PlwtosynthesisResearch 48: 41--46, 1996. (~) 1996 KluwerAcademic Publishers. Printedin the Netherlands. Reflection
Bill Arnold and calorimetric measurements of the quantum requirement of photosynthesis - once again ahead of his time* S h m u e l M a l k i n I & D a v i d C. F o r k 2 l The Weizmann Institute of Science, Rehovot 76100 Israel; 2Department of Plant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford CA 94305-1297, USA Received 26 October 1995; accepted in revised form 17 November1995
Key words: Emerson, energy-storage, photoacoustic(s), photocalorimetry, photothermal radiometry, probe beam deflection, Warburg
Abstract The approach of photocalorimetry to decide on the true quantum requirement of photosynthesis - one of the main issues of the research in the first half of the century and a source of a bitter debate - is described. Bill Arnold's original approach to get into the true answer is reflected from the point of view of present day calorimetric techniques.
Abbreviations: PA-photoacoustic(s); PTR-photothermal radiometry; P B D - p r o b e beam deflection (thermal measurement); E S - energy storage; Q R - q u a n t u m requirement
Introduction The beginning of the modern period of photosynthesis research in the first half of the century was marked by bold speculative theories, most of which were quite far from reality as we know it today. To resolve the complicated jigsaw puzzle of photosynthesis required a long and tedious trial and error process, along with the development and refinement of background sciences and technological advances. It took decades before secure lines of research with a defined direction began, when manageable questions could be asked and answered and details filled in. One of the difficulties facing early researches was that a comprehensive understanding of photosynthesis, even in its broadest outlines, required the involvement of a broad spectrum of scientific disciplines. Physicists, chemists and biologists had to come together and seek a common language. This was difficult at first since there were natural barriers such as the lack of profes-
* This is CIW/DPB Contribution No. 1286.
sional understanding and sometimes even suspicion and misunderstanding between the different parties. Fortunately, the scientific community was given a bright example that beautifully illustrated how an interdisciplinary approach could lead to corner-stone advances. The concepts of the photosynthetic unit, the reaction center, excitation energy transfer - all these bread-and-butter concepts of today arose from experiments performed in the thirties via a collaboration between an experimental physicist (William Arnold) and a plant physiologist (Robert Emerson). Although the impact of their experiments was not immediately apparent, the results of this collaboration opened the door to our modern knowledge and understanding of how photosynthesis operates (Myers 1994).
The quantum requirement problem In this article we would like to reflect on a less known work of Arnold, concerning calorimetric measurements of how much light energy is actually stored by the photosynthetic process. Knowing the caloric
42 content (heat of combustion) of the principal products of photosynthesis (i.e. sugars) allows the estimation of the quantum yield of photosynthesis. The correct assessment of the quantum yield had occupied the best researchers for decades. It was not a trivial question then, both experimentally and theoretically. The correct value of the maximum quantum yield (i.e., the quantum yield measured under light limiting conditions) could point to the mechanism, or at least would eliminate inconsistent mechanisms for photosynthesis. In fact, the relevant term used was (minimum) quantum requirement, the reciprocal of the (maximum) quantum yield - the number of absorbed photons necessary to complete a unit of reaction, namely to bring about the evolution of a molecule of 02 and a concomitant fixation of CO2. The idea was that this number, rather than reflecting the efficiency of the primary reaction itself, could provide the overall efficiency of the photosynthetic process. From the experimental point of view, the measurement of the quantum yield was a difficult and tedious task in those days since it involved two types of parallel measurements: the determination of the absorbed photon flux (usually obtained with a bolometer) and manometric measurements of the change of pressure of the gas evolved, which was then the only way to measure oxygen evolution. One had to read the difference between two water columns with a precision of less than a 0.1 mm Hg (Emerson had a reputation for his ability to read a hundredth of a mm using a cathetometer, cf. Myers 1994). The sensitivity of the measurement, compared to present day techniques, was relatively low. A typical measurement required a relatively long exposure to light (many minutes at least) and was done using whole organisms (the most popular was the green alga C h l o r e l l a - no partial reaction was then sufficiently efficient or stable enough to use). Furthermore, it was difficult to account for respiration and induction effects and to determine the extent of light absorption. No wonder that the results of many experiments varied over a very large range (between about 2.5 to 12 hv/O2 evolved - Rabinowitch 1951). The problem was that a leading laboratory, Otto Warburg's in Berlin, got consistently low values of the quantum requirement and Warburg suggested what turned out to be an incorrect idea in an attempt to explain his low values. However, results from other laboratories, most notably Emerson's, did not agree with Warburg but yielded values centering around about 8-10 hv / O2 instead. This difference started a bitter debate, lasting for decades, concerning the
correct value for the quantum requirement of photosynthesis. This debate was driven by the strong personality and insistence of Warburg who was one of the most prominent and influential biochemists of his time (Krebs 1972). Warburg had acquired most of his fame through his pioneering investigations on the enzymes of cellular respiration, for which he won the Nobel Prize in 1931. Warburg also made significant advances in photosynthesis research. He was the first to choose Chlorella as an organism well suited for measurements of photosynthesis and to perfect the manometric method to measure gas exchange for the determination of the quantum requirement of photosynthesis. His quantum requirement measurements were motivated very much by Einstein's law of photochemical equivalence. He was very familiar with this concept when it was already in its early stage, since his father, the physicist Emil Warburg, had a close relationship with Albert Einstein and was the first to do specific experiments to verify and substantiate Einstein's concept for photochemical reactions. Through his father, Otto Warburg himself acquired the knowledge and the best physical equipment of the time that enabled him to do this kind of experiment. Warburg made successful use of this concept to investigate the action spectrum for the photochemical decomposition of the cytochrome oxidase complex with CO, and obtained its absorption spectrum, a critical step towards its isolation.
