed by the enzyme RuBISCO (Ribulose Bisphosphate Carboxylase Oxygenase), which, at 550 kDa per molecule, is probably the world's most abundant protein. Raven and Johnston (1991) point out that RuBISCO is a particularly inefficient enzyme, as it is large, has a high N:C ratio, and also catalyses a backwards reaction with oxygen leading to wasteful photorespiration. Therefore, it is particularly important to maintain a high CO2 / O2 ratio in the chloroplast near RuBISCO, and CA can serve this purpose.

External CA is generally only produced as needed, when CO2 supply to the cell becomes growth-limiting (see next section). In typical seawater about 99% of the dissolved inorganic carbon is in the form of bicarbonate ions (HCO3-, see section 1.1.2), which, being charged, cannot cross the non-polar lipid cell membrane. To enter the cell, the bicarbonate must be converted to CO2, but this uncatalysed reaction is slow compared to diffusion on this microscale (as it is for air-sea CO2 transfer, see section 1.5.1). Riebesell et al (1993) showed using a simple diffusion-reaction model around a "typical diatom" that CO2 supply can be rate limiting for photosynthesis (such models will be discussed and developed further in section 3.2). Some species solve this limitation by producing external CA to convert bicarbonate to CO2 outside the cell, whereas others have developed a different "Carbon Concentrating Mechanism" involving active transport of bicarbonate ions across the cell membrane (for instance, by an electrostatic attraction or anion-exchange mechanism -see for example Nimer at al 1997, or Tortell et al 1997). Some species use both mechanisms in different conditions. However, Raven (1995) pointed out that when there is active bicarbonate uptake, membrane bound CA may actually increase the "leak" of CO2 back out of the cell, since the catalytic process works in both directions.

CA may have an additional role in the process of calcification. Recently several studies have shown that Emiliana Huxleyi and some other coccolithophores produce CA during calcification, also in response to low CO2 (for references see Table 2-1 ). The purpose of this CA is the subject of much debate since it is generally assumed that calcification provided sufficient CO2 for photosynthesis. Perhaps the CA is needed to catalyse the calcification process itself? Indeed, Dreybrodt et al (1997) found experimentally that adding CA could increase the rate of precipitation of CaCO3 by a factor of 15. The importance of buffer ions in proton transport was pointed out above in the discussion of the mechanism of catalysis (section 2.2.2). In seawater, however, the major buffer ion is the substrate itself, bicarbonate ions. If the "buffer" in figure 2-1b is replaced by HCO3- (as observed by Silverman and Tu 1975), then the overall reaction catalysed by carbonic anhydrase becomes:

2HCO3- => CO2 + CO32- + H2O

If the CO32- then precipitates with Ca2+ or Mg2+ ions, and the CO2 is used mainly for photosynthesis, then we have the process of calcification. On the other hand, Nimer et al (1994) observed in cultures of Emiliana Huxleyi, that most calcification took place in the "exponential growth" phase, whereas most CA was produced in the stationary phase at the end of the bloom. This suggests that CA is produced to increase the availability of CO2 for photosynthesis, as an alternative mechanism to calcification .

Another intriguing process is the recently discovered role of CA in the uptake of the Carbonyl Sulphide (COS) by Chlamydomonas (Protoschillkrebs et al 1995), which suggests a possible link between the air-sea fluxes of CO2 and the sulphur gases.

2.3.3 Species of marine microalgae with carbonic anhydrase

Table 2-1 illustrates the variety of species of marine microalgae in which carbonic anhydrase activity has now been identified. There are now hundreds of papers reporting detection of CA in specific species of microalgae, so it would not be appropriate here to attempt to list them all. Some of the references in the table are to review papers (as indicated) rather than to the original source. A few of the species listed in Table 2-1 are freshwater algae, but have been included because they are easy to culture and investigate, and therefore have yielded useful physiological and kinetic data (notably the green alga Chlamydomonas Reinhardii).

A few macroalgae (seaweeds) have also been included in the table, as they may be very abundant in some regions of the sea. CA has also been detected in various species of fish, shellfish, corals, and other marine organisms, but there is not space to list examples here. I assume that larger organisms contribute little to the total pool of CA in the surface microlayer of the ocean, compared to phytoplankton.

All the major families of marine microalgae are represented in Table 2-1, and it is possible that all microalgae may produce CA if the cultures are prepared in "low CO2" conditions. Often, earlier reports that a species does not possess CA were due to the use of "high-CO2" culture media in which there is less physiological need for the enzyme. Although in some cases investigators found evidence for "internal" CA when they could not detect "external" CA (see previous section), both types were found in most of the species referenced in this table.

2.3.4 Physiological response to low pCO2, and the "Zinc hypothesis".

Many recent investigations with cultures of marine microalgae (references in Table 2-1 ), have shown that the production of external CA is triggered by a low concentration of dissolved CO2 in the surrounding water. This is also supported by the recent observations of Berman-Frank et al (1994, 1995) during the spring bloom in lake Kinneret (= Sea of Galilee, Israel): they measured CA in the cells of the Dinoflagellate "Peridinium Gatunense" and found a clear inverse relationship between pCO2 in the lake and CA in the cells. This response to low pCO2 is predictable, since CA is a large molecule whose production requires considerable resources, so the algae will not make it unnecessarily. Section 3.2 will show how it is possible to model the uptake of CO2 catalysed by cell-surface CA, by adapting the reaction-diffusion model of Riebesell et al (1993), and thereby calculating the relative physiological benefit of the CA over a range of external conditions (pCO2, temperature).