The photocalorimetric approach of Bill Arnold In order to resolve the debate on the quantum requirement it was necessary to use a different approach. This was actually achieved by the photocalorimetric experiments of Arnold. At the time they should have made a bigger impact since they clearly provided the correct answer of about 8 h v / 0 2 . As with so many of the measurements done by Arnold, his early use of the calorimetric method in 1936-37 to determine the quantum yield of photosynthesis was ahead of his times. Herman Spoehr, who headed Carnegie Institution's Department of Plant Biology predicted in 1926 (Spoehr 1926) that small heat differences between photosynthetically active and inactive leaves could be used to quantify photosynthetic efficiency. Arnold adapted for biological use Callendar's 'radio balance' (Callendar 1911) that was developed to measure heat emission from radioactive materials. This apparatus was based on the measurement of temperature change of a small amount of irradiated sample resting in a sil-
43 Table 1. Representative maximum energy storage efficiency determinations and the corresponding calculated quantum requirements (from Arnold's data (1949)) Organism
ES (at)~ = 660nm)
QR
Chlorella pyrenoidosa Chlorella vulgaris Scenedesmus sp. Avocado leaf
0.279 0.232 0.259 0.165
9.2 11.1 10.3 15.6
ver cup, by the thermoelectric effect, through use of a network of thermocouple junctions, communicating to the main body of the apparatus. It essentially acted as a null instrument, opposing the temperature changes by passing an electric current through one of the thermocouples and cooling the sample by the resulting Peltier effect. Arnold was able to measure the small heat difference between a photosynthetically active and an inactive sample, with a measurement time of about a minute. Chlorella pyrenoidosa, Chlorella vulgaris, Scenedesmus sp. and an avocado leaf were used as samples. To inactivate his samples Arnold preexposed them to ultra-violet light. While the results varied quite a bit between the different experiments, Arnold found that the maximum efficiency of light energy storage, using a wavelength about 660 nm, was around 28% in Chlorella pyrenoidosa. This number is equivalent to a quantum requirement of 9.2, sufficiently close to the theoretical value of 8 quanta, which is the minimum requirement for two photochemical systems acting in series (see Appendix for the details of the conversion between energy storage and quantum requirement). This, and similar data are summarized in Table 1. His higher numbers for the quantum requirement (not shown) can be accounted by less competent samples, but it is also possible that the UV treatment in some cases (particularly for the case of the leaf) was not sufficient for complete inactivation, resulting in smaller differences of heat emission between the sample and its reference (Bell 1985). Unfortunately, it seems that Arnold was not sufficiently sure that he obtained the right result, since he was himself influenced by the vigor by which Warburg insisted upon quantum requirements of 4 and even less. Therefore, the publication of his article was deferred for twelve (!) years, when finally Hans Gaffron convinced Arnold to publish the results (Arnold 1949).