It is also possible to demonstrate experimentally the importance of CA in low pCO2 conditions, by limiting the availability of zinc in a culture medium. Morel et al (1994) showed that growth of the diatom "Thalassiosira Weissflogii" could be limited by zinc when pCO2 was low. On this basis they proposed a "zinc hypothesis" along the same lines as the "iron hypothesis" of John Martin, whose original suggestion that iron could be the limiting nutrient in certain "high-nutrient low-chlorophyll" regions of the ocean was derived from similar culture experiments (the history is told by Chisholm and Morel 1991 and papers therein) and later shown to be valid during pioneering lagrangian field experiments in the equatorial Pacific (Coale et al 1996). However, there have been no similar experiments to confirm the "zinc hypothesis", and Morel et al (1994) suggest that zinc limitation may have been more important in glacial periods (when pCO2 was lower) than at present.

Further work with the same diatom Thalassiosira showed that cadmium (Lee et al 1995) and cobalt (Yee and Morel 1996) could partially replace zinc in carbonic anhydrase when the metals were present at concentrations typical of surface seawater, but work with other species (Lee and Morel 1995) showed that cadmium only acted as a nutrient in a narrow species-specific concentration range. A more general discussion of trace metal limitation in seawater is given by Morel et al (1991).

Other recent experiments on lakes have also shown that when the water pCO2 falls (pH rises), CO2 supply can become rate-limiting for growth (Hein 1997). Some species adapt better than others, with blue-green microalgae being the most favoured at high pH due to their ability to take up bicarbonate (Shapiro 1997). Hein and Sand-Jensen (1997) found a similar species shift in incubated seawater samples when the pH was deliberately altered to cause a change in pCO2 without changing TCO2. They found faster uptake of labelled 14C at lower pH (higher pCO2) -implying carbon limitation of growth.

2.4 The potential for enrichment of carbonic anhydrase in the sea-surface microlayer

2.4.1 Introduction to the problem

The hypothesis that CA may catalyse air-sea CO2 exchange assumes a significant concentration of the enzyme, not in algal cells, but in the sea surface microlayer which forms the rate-limiting barrier for air-sea CO2 transfer (see section 1.2.1). We might question firstly whether CA would be released by algal cells at all, as losing such a large molecule would be a considerable loss of energy and resources for the cell. On the other hand, many useful metabolites are released when algal cells eventually die and break up, including various small molecules such as DMSP which may have a significant impact on atmospheric chemistry and climate (see section 1.1.5, and also further discussion of this issue in section 10.6). So, if some CA is released, then the next question is, can it remain active independently of the cell. Such large proteins are easily denatured and their catalytic activity depends on their weak quaternary structure (protein folding) which in turn depends on the molecule's microenvironment. Indeed, Kanwisher (1963) observed that bovine CA seemed to be rapidly denatured in seawater, and experimental measurements reported later in thesis will confirm this (section 3.5 and section 7.6). Many varieties of CA are also inhibited by the salt ions of seawater -as discussed in section 2.7.3. On the other hand, the extracellular CA of marine microalgae has evolved to function in seawater outside the cell, so it might be expected to survive better in this environment, although the structure and activity of the molecule may still depend on a non-polar section being embedded in the cell wall.

Previous investigations have focussed on measuring the activity of CA in concentrated extracts of algae, either from laboratory cultures or lake samples, not on measuring the activity of CA dissolved in natural seawater or lake-water itself. In natural waters concentrations are likely to be much lower than in culture extracts, and would therefore call for much more sensitive measurement techniques. In the absence of such measurements, we cannot directly answer the two questions above. However, there have been many measurements of dissolved organic matter in seawater, and more specifically of dissolved proteins. These measurements may give us a rough indication, of how much CA might possibly be present in seawater. We may also consider the concentration of zinc, since this is an essential component of the enzyme.

A further complication is that the concentration of CA in the sea-surface microlayer may be very different from its concentration in the bulk water. Organic matter is continuously brought to the microlayer by vertical overturning (surface renewal cells) and rising bubbles, and surface active molecules will remain there. Many studies of the sea-surface microlayer have shown that the concentration of proteins, as well as lipids and carbohydrates, can be more than an order of magnitude greater in the microlayer compared to the bulk surface water. Extracellular CA, in particular, is likely to be highly surface-active, as part of the molecule is normally positioned in the lipid cell membrane.

The direct physical effects of such organic surface films on air-sea gas exchange have already been introduced in section 1.4.5. I will return later to these physical effects, and also discuss the possibility that plankton within the microlayer may absorb or release a significant amount of CO2, after considering the experimental results in chapter 8 (section 8.5). In the remainder of this section, the topic of microlayer enrichment will be introduced from a chemical and biological viewpoint, with particular emphasis on the amino acids and zinc which make up CA. Note that crude experimental measurements of microlayer enrichment of bovine CA will also be reported later in section 3.5.

2.4.2 Collecting samples for measuring surface microlayer enrichment

All microlayer sampling methods work by exploiting the surface tension of water, and most methods are only effective in relatively calm sea conditions. One of the earliest methods is the Garrett screen (Garrett 1967), a fine mesh held in a frame, which picks up a film as it is pulled out of the water, although it will also act like a sieve, collecting any large plankton from below the surface. The rotating drum method (e.g. Hardy et al 1988) is perhaps more convenient for collecting large samples. The drum turns around a horizontal axis, held between the hulls of a small catamaran, and the film which adheres to it is scraped off into a collecting bottle. For laboratory work, a glass plate can be withdrawn vertically, collecting a film which is then scraped off (Harvey & Burzell 1972). Hardy and Cleary (1992) compared several of these sampling methods when measuring microlayer enrichments of toxic heavy metals and organotin in North Sea samples, and found they gave similar results.

Microlayer enrichments for a substance or organism are often reported as the ratio of the concentration in the microlayer sample, divided by the concentration in bulk water. However it is difficult to compare enrichments measured by different workers, not only because they cover a wide range of locations and sea conditions, but also because each sampling method collects a different thickness of water. Typically this is of the order of 20-500mm, but an accurate figure is often not known. Hunter and Liss (1981) consider this problem in a comprehensive review of microlayer chemistry. Sieburth et al (1976) suggested that most of the surface-active molecules were contained in a layer only 0.1mm at the surface, and most of the microorganisms collected in a layer about 1mm thick. In this case, although they measured the concentration of dissolved organic carbon in a sampled layer 150mm thick to be only 1.73mgl-1 compared with 1.10mgl-1 in the bulk seawater, they calculated the concentration in the 0.1mm microlayer to be 1336mgl-1! It is important to be aware of such assumptions when considering reported enrichment factors.

2.4.3 Measurements of enriched biological activity the sea-surface microlayer

The earliest investigations of biological enrichment in the sea-surface microlayer, such as those of Zaitsev (1971), focussed on the eggs and larvae of fish, crustaceans and other large marine organisms. Many of these eggs and larvae are inherently buoyant, using the densely populated, nutrient-rich, and viscous environment of the microlayer as a convenient nursery. Many types of zooneuston were also discovered, which had evolved specifically to exploit this abundant food source (note that neuston is a general term for organisms inhabiting the microlayer).

The vast blooms of the filamentous cyanobacteria Trichodesmium which dominate some areas of the tropical North Atlantic, were also an obvious focus for early investigations. Sieburth and Conover (1965) observed that slicks in the Sargasso Sea tended to be associated with these blooms. More recently Hardy et al (1988) took microlayer samples from this region using a rotating drum, and found that Trichodesmium was up to 400 times more abundant in the microlayer, compared to the bulk water.

Sieburth et al (1976) carried out a detailed study at various locations in the North Atlantic, using a screen which sampled a microlayer about 150m m thick, and measuring biomass, pigments, carbohydrates, and dissolved organic carbon (DOC). Measured enrichments were generally in the range 1-3, but the values for biomass were much higher in some stations, although at others no significant enrichment was detected. Carbohydrates formed a greater proportion of DOC in the microlayer than in the bulk. Cultivatable bacteria and amoebae were also measured at two stations: the bacteria were enriched by a factor of 1000 in one case, and were depleted by half in the other, but this latter sample was from a shallow water station dominated by a bloom of the diatom Nitzschia, which was itself enriched, as indicated by the chlorophyll data. Many copepods were also observed at the surface, infested with suctorians.

Based on such results Sieburth (1983) painted a picture of the "skin of the sea" as a gel of complex tangled molecules, inhabited by many diverse microrganisms ranging from 1m m to 1mm, in concentrations more typical of intense laboratory cultures than normal bulk seawater samples. This remains the prevailing viewpoint, as illustrated by Hardy et al (1997), although it is still based on analysis of collected samples, rather than on observations of the microlayer in situ.

Another detailed study of biological enrichment in the microlayer, also using a screen sampler, was made by Williams et al (1986) off the coast of Baja California. Here, the phytoneuston were dominated by dinoflagellates, with the balance of species being quite different from that in the bulk water. Sampled enrichment factors were from 1.3-2.0 for neuston, and from 1.1 to 3.7 for dissolved proteins, although the enrichments in the microlayer itself may be much higher. Indeed, Brockmann et al (1976) measured microlayer enrichment factors of over 1000 for the dinoflagellate Prorocentrum micans in coastal North Sea samples.

Hardy and Apts (1984) collected microlayer samples from Puget Sound with a membrane filter. They measured bacterial activity directly in the original samples, rather than growing cultures from them, and found microlayer enrichment factors from 102 to 106! Hardy and Apts (1989) investigated the activity of phytoneuston by using microcosms to incubate both microlayer and subsurface samples, taken from sea areas both with and without slicks. In slick samples, the enrichment factors were 154 for phytoneuston population, 18 for chlorophyll, 52 for particulate carbon fixation, and 63 for dissolved carbon excretion, while the equivalent figures outside of slicks were 37,1.3,2 and 17. This suggests that the activity per cell is greater in the microlayer.

Carlucci et al (1992) investigated bacteria in the microlayer off the coast of Southern California using a screen sampler, and reported sampled enrichment factors from 1.4 to 3.2 (several previous papers of these authors also reported similar results). However, measurements of glutamic acid and ATP again suggested that the activity and turnover rate of the bacteria were much greater in the film samples. They also measured the concentrations of individual amino acids, and found that the distribution was significantly different in the microlayer compared to the bulk water, perhaps suggesting a greater proportion of animal rather than plant protein.