From the radio-balance period to modern photoacoustic and other photothermal methods Today, by using the photoacoustic (PA) method we can make photocalorimetric measurements in a much easier manner, and be more assured about the results. This method is based on using pulsed or periodically modulated light and monitoring pressure changes which result from temperature changes caused by heat evolution (Fork and Herbert 1993; Malkin and Canaani 1994). For this, a microphone or piezoelectric device is used and the method is so sensitive that a difference of temperature of the order of a millidegree Celsius can be detected easily. There are other ways to sense temperature changes induced by the modulated light: 'photothermal radiometry' (PTR), by which one detects the modulated part of the infra-red radiation emitted from the sample in a strength related to the amplitude of the temperature changes (Kanstad et al. 1983), and 'probe beam deflection' (PBD - also known as the 'mirage' effect method), where refractive index changes are induced and alter the trajectory of a probe laser beam (Havaux et al. 1990). These last two techniques have been used only infrequently in photosynthesis. The photoacoustic and the related methods, at least in principle, can yield information on the time characteristics of the heat evolution and differentiate between different phases in the kinetics of the heat emission. Using light whose intensity is modulated at a certain frequency f, only heat which is evolved in a time lapse shorter than roughly a cycle time (i.e. I/f), will give rise to synchronous pressure modulation. Calibration of the pressure changes with respect to the emitted energy can be done easily by using strong non-modulated light, that is added on top of the existing modulated light. The strong light brings the photosynthetic system to a state of over-saturation and inefficient use of light quanta (i.e. the quantum yield tends to zero). In this state modulated light produces modulated heat in its full equivalence of energy. Using an appropriate frequency to match any particular stage in the chain of photosynthetic reactions, one can find the energy status of that particular stage. Figure 1 shows two experimental records measured according to the above protocol. One was obtained with a water infiltrated leaf, taken at a low frequency limit in the possible frequency range (ca. 12 Hz), using photoacoustic detection and the other was obtained with photothermal radiometry with an ordinary intact leaf. Comparing the signal levels with (S +) and without ( S - ) background strong light one can
44 Table 2. Representative energy storage efficiency determinations and calculated quantum requirements (from photoacoustic and photothermal calorimetry) Sample
Method
Central A (nm)
f (Hz)
ES
QR
Reference and remarks
Porphyra perforata (thallus)
PA PBD PA PTR PTR PA
580 685 685 615 615 630
35 15 12 2 14 15
0.32 0.32 0.29 0.26 0.28 0.27
7.9 9.3 10.3 10.4 9.7 10.2
Malkin et al. (1991) Havaux et al. (1990) Malkin et al. (1992) Kanstad et al. (1983) Kanstad et al. (1983) Carpentier et al. (1984) (Reference sample was a DCMU treated leaf)
Pea leaf Pea leaf (water infiltrated) Siberian pea Wheat
Anacystis nidulans
A
!
B
< Q
Figure 1. A. Experimental records of the signal from a microphone, placed in the gas phase around a water infiltrated leaf, which is illuminated by modulated light (photoacoustic signal). The sensor output was processed by a lock-in amplifier, which is only sensitive to signals appearing in the same frequency as that of the modulation frequency and allows adjustment oftbe phase angle. The two records are from two orthogonal phase setting named I and Q. The similar pattern of the recordings is typical for a single contribution (compare to Figure 2), arising from the conversion of the light energy to thermal energy (from Malkin et al. 1992). B. Experimental record of the signal amplitude from an infra-red detector (Pb/Sn/Te) optimized for room-temperature black-body radiation at 10/zm, measuring the modulated part of the thermal radiation from an intact leaf, which is illuminated by modulated light. The phase setting was adjusted to maximize the signal (from Kanstad et al. 1983). Wavy arrows switch on of the modulated light (660 nm, 13Wm -2, 26Hz in A, 570--660 nm, 25 Wm -2, 23 Hz in B). Straight arrows - switch on (up) and off (down) of a strong saturating (non-modulated) light.
calculate the effeciency of energy storage, ES, as [S +S - ] / S +. The reason to use a water infiltrated leaf in the experiment of Figure 1A is because in leaves and lichens the photoacoustic signal at low frequencies is usually a composite of two contributions: pressure changes caused by heat emission and pressure changes produced by photosynthetic oxygen evolution. The contribution of the latter can be quite strong, and different from the heat contribution signal and it often dominates the behavior of the photoacoustic signal. The
Figure 2. Experimental record of photoacoustic signals from an intact leaf. Simultaneous signals from two orthogonal phase settings (I and Q) of the lock-in amplifier are exhibited. The part of the signal which is eliminated by the strong saturating (non-modulated) light is due to oxygen evolution. The phase adjustment was done so that the Q signal will contain only the oxygen evolution component. Other details are as in Figure 1A (from M a l l ~ et al. 1992).