Many researchers have suggested that there will be greater enrichment of bacteria and zooplankton than of phytoplankton in the microlayer, because bubble-mediated transport of surface-active molecules and particles to the surface may provide a food source for heterotrophic organisms, while on the other hand, the photosynthetic activity of many species of marine algae may be suppressed by the unusually high levels of short-wavelength u.v. radiation in the microlayer. For example, Hardy and Apts (1984) found that carbon reduction rates were 20-150 times greater in the microlayer in winter and spring, but the enhancement was much less in summer, which they attributed to photoinhibition. Williams et al (1986) report similar inhibition. Carlsson et al (1988) made continuous measurements of chlorophyll and u.v. absorbtion in the microlayer, sampled with a rotating drum, and found that chlorophyll could even be depleted in the microlayer, although there was great variability. Compiano et al (1993) found that the relative proportion of phytoplankton pigments was reduced in the microlayer, although total particulate organic carbon and nitrogen were enriched.

It has been suggested (e.g. Hardy et al 1997) that if respiration is enhanced in the microlayer more than photosynthesis, then the consequence would be to increase pCO2 locally in the microlayer, which might lead to a decrease in the net global air-sea CO2 flux. On the other hand, the results from laboratory experiments reported later in this thesis (section 8.5) suggest that the opposite may also be possible. Clearly the net effect depends on the balance of species and more investigation is needed. There have been few direct measurement of CO2 uptake or release in the microlayer, with the notable exception of Garabetian (1990), who investigated the respiratory activity of the neuston by measuring the uptake of glucose labelled with 14C, and observed increased respiration in the microlayer. This was attributed mainly to bacteria based on an experiment with cycloheximide. The net release of 14CO2 was reduced by 50% when samples were incubated in the light (Garabetian 1991), perhaps indicating photosynthetic uptake, and the same result was obtained using both glass and quartz tubes, the latter allowing penetration of short-wavelength u.v. light.

2.4.4 Measured enrichments of proteins and zinc in the microlayer

Whatever the balance of phytoplankton, bacteria and larger zooplankton in the microlayer, with such intense biological activity it is not surprising that the observed enrichments of DOC have been found to be primarily due to biological metabolites -principally carbohydrates and proteins, rather than either the fatty acids originally assumed to dominate sea-surface slicks, or the large polyphenolic humic molecules which make up a large proportion of marine DOC. For reviews of many early investigations see Hunter and Liss (1981), Lion and Leckie 1981, Liss (1989). More recently, for example, Garabetian et al (1993) reported microlayer enrichments for many groups of compounds in samples collected with a rotating drum, and came to similar conclusions.

Some illustrative figures for measured enrichment factors follow, focussing on proteins, since these might include carbonic anhydrase. Henrichs and Williams (1985) reported enrichment factors between 1 and 12 for hydrolyzeable amino acids. Saliot et al (1991) measured enrichment factors for proteins between 1 and 18, and up to 45 for carbohydrates and lipids. Carlucci et al (1992) reported that free amino acids were enriched by a factor ranging from 3 up to 53 in microlayer samples taken off the coast of southern California, with a greater enrichment in samples taken further from the coast. Enrichments of combined amino acids were smaller -from 1.3 to 3.1. Note that all these figures are of course dependent on the thickness of the sampled layer -the actual enrichments may be much higher than the sampled enrichments reported here.

Although proteins are more concentrated in the microlayer, we must bear in mind that their lifetime there may be shorter than in bulk water -both due to consumption by bacteria, and degradation into a refractory form by sunlight and chemical interaction with other molecules, all of which are more abundant in the microlayer. These destructive processes are discussed by Keil and Kirchman (1993, 1994).

The concentration of zinc in the sea-surface microlayer may also be considered as a constraint on the possible concentration of carbonic anhydrase, as discussed by Emerson (1995) and Keller (1994). The concentration of zinc in subsurface seawater is only between 0.1nM and 10nM (e.g. Bruland 1980, Danielsson et al 1985, Yeats and Campbell 1983, Nolting 1986), the higher values being found nearer coasts due to inputs from rivers or terrestrial dust. However, many studies have shown that trace metal concentrations may be greatly enriched in the microlayer. The table of results from several authors given by Hardy (1997) shows a range of sampled enrichment factors for zinc between 2 and 22. Most of the zinc seems to be bound by complex organic ligands, which may include carbonic anhydrase. If the extra zinc is actually contained in a much thinner layer than that sampled, the concentration at the surface may be of the order of 100nM or more. Some early measurements reported even higher concentrations in the microlayer, but since these were made before the advent of the clean sampling techniques (see Bruland 1980), they may not be reliable.

2.5 Enzyme kinetics and CA activity

To calculate whether the hypothesis that carbonic anhydrase may significantly catalyse air-sea CO2 exchange could be realistic, we need to know the effect that a given concentration of enzyme in the microlayer could have on the CO2 hydration rate. It is not easy to measure catalysis of the hydration / dehydration of CO2 at the surface of an algal cell in culture, and it is even more difficult to measure this in the sea-surface microlayer which is the focus of interest in this study. It is, however, possible to measure the catalytic activity of CA in solutions extracted from algal cultures, or purified on gels. From this, we can derive enzyme kinetic data by setting up initial-velocity experiments, and from this data we can attempt to predict the catalysis in vivo or in situ in the microlayer.

As those who study the global carbon cycle may not be familiar with enzyme kinetics, I will develop the equations here from basic principles. The first stage, explaining the basic principles behind the initial-velocity experiments which are used to measure enzyme kinetic constants, is standard textbook material. However, it is important to note that these initial-velocity experiments are very different from the steady-state reaction-diffusion system of CO2 transfer across the sea-surface microlayer, or indeed around an algal cell, where different simplifying assumptions must be made. This point was not recognised by Emerson (1995) whose calculations of the possible effect of carbonic anhydrase on gas exchange are, I believe, invalid for this reason (more on this in section 2.5.5).