photoacoustic oxygen evolution signal can be eliminated by the strong light, rather than enhanced by it. Fortunately for calorimetric measurements, it is eliminated by water infiltration and high frequency, because of the strong damping of oxygen evolution pressure modulations as oxygen diffuses through the aqueous phase. Figure 2 shows an example of the composite photoacoustic signal in an ordinary leaf, and demonstrates how one can also use this effect to monitor photosynthetic oxygen evolution continuously with a time resolution of a fraction of a second. This brings us to another connection to the past. The instrument to measure photosynthesis at that time was the manometric 'Warburg apparatus', sometimes just abbreviated 'Warburg', which measured the pressure increase over a reaction vessel following a light expo-
45 sure. Thus photoacoustics, in the sense that it measures photosynthetic oxygen evolution by pressure modulation, can be considered as a m o d e m version of the 'Warburg', perhaps appropriately named 'modulated Warburg'. However, while there is an enormous gain in sensitivity and speed, there is no absolute calibration. Methods to calibrate the photoacoustic oxygen evolution signal must still be worked out. As a result, one can only measure relative values for the quantum yield and one cannot use the 'modulated Warburg' aspect of the photoacoustic method to measure absolute quantum yields, as was attempted in the past. Nevertheless, the above does not apply to the thermal measurement aspect of photoacoustics. One can use the above formula for the fraction of energy storage (Appendix) both ways: to either find the actual energy stored in the photosynthetic intermediates, or to find the quantum yield if this energy is k n o w n a priori from calorimetric data. Let us do this last exercise, as if we are now fifty years ago, but have all the data presented by the photoacoustic experiments (including other photothermal methods, mentioned above). In this we assume a value AE, for the energy of the 'final' product (i.e. glucose), which is about 2818 kJ/mole. Assuming an overall turn-over time of around 10 ms at room temperature, which results from another work of Arnold (Emerson and Arnold 1932a,b) that was confirmed later (Kok 1956), the most appropriate frequencies to use must be below about 15 Hz. However, experience shows that usually there is no significant change in the results over a wide frequency range. Some results are presented in Table 2 (note in this table the experiment at very low frequency [2Hz]). One can determine from Table 2 an average value for the quantum requirement of 9.6, which, if found fifty years ago, would have provided solid support for the quantum requirement measurements of Emerson et al. and other investigators (Rabinowitch 1951). The values in Table 2 are not much different than those of Arnold in Table 1, consistent with the idea of two photosystems acting in series. Thus, once again, as with so many of his experiments, Bill Arnold was ahead of his time and his results looked at from what we know today could have provided at a much earlier time the correct interpretation of the basic operation of the photosynthetic process.
Acknowledgement The authors wish to thank Professor Jack Myers for providing several useful references.
Appendix The connection between the efficiency of energy storage (ES ) and quantum requirement ( QR ) Experimentally: E S = 1-Hs/Hr where Hs is the heat evolved from the sample under study and Hr is the heat evolved from a reference (inactivesample). Theoretically: ES =- dpAE/Nhv, hence qb= ES Nhv/AE orQR ----1/q~= AE/[ES Nhv]. (If losses occur in the photochemistry,then QR < AE/[ES Nhv].) ~b- the quantumyield; AE - the energy gain of photosynthesis per mole product; N - Avogadro number;h - Planck constant;v - the electromagneticfrequency.Nhv - the energycontentof an Einstein (mole photons). The values involvedare: heat of combustionof glucose 2812 kJ/mole. HenceAE~ 469 kJ/mole of fixed CO2 (neglectingheats of solutionand enthalpy/energydifference).