I did not find any appropriate "textbook" formula relating the standard enzyme kinetic constants to catalysis of CO2 transfer in such a system, and therefore the derivation of this formula presented in the second stage below is my own. This derivation is placed here in this "introductory" chapter for convenience, since it follows naturally from the definition of the kinetic constants, and since it also determines which of the kinetic constants we need to know when seeking data from the literature on measurements of CA activity. A selection of such data will be summarised in section 2.7, following a brief introduction to practical methods of measuring CA activity in section 2.6. However, the application of the formula in calculating the possible catalysis of air-sea gas exchange, will be postponed until the next chapter, section 3.2 and section 3.3.

2.5.1 Definitions

Although the importance of buffer ions in carrying protons to or from the enzyme was stressed in section 2.2.2, and may be the rate-limiting process at low buffer concentrations, there is insufficient data to account for this since most measurements were made in well buffered solutions. If we ignore proton transport, then the catalysis process can be summarised as:

A + E <==> EA <==> EB <==> B + E

where E = free enzyme, and (in this case), A= CO2(aq), B = HCO3-

(A, B, E etc. are used in this general derivation, since it could apply to many enzyme-substrate systems)

It can be shown that this is effectively equivalent to

ka ® kb ®

A + E <===> ES <===> E + B

ka kb

Where: ES = enzyme substrate complex,

ka, and kb are rate constants with units mol-1 s-1

ka and kb are rate constants with units s-1

Also we will designate: E0 = ES + E = total enzyme concentration.

A0 = EA + A = total A concentration

B0 = EB + B = total B concentration

2.5.2 Stage 1: Initial Velocity experiments

The individual rate constants ka etc. cannot be measured directly. Instead, "Initial Velocity" experiments are used to derive the kinetic data. The initial velocity "V" is the rate of production of B (or loss of A) when the concentration of A is much greater than either B or E. For a short while, therefore, A can be taken as constant, such that:

A @ A0 >> B or E

The initial velocity is then given by

V = kb ES

As A0 ® then ES ® E0 and V ® Vmax = kb Eo

To go further we need to make the "steady-state" assumption of Briggs and Haldane, that dES/dt = 0. This is valid so long as there is much less enzyme than total substrate (even when dA/dt or dB/dt are not zero, because we are still considering initial velocity experiments). Two alternative assumptions which lead to the same result are considered later. Note that this "steady-state" assumption should not be confused with the steady-state flux in a reaction-diffusion system, which we will consider in stage 2.

When B is very small and A @ A0 then:

dES / dt = ka E A0 - (ka + kb) ES = 0

substituting E0 = E + ES and rearranging gives:

ES = ka A0 E0 / (ka + kb + ka A0)

substituting V = kb ES and rearranging gives:

V = Vmax A0 / (Km + Ao )

where: Km = (ka + kb) / ka

Km is known as the Michaelis-Menten constant and has the units of concentration (mol).

By measuring V at different concentrations of A0 , both Vmax and Km can be derived from a series initial velocity experiments with constant enzyme concentration, plotting 1/V against 1/A0.

If Vmax is divided by the total enzyme concentration E0 this gives the kinetic constant kb. In the literature this is usually written as "kcat". At very high substrate concentrations (i.e. as A ® ) then it can be considered as a "pseudo first-order" rate constant for the reaction,

as V= kcat E , and is also known as the "turnover number" (number of substrate molecules transformed per enzyme molecule).

The "second order" rate constant is sometimes written as kenz, such that V = kenz A E. To see the relation between these, consider that V is the product of the rate of production of ES multiplied by the fraction of that ES which goes on to form B, i.e.

V = kenz A E= ka A E kb / (kb + ka)

Substitution shows that kenz = kcat / Km

It is important to remember that these "first order" and "second order" interpretations only apply to initial velocity experiments.

(Note that there are two alternatives to the "steady state" assumption used above:

If we had assumed that ka >> kb , then E + A <==> ES will be in equilibrium, so ES = E A0 ka / ka and substituting E0 = ES + E gives the same formula for V with Km = ka / ka ,

If instead we assumed that ka << kb then there is no back reaction so V = E A0 ka = ES kb and again substituting E0 = ES + E gives the same formula for V with Km = kb / ka .

It is apparent that these results are specific cases of the more general formula derived from the "steady-state" assumption).

2.5.3 Equilibrium conditions

We will now leave the conditions of initial velocity experiments, where A >> B.

Consider first the equilibrium condition, dA/dt = dB/dt = 0,

which can be expanded to:

ka ES - ka A E = kb ES - kb B E = 0

Rearranging gives:

ka kb / kb ka = B / A = Keq.

Where Keq is the thermodynamic equilibrium constant.

If Km and kcat are the equivalent of Km and kcat for the reaction B ® A, i.e. the constants derived from the initial velocity experiment with B >> A, then it is possible to rearrange this result to give:

kcat Km / kcat Km = Keq , which is known as the "Haldane relationship".

2.5.4 Stage 2: CO2 transfer in a steady state reaction-diffusion system

In a diffusion-reaction system such as the surface of a photosynthesising cell, or the sea-surface microlayer, neither initial velocity nor equilibrium conditions are appropriate. A formula specifically for such a system will be derived below (key results in purple).