References ArnoldW (1949) A calorimetricdeterminationof the quantumyield in photosynthesis. In: Frank J and Loomis WE (eds) Photosynthesis in Plants, pp 273-276. The IA State Press, Ames, IA Bell LN (1985) Energetics of the photosynthesizingplant cell (cf. chapter 12 and references therein). Soviet Scientific Reviews, Supplement Series Physico Chemical Biology, Vol V, VP Skulachev (ed), originally in Russian (1980). Bell and Bain Ltd, Glasgow Callendar HL (1911) The radio-balance.A thermoelectric balance for the absolute measurementof radiation, with applicationsto radium and its emanation.Proc Physical Soc London23:1-34 CarpentierR, LaRueB and LeblancRM (1984) Photoacousticspectroscopy of Anacystis nidulans III. Detection of photosynthetic activities. Arch BiochemBiophys 228:534-576 Emerson R and ArnoldW (1932a) A separation of the reactions in photosynthesis by means of intermittentlight. J Gen Physiol 15: 391-420 Emerson R and Arnold W (1932b) The photochemical reaction in photosynthesis. J Gen Physiol 16:191-205 Fork DC and Herbert SK (1993) The application of photoacoustic techniquesto studiesof photosynthesis.PhotochemPhotobio157: 207-220 Havaux M, LorrainL and LeblancRM (1990) Photothermal beam deflection:A new method for in in vivo measurementsof thermal energydissipationand photochemicalenergyconversionin intact leaves. PhotosynthRes 24:63-73 Kanstad SO, Cahen D and Malkin S (1983) Simultaneousdetection of photosynthetic energy storage and oxygen evolution in
46 leaves by photothermal radiometry and photoacoustics. Biochim Biophys Acta 722:182-189 Kok B (1956) Photosynthesis in flashing light. Biochim Biophys Acta 21: 245-258 Krebs H (1972) Otto Heinrich Warburg. Biogr. Memoirs R Soc 18: 629-699 Malkin S and Canaani O (1994) The use and characteristics of the photoaocustic method in the study of photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 40:493-526 Malkin S, Charland M and Leblanc RM (1992) A photoacoustic study of water infiltrated leaves. Photosynth Res 33:37-50
Malkin S, Herbert SK and Fork DC (1990) Light distribution, transfer and utilization in the marine red alga Porphyraperforata from photoacoustic energy-storage measurements. Biochim Biophys Acta 1016:177-189 Myers J (1994) The 1932 experiments. Photosynth Res 40:303-310 Rabinowitch EI (1951) Photosynthesis and Related Processes. Vol II, Part I (see chapter 29 and references therein, pp 1083-1141). Interscience Publishers, New-York Spoehr HA (1926) Photosythesis. The Chemical Catalog Co, New York
Bill Arnold and calorimetric measurements of the quantum requirement of photosynthesis - once again ahead of his time* S h m u e l M a l k i n I & D a v i d C. F o r k 2 l The Weizmann Institute of Science, Rehovot 76100 Israel; 2Department of Plant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford CA 94305-1297, USA Received 26 October 1995; accepted in revised form 17 November1995
Key words: Emerson, energy-storage, photoacoustic(s), photocalorimetry, photothermal radiometry, probe beam deflection, Warburg
Abstract The approach of photocalorimetry to decide on the true quantum requirement of photosynthesis - one of the main issues of the research in the first half of the century and a source of a bitter debate - is described. Bill Arnold's original approach to get into the true answer is reflected from the point of view of present day calorimetric techniques.
Abbreviations: PA-photoacoustic(s); PTR-photothermal radiometry; P B D - p r o b e beam deflection (thermal measurement); E S - energy storage; Q R - q u a n t u m requirement
Introduction The beginning of the modern period of photosynthesis research in the first half of the century was marked by bold speculative theories, most of which were quite far from reality as we know it today. To resolve the complicated jigsaw puzzle of photosynthesis required a long and tedious trial and error process, along with the development and refinement of background sciences and technological advances. It took decades before secure lines of research with a defined direction began, when manageable questions could be asked and answered and details filled in. One of the difficulties facing early researches was that a comprehensive understanding of photosynthesis, even in its broadest outlines, required the involvement of a broad spectrum of scientific disciplines. Physicists, chemists and biologists had to come together and seek a common language. This was difficult at first since there were natural barriers such as the lack of profes-
* This is CIW/DPB Contribution No. 1286.
sional understanding and sometimes even suspicion and misunderstanding between the different parties. Fortunately, the scientific community was given a bright example that beautifully illustrated how an interdisciplinary approach could lead to corner-stone advances. The concepts of the photosynthetic unit, the reaction center, excitation energy transfer - all these bread-and-butter concepts of today arose from experiments performed in the thirties via a collaboration between an experimental physicist (William Arnold) and a plant physiologist (Robert Emerson). Although the impact of their experiments was not immediately apparent, the results of this collaboration opened the door to our modern knowledge and understanding of how photosynthesis operates (Myers 1994).