We want to know the net rate of reaction A ® B, i.e. the rate of loss of A due to reaction rather than diffusion. This calculation should also include the uncatalysed reaction.

net rate = - dA / dt

= ka A E - ka ES + (kCO2 + OH kOH) A - kCO2 B

where kCO2 and kCO2 are the forward and reverse rate constants for the uncatalysed reaction of CO2 with water, kOH is the rate constant for the reaction of CO2 with OH- , and OH is the concentration of OH-. (Using the same notation as before in section 1.5.2).

substituting ES = E (ka A + kb B ) / (ka + kb )

which derives from the "steady-state assumption" dES/dt = 0

gives: net rate = [A E ka (ka + kb ) - ka (ka A E + kb B E )] / (ka + kb ) + (kCO2 + OH kOH) A - kCO2 B

= E [A ka kb - B kb ka] / (ka+ kb ) + (kCO2 + OH kOH) A - kCO2 B

substituting Keq = ka kb / kb ka = (kCO2 + OH kOH) / kCO2

gives net rate = [ E ka kb / (ka + kb) + (kCO2 + OH kOH) ] [A - B / Keq ]

substituting Km = (ka + kb) / ka

and kcat = kb

gives net rate = [ E kcat / Km + kCO2 + OH kOH ] [A - B / Keq ]

Now the constants in this formula are all measurable quantities, except the concentration of free enzyme E which we need in terms of Eo. To determine this, we return to the "steady state assumption":

(Eo - E) / E = (ka A + kb B ) / (ka + kb )

substituting Km = (ka + kb) / ka and Km = (ka + kb) / kb

gives (E0 - E) / E = (A / Km + B / Km)

or E = E0 / (A / Km + B / Km + 1)


Incorporating this into the main formula above gives:

net rate = [ E0 kcat / (A + B Km / Km + Km) + kCO2 + OH kOH ] [A - B / Keq ]

(Note that if we ignore the uncatalysed reaction and consider the condition that B ® 0 then this expression simplifies to: net rate = -dA/dt = E0 kcat A / (A + Km ), which is the initial velocity equation for V as we had before)

In practice, the existing formulae for calculating the net rate of transport of CO2 across the sea surface microlayer (e.g. the formula of Hoover and Berkshire 1969 -see section 1.5.4) or into an algal cell (e.g. Riebesell et al 1993 -see section 3.2) already include the factor [A - B / Keq] in the reaction - diffusion equations. The critical quantity for which we are searching is therefore the left hand bracket, which is the catalysed equivalent of the rate constant ktot already used in section 1.5.2

So to conclude:

ktot = E0 kcat / (A + B Km / Km + Km) + kCO2 + OH kOH

2.5.5 Calculations used by other investigators

Emerson (1995) considered only the "turnover number" in his crude calculation of the possible effect of carbonic anhydrase on gas exchange. His calculation procedure is not given in full, however it appears that his assumption (using the same notation as above) was that

net rate = E0 kcat + (kCO2 + OH kOH) A

This leads to a great overestimation of the effectiveness of the enzyme, although the figures thus derived do not seem to be carried through into his subsequent discussion.

The formula used by Keller (1994 -in the appendix) is more complex. He considers both forward and back reactions, but assumes that Km = Km , (actually Km is probably higher -see data in section 2.7.1). He also considers that the rate is limited by proton removal in the hydration direction, but not in the dehydration direction. The hydration rate constant is thus multiplied by a factor

1 / (1+ [H+]/ KCA)

where KCA (about 10-7.5) is the acid-base ionisation constant for carbonic anhydrase.

This factor is not much less than unity at the pH of seawater. Qualitatively this pH effect appears similar to that measured by Khalifah (1971) for Human CA, although the measurements of Pocker for spinach CA (shown graphically in Silverman 1991) show a somewhat different response (recall that the CA of microalgae is closer to that of humans than that of spinach).

Using my notation, Keller's formula is

-dA/dt = E0 kcat A / (Km + A) - E0 kcat B / (Km + B) + (kCO2 + OH kOH ) [A - B / Keq]

where kcat is derived from the condition that -dA/dt = 0 at equilibrium, which can be reduced to:

kcat = kcat (Km + A Keq ) / (KmKeq + A Keq)

I believe this formula is also wrong, as it's only justification is an allusion to "Michaelis-Menten" kinetics, which were shown earlier to apply to initial velocity experiments rather than steady-state reaction-diffusion conditions. Keller's figure 4, produced with this formula and a ratio of kcat / km of 1.7x107, predicts an enhancement factor of 1.6 for CO2 gas exchange across a film of thickness 50mm where the CA concentration is 10-7 M.

In section 7.6.4, the measured catalysis of the CO2 transfer velocity in the laboratory tank with added bovine carbonic anhydrase, is compared the catalysis predicted using the formula derived in section 2.5.4 above, combined with the formula of Hoover and Berkshire for chemical enhancement. The measured and predicted enhancements agree remarkably well, considering all the simplifying assumptions and the uncertainties in the rate constants. This result gives some confidence in the formula derived above, rather than the simpler formulae used by Emerson (1995) and Keller (1994).

2.6 Measurement of carbonic anhydrase and its activity

A brief introduction to practical methods of measuring CA activity is given below, followed in the next section by some specific values for the enzyme kinetic constants reported in the literature.