The quantum requirement problem In this article we would like to reflect on a less known work of Arnold, concerning calorimetric measurements of how much light energy is actually stored by the photosynthetic process. Knowing the caloric
42 content (heat of combustion) of the principal products of photosynthesis (i.e. sugars) allows the estimation of the quantum yield of photosynthesis. The correct assessment of the quantum yield had occupied the best researchers for decades. It was not a trivial question then, both experimentally and theoretically. The correct value of the maximum quantum yield (i.e., the quantum yield measured under light limiting conditions) could point to the mechanism, or at least would eliminate inconsistent mechanisms for photosynthesis. In fact, the relevant term used was (minimum) quantum requirement, the reciprocal of the (maximum) quantum yield - the number of absorbed photons necessary to complete a unit of reaction, namely to bring about the evolution of a molecule of 02 and a concomitant fixation of CO2. The idea was that this number, rather than reflecting the efficiency of the primary reaction itself, could provide the overall efficiency of the photosynthetic process. From the experimental point of view, the measurement of the quantum yield was a difficult and tedious task in those days since it involved two types of parallel measurements: the determination of the absorbed photon flux (usually obtained with a bolometer) and manometric measurements of the change of pressure of the gas evolved, which was then the only way to measure oxygen evolution. One had to read the difference between two water columns with a precision of less than a 0.1 mm Hg (Emerson had a reputation for his ability to read a hundredth of a mm using a cathetometer, cf. Myers 1994). The sensitivity of the measurement, compared to present day techniques, was relatively low. A typical measurement required a relatively long exposure to light (many minutes at least) and was done using whole organisms (the most popular was the green alga C h l o r e l l a - no partial reaction was then sufficiently efficient or stable enough to use). Furthermore, it was difficult to account for respiration and induction effects and to determine the extent of light absorption. No wonder that the results of many experiments varied over a very large range (between about 2.5 to 12 hv/O2 evolved - Rabinowitch 1951). The problem was that a leading laboratory, Otto Warburg's in Berlin, got consistently low values of the quantum requirement and Warburg suggested what turned out to be an incorrect idea in an attempt to explain his low values. However, results from other laboratories, most notably Emerson's, did not agree with Warburg but yielded values centering around about 8-10 hv / O2 instead. This difference started a bitter debate, lasting for decades, concerning the
correct value for the quantum requirement of photosynthesis. This debate was driven by the strong personality and insistence of Warburg who was one of the most prominent and influential biochemists of his time (Krebs 1972). Warburg had acquired most of his fame through his pioneering investigations on the enzymes of cellular respiration, for which he won the Nobel Prize in 1931. Warburg also made significant advances in photosynthesis research. He was the first to choose Chlorella as an organism well suited for measurements of photosynthesis and to perfect the manometric method to measure gas exchange for the determination of the quantum requirement of photosynthesis. His quantum requirement measurements were motivated very much by Einstein's law of photochemical equivalence. He was very familiar with this concept when it was already in its early stage, since his father, the physicist Emil Warburg, had a close relationship with Albert Einstein and was the first to do specific experiments to verify and substantiate Einstein's concept for photochemical reactions. Through his father, Otto Warburg himself acquired the knowledge and the best physical equipment of the time that enabled him to do this kind of experiment. Warburg made successful use of this concept to investigate the action spectrum for the photochemical decomposition of the cytochrome oxidase complex with CO, and obtained its absorption spectrum, a critical step towards its isolation.
The photocalorimetric approach of Bill Arnold In order to resolve the debate on the quantum requirement it was necessary to use a different approach. This was actually achieved by the photocalorimetric experiments of Arnold. At the time they should have made a bigger impact since they clearly provided the correct answer of about 8 h v / 0 2 . As with so many of the measurements done by Arnold, his early use of the calorimetric method in 1936-37 to determine the quantum yield of photosynthesis was ahead of his times. Herman Spoehr, who headed Carnegie Institution's Department of Plant Biology predicted in 1926 (Spoehr 1926) that small heat differences between photosynthetically active and inactive leaves could be used to quantify photosynthetic efficiency. Arnold adapted for biological use Callendar's 'radio balance' (Callendar 1911) that was developed to measure heat emission from radioactive materials. This apparatus was based on the measurement of temperature change of a small amount of irradiated sample resting in a sil-
43 Table 1. Representative maximum energy storage efficiency determinations and the corresponding calculated quantum requirements (from Arnold's data (1949)) Organism
ES (at)~ = 660nm)
QR
Chlorella pyrenoidosa Chlorella vulgaris Scenedesmus sp. Avocado leaf
0.279 0.232 0.259 0.165
9.2 11.1 10.3 15.6
ver cup, by the thermoelectric effect, through use of a network of thermocouple junctions, communicating to the main body of the apparatus. It essentially acted as a null instrument, opposing the temperature changes by passing an electric current through one of the thermocouples and cooling the sample by the resulting Peltier effect. Arnold was able to measure the small heat difference between a photosynthetically active and an inactive sample, with a measurement time of about a minute. Chlorella pyrenoidosa, Chlorella vulgaris, Scenedesmus sp. and an avocado leaf were used as samples. To inactivate his samples Arnold preexposed them to ultra-violet light. While the results varied quite a bit between the different experiments, Arnold found that the maximum efficiency of light energy storage, using a wavelength about 660 nm, was around 28% in Chlorella pyrenoidosa. This number is equivalent to a quantum requirement of 9.2, sufficiently close to the theoretical value of 8 quanta, which is the minimum requirement for two photochemical systems acting in series (see Appendix for the details of the conversion between energy storage and quantum requirement). This, and similar data are summarized in Table 1. His higher numbers for the quantum requirement (not shown) can be accounted by less competent samples, but it is also possible that the UV treatment in some cases (particularly for the case of the leaf) was not sufficient for complete inactivation, resulting in smaller differences of heat emission between the sample and its reference (Bell 1985). Unfortunately, it seems that Arnold was not sufficiently sure that he obtained the right result, since he was himself influenced by the vigor by which Warburg insisted upon quantum requirements of 4 and even less. Therefore, the publication of his article was deferred for twelve (!) years, when finally Hans Gaffron convinced Arnold to publish the results (Arnold 1949).