2.6.1 Activity measurements (electrode, spectrophotometric, manometric)

Roughton and Booth (1933) first measured the activity of carbonic anhydrase using a manometric method, i.e. observing the volume of CO2 gas evolved after a perturbation of a carbonate solution. Later they developed a spectrophotometric method, whereby the rate of change of pH is monitored using an indicator dye (bromthymol blue) following a perturbation of the CO2 system due to mixing two different (buffered) solutions, one of them saturated with CO2. As the CO2 gradually converts to HCO3-, so the pH falls. Wilbur and Anderson (1948) pointed out that the indicator dye and phosphate buffers inhibited the enzyme, so adapted the method using a glass pH electrode. This method is still used today, and also defines the commonly used "Wilbur Anderson unit" for CA activity. One WA unit will reduce the pH of a 20mM Trizma buffer from 8.3 to 6.3 in one minute at 0o C, whereas this change takes about 2 minutes uncatalysed. It is necessary to calibrate the slow response of the electrode. Other investigators report the ratio of the catalysed and uncatalysed times as an arbitrary "enzyme unit", but these units are only comparable between different papers if the same buffers, temperatures and pH changes are used.

A similar pH electrode method can be used to determine true enzyme kinetic data during initial velocity experiments (kcat and Km : as in section 2.5.2) if the initial rate of production of hydrogen ions is calculated from the pH change and a pH-dependent buffer factor specific to the experimental solution. This buffer factor accounts for the protons which do not remain free in solution (see Khalifah 1971). The "initial velocity" must be determined for a range of initial substrate (CO2) concentrations, as described in section 2.5.2, to derive the constants. This is essentially the method used by Sanyal and Maren (1981) and Bundy (1986) whose enzyme kinetic data will be referred to in Table 2-2. It should be noted that the rate constants are highly dependent on temperature, as well as on the buffer used.

Khalifah (1971) instead used the "stopped-flow" pH indicator method, as did Williams (1983) who was looking for evidence of CA activity in seawater and lake water. Williams reported a precision of 0.01 s-1, which he says corresponds to a CA concentration of about 5 mg l-1. Shingles and Moroney (1997) adapted this technique using fluorescent pH indicator dyes, and claim they can measure initial velocities with as little as 65 ng l-1 CA.

Silverman and Tu (1975) used an 18O isotope technique to study human CA "C", in order to determine the effect of low buffer concentrations and pH on the dehydration rate. The advantage of this approach is that the solution remains in chemical equilibrium, although it is in isotopic disequilibrium. They measured the rate at which 18O transferred from initially labelled bicarbonate to water, for a variety of pH and buffer concentrations. The actual reaction rates, rather than the enzyme kinetic constants kcat and Km (see section 2.5.2) were reported, which hinders comparison with other data.

2.6.2 Measurements of detecting catalysed CO2 uptake by algal cells

CA activity has often been determined in algal cells in vivo by measuring the rate of CO2 uptake by the cells, before and after the addition of an inhibitor of CA activity such as ethoxyzolamide, acetazolamide, or dextran bound sulphonamide (the latter only affecting external CA since it cannot pass through the cell membrane). The rate of CO2 uptake by the algal cells can be measured by various methods, including determination of the total carbon content of the cells by the (destructive) silicone oil centrifugation technique, the detection of 14C or 13C labelled substrates taken up by the cells (e.g. Aizawa and Miyachi 1986), or the evolution of oxygen due to photosynthesis, as recently applied to marine macroalgae by Mercado et al (1997).

An alternative approach for observing CA in natural systems was proposed by Drescher (1978), using the fluorescent probe "Dansyl Amide", or dimethylaminonapthalene-1-sulphonamide (DNSA). The highly-efficient quantum energy transfer from the tryptophan units in CA to the DNSA molecule was investigated in some detail by Chen and Kernohan (1967), and subsequently used by Newman and Raven (1993) to show the location of CA in vivo in a freshwater macroalga. Some preliminary experiments using this fluorescent technique for direct measurement of CA in the sea surface microlayer will be presented later in section 3.5.

2.6.3 Purification of CA on gels and determination of molecular mass

Carbonic anhydrase can be purified using polyacrylamide gel electrophoresis, but this enzyme must then be distinguished from many other similar proteins which might be found in a plant or animal extract. Graham et al (1984) describe one such method, which was also used by Morel et al (1994) investigating zinc limitation of marine algae. A gel slice is rinsed with a buffered solution of the pH indicator dye bromocresol purple, then briefly exposed to CO2 gas, then rapidly frozen using solid CO2. Yellow bands (which fluoresce under UV light) then indicate the increased proton concentration released by enzyme-catalysed hydration of CO2.

In order to report values for the kinetic constant kcat from initial velocity experiments, the amount of enzyme used (usually measured by weight) must be converted to a concentration, so the molecular mass must be known. Most "eukaryotic" CA types (see section 2.3.1) have a mass in the range 30 - 40 kDa. CA extracted from higher plants sometimes has a much greater mass (the review of Graham et al 1984 gives a range of 140-250 kDa for CA from various common vegetables), but these are probably polymers of several active units (for example, CA from Spinach has a mass of 180kDa made up of 6 monomers of 30kDa each). The molecular weight can be determined by polyacrylamide gel electrophoresis techniques (as above), or even analysis of the amino acid sequence. However, if such data is not available it is also possible to estimate the concentration of active sites by assuming non-competitive inhibition by a sulphonamide inhibitor, if Vmax (=kcatE0) is measured over a range of inhibitor concentrations.

Bundy (1986) used both electrophoresis and inhibitors, but used the concentration derived from the inhibitor experiments to determine kcat. He found that the chlamydomonas CA seemed to have a weight of 165kDa, but that this contained polypeptides of weight 42kDa and 76kDa. If the protein is a tetramer with four active sites, then the kcat refers to the concentration of the monomer, not the protein.