From the radio-balance period to modern photoacoustic and other photothermal methods Today, by using the photoacoustic (PA) method we can make photocalorimetric measurements in a much easier manner, and be more assured about the results. This method is based on using pulsed or periodically modulated light and monitoring pressure changes which result from temperature changes caused by heat evolution (Fork and Herbert 1993; Malkin and Canaani 1994). For this, a microphone or piezoelectric device is used and the method is so sensitive that a difference of temperature of the order of a millidegree Celsius can be detected easily. There are other ways to sense temperature changes induced by the modulated light: 'photothermal radiometry' (PTR), by which one detects the modulated part of the infra-red radiation emitted from the sample in a strength related to the amplitude of the temperature changes (Kanstad et al. 1983), and 'probe beam deflection' (PBD - also known as the 'mirage' effect method), where refractive index changes are induced and alter the trajectory of a probe laser beam (Havaux et al. 1990). These last two techniques have been used only infrequently in photosynthesis. The photoacoustic and the related methods, at least in principle, can yield information on the time characteristics of the heat evolution and differentiate between different phases in the kinetics of the heat emission. Using light whose intensity is modulated at a certain frequency f, only heat which is evolved in a time lapse shorter than roughly a cycle time (i.e. I/f), will give rise to synchronous pressure modulation. Calibration of the pressure changes with respect to the emitted energy can be done easily by using strong non-modulated light, that is added on top of the existing modulated light. The strong light brings the photosynthetic system to a state of over-saturation and inefficient use of light quanta (i.e. the quantum yield tends to zero). In this state modulated light produces modulated heat in its full equivalence of energy. Using an appropriate frequency to match any particular stage in the chain of photosynthetic reactions, one can find the energy status of that particular stage. Figure 1 shows two experimental records measured according to the above protocol. One was obtained with a water infiltrated leaf, taken at a low frequency limit in the possible frequency range (ca. 12 Hz), using photoacoustic detection and the other was obtained with photothermal radiometry with an ordinary intact leaf. Comparing the signal levels with (S +) and without ( S - ) background strong light one can
44 Table 2. Representative energy storage efficiency determinations and calculated quantum requirements (from photoacoustic and photothermal calorimetry) Sample
Method
Central A (nm)
f (Hz)
ES
QR
Reference and remarks
Porphyra perforata (thallus)
PA PBD PA PTR PTR PA
580 685 685 615 615 630
35 15 12 2 14 15
0.32 0.32 0.29 0.26 0.28 0.27
7.9 9.3 10.3 10.4 9.7 10.2
Malkin et al. (1991) Havaux et al. (1990) Malkin et al. (1992) Kanstad et al. (1983) Kanstad et al. (1983) Carpentier et al. (1984) (Reference sample was a DCMU treated leaf)
Pea leaf Pea leaf (water infiltrated) Siberian pea Wheat
Anacystis nidulans
A
!
B
< Q
Figure 1. A. Experimental records of the signal from a microphone, placed in the gas phase around a water infiltrated leaf, which is illuminated by modulated light (photoacoustic signal). The sensor output was processed by a lock-in amplifier, which is only sensitive to signals appearing in the same frequency as that of the modulation frequency and allows adjustment oftbe phase angle. The two records are from two orthogonal phase setting named I and Q. The similar pattern of the recordings is typical for a single contribution (compare to Figure 2), arising from the conversion of the light energy to thermal energy (from Malkin et al. 1992). B. Experimental record of the signal amplitude from an infra-red detector (Pb/Sn/Te) optimized for room-temperature black-body radiation at 10/zm, measuring the modulated part of the thermal radiation from an intact leaf, which is illuminated by modulated light. The phase setting was adjusted to maximize the signal (from Kanstad et al. 1983). Wavy arrows switch on of the modulated light (660 nm, 13Wm -2, 26Hz in A, 570--660 nm, 25 Wm -2, 23 Hz in B). Straight arrows - switch on (up) and off (down) of a strong saturating (non-modulated) light.
calculate the effeciency of energy storage, ES, as [S +S - ] / S +. The reason to use a water infiltrated leaf in the experiment of Figure 1A is because in leaves and lichens the photoacoustic signal at low frequencies is usually a composite of two contributions: pressure changes caused by heat emission and pressure changes produced by photosynthetic oxygen evolution. The contribution of the latter can be quite strong, and different from the heat contribution signal and it often dominates the behavior of the photoacoustic signal. The
Figure 2. Experimental record of photoacoustic signals from an intact leaf. Simultaneous signals from two orthogonal phase settings (I and Q) of the lock-in amplifier are exhibited. The part of the signal which is eliminated by the strong saturating (non-modulated) light is due to oxygen evolution. The phase adjustment was done so that the Q signal will contain only the oxygen evolution component. Other details are as in Figure 1A (from M a l l ~ et al. 1992).
photoacoustic oxygen evolution signal can be eliminated by the strong light, rather than enhanced by it. Fortunately for calorimetric measurements, it is eliminated by water infiltration and high frequency, because of the strong damping of oxygen evolution pressure modulations as oxygen diffuses through the aqueous phase. Figure 2 shows an example of the composite photoacoustic signal in an ordinary leaf, and demonstrates how one can also use this effect to monitor photosynthetic oxygen evolution continuously with a time resolution of a fraction of a second. This brings us to another connection to the past. The instrument to measure photosynthesis at that time was the manometric 'Warburg apparatus', sometimes just abbreviated 'Warburg', which measured the pressure increase over a reaction vessel following a light expo-
45 sure. Thus photoacoustics, in the sense that it measures photosynthetic oxygen evolution by pressure modulation, can be considered as a m o d e m version of the 'Warburg', perhaps appropriately named 'modulated Warburg'. However, while there is an enormous gain in sensitivity and speed, there is no absolute calibration. Methods to calibrate the photoacoustic oxygen evolution signal must still be worked out. As a result, one can only measure relative values for the quantum yield and one cannot use the 'modulated Warburg' aspect of the photoacoustic method to measure absolute quantum yields, as was attempted in the past. Nevertheless, the above does not apply to the thermal measurement aspect of photoacoustics. One can use the above formula for the fraction of energy storage (Appendix) both ways: to either find the actual energy stored in the photosynthetic intermediates, or to find the quantum yield if this energy is k n o w n a priori from calorimetric data. Let us do this last exercise, as if we are now fifty years ago, but have all the data presented by the photoacoustic experiments (including other photothermal methods, mentioned above). In this we assume a value AE, for the energy of the 'final' product (i.e. glucose), which is about 2818 kJ/mole. Assuming an overall turn-over time of around 10 ms at room temperature, which results from another work of Arnold (Emerson and Arnold 1932a,b) that was confirmed later (Kok 1956), the most appropriate frequencies to use must be below about 15 Hz. However, experience shows that usually there is no significant change in the results over a wide frequency range. Some results are presented in Table 2 (note in this table the experiment at very low frequency [2Hz]). One can determine from Table 2 an average value for the quantum requirement of 9.6, which, if found fifty years ago, would have provided solid support for the quantum requirement measurements of Emerson et al. and other investigators (Rabinowitch 1951). The values in Table 2 are not much different than those of Arnold in Table 1, consistent with the idea of two photosystems acting in series. Thus, once again, as with so many of his experiments, Bill Arnold was ahead of his time and his results looked at from what we know today could have provided at a much earlier time the correct interpretation of the basic operation of the photosynthetic process.
Acknowledgement The authors wish to thank Professor Jack Myers for providing several useful references.
Appendix The connection between the efficiency of energy storage (ES ) and quantum requirement ( QR ) Experimentally: E S = 1-Hs/Hr where Hs is the heat evolved from the sample under study and Hr is the heat evolved from a reference (inactivesample). Theoretically: ES =- dpAE/Nhv, hence qb= ES Nhv/AE orQR ----1/q~= AE/[ES Nhv]. (If losses occur in the photochemistry,then QR < AE/[ES Nhv].) ~b- the quantumyield; AE - the energy gain of photosynthesis per mole product; N - Avogadro number;h - Planck constant;v - the electromagneticfrequency.Nhv - the energycontentof an Einstein (mole photons). The values involvedare: heat of combustionof glucose 2812 kJ/mole. HenceAE~ 469 kJ/mole of fixed CO2 (neglectingheats of solutionand enthalpy/energydifference).
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