2.7 Enzyme kinetic constants for CA reported in the literature

2.7.1 Kinetic constants kcat and Km

Returning to the formula derived in section 2.5.4 above for catalysis of CO2 transfer in a steady-state reaction-diffusion system,

ktot = E0 kcat / (A + B Km / Km + Km) + kCO2 + OH kOH

to calculate ktot we need to know figures for kcat Km, Km, kCO2, and kOH. The uncatalysed rate constants kCO2 and kOH have already been discussed in section 1.5.2, but for comparison we should recall here that kCO2 dominates at normal seawater pH, and is typically about 0.03 s-1, although highly temperature dependent.

Many measurements of carbonic anhydrase activity are expressed in arbitrary "enzyme units" based on the Wilbur Anderson method (see previous section). These are useful for comparison between different extracts, but are not easily extrapolated to fundamental units as each investigator used different experimental conditions. The only true enzyme kinetic data which I have found for CA extracted from microalgae is by Bundy (1986) for chlamydomonas reinhardii. This freshwater green alga has been much studied, because it is easily cultured and also has some mutant forms which can be used for comparative experiments. Many other papers also report kinetic measurements for CA from chlamydomonas (for example, Karlsson et al 1995), but only using the arbitrary "enzyme units".

Table 2-2 compares the values of kcat and Km from Bundy (1986) with some other measurements for human and bovine CA.

2.7.2 Effect of temperature

Bundy (1986) pointed out that the seemingly low value he measured for kcat is in part due to the low temperature. Sanyal and Maren (1981) measured the effect of a range of temperatures on kcat and Km for Human CA, and found that kcat rose significantly with temperature whereas Km was not affected. They applied the well-known Arrhenius equation

rate = Const * exp [-Ea / RT]

to determine the activation energies of hydration and dehydration:

For Human CA "C": DGhydration = 38.1 kJ mol-1

They also measured the uncatalysed reaction, for which DGhydration =81.7 kJ mol-1

Values for dehydration were similar to those for hydration (as expected).

In the absence of such data for algal CA, I will apply the value of Sanyal and Maren (1981) for the activation energy for Human CA to Bundy's kinetic constants for chlamydomonas. The formula therefore becomes:

kcat = 44800 * exp [16.8 - 38100 / (8.3143 * T)]

at T = 298 K this gives kcat = 186 x103 s-1 .

We also need a value for Km, for which there have been fewer measurements. The measurements of Sanyal and Maren (1981) given above suggest that Km is about 4 times Km for human CA. Maren et al (1976) measured Km / Km = 7.5, also for Human CA, whereas the review of Sultemeyer (1991) gives a range of Km from 30 to 60, and of Km from 1.5 - 40 for CA from C3 plants (polymeric enzymes). Here I will assume that the ratio is 4, i.e. Km is 12 mM and Km is 3.1 mM (Bundy's measurement).

2.7.3 Anion Inhibition

There is a further complication, which is the effect of anion inhibitors. All carbonic anhydrases are strongly inhibited by sulphonamides, which are sometimes used for kinetic measurements and photosynthetic experiments. However, many other anions also inhibit the enzyme, the strength of the inhibition varying greatly with the source of the enzyme. Wilbur and Anderson (1948) noted that the phosphate buffers and dyes used in early kinetic studies inhibited catalysis by about 10%. Maren et al (1976) pointed out that the concentration of Cl- ions in human blood strongly inhibited human CA "I", and human CA "II" to a lesser extent. This might imply that the enzyme would not work efficiently in seawater.

Bundy (1986) found that 0.049M NaCl inhibited 50% of the activity of chlamydomonas CA. The equivalent figure (usually designated I50 ) for Human CA "C" is 0.2M (Sanyal and Maren 1981). It can be shown that for "non-competitive" inhibition, the rate of reaction is reduced by a factor (1+ I / I50), where I is the actual concentration of inhibitor. Therefore if Bundy's chlamydomonas figure applied to CA from algae in the sea, where [Cl-] = 0.6M, the reaction rate would only be 1/13th of that in freshwater. However, it seems reasonable to assume that the extracellular CA of marine microalgae would have evolved to be less affected by anions in seawater. Okazaki (1974) gives a value of I50 for NaCl inhibiting CA from the microalga Serratacardia Maxima of 0.9M, in which case the reduction would be only 40%. This figure was used in most subsequent calculations of enzyme catalysis in this thesis.

Note also that inhibition by some of the sulphonamides is much stronger than NaCl. For example, I50 for exthoxyzolamide is only 5.3 nM for chlamydomonas and 2nM for Human CA "C". Figures for other sulphonamides are slightly higher, a table is given by Bundy (1986).

More recently, Dionosio-Sese and Miyachi (1992) investigated the effect of NaCl on CA activity in several marine microalgae, and found that in some species CA activity was inhibited whereas in others it was actually enhanced. Amoroso et al (1996) found that added MgSO4 increased the activity of CA from chlamydomonas more than 3 fold. Fisher et al (1996) showed that Dunaliella Salina, which thrives in very salty lakes, increases CA production as the salt concentration rises.

Clearly, inhibition or even enhancement of CA activity by the common anions of seawater is very species-specific, and should be borne in mind as one of many sources of uncertainty in later calculations, where the formulae for enzyme kinetics and air-sea CO2 exchange will be combined. The next chapter will introduce such calculations, and also demonstrate the importance of considering the physiological response of marine algae to water pCO2 when calculating the possible effect of catalysis on the net global air-sea CO2 flux.

Continue to Chapter 3:
Early Calculations and Experimental Observations

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