Importance of the time scale of the measurement apparatus for time resolution of an oxidation-reduction biological event

101 the various amino acid residues found in proteins. The starting point is at the bottom left and the stopping point is at top left. Three kinds of structures ie helices, arrows and turned arrows represent the different kinds of secondary structures found in proteins namely a-helix, 13-sheet and [3-turn, respectively. One dice and four counters are required.

How to Play (Rules) (i) A maximum of four persons can play. (ii) Counters are distributed among the players. (iii) Player number 1 throws the dice. (iv) The game starts if the dice displays 1 or 6. The player then moves a counter from the start point to its destination according to the number. (v) Subsequent movements of counters on the board are such that one number moves a counter one square ahead, and counter movements in different rows are depicted by arrows. (vi) If a counter arrives at the tail of an arrow or spiral it will jump from tail to head. (vii) The player whose counter first reaches the stop point is the winner. Accordingly, positions of others will be decided.

Conclusion Playing this game several times has been found beneficial for memorizing amino acids favouring different types of secondary structures of proteins.

Acknowledgement Financial assistance from Aligarh Muslim University is gratefully acknowledged.

Analysis of Experimental Data

0307-4412(94)00179-0 Importance of the Time Scale of the Measurement Apparatus for Time Resolution of an OxidationReduction Biological Event M PRATS* and F RODRIGUEZ§ * Universit~ Paul Sabatier, L P T F du CNRS and § I B C G du C N R S 118, Route de Narbonne 31062 Toulouse Cedex, France

Introduction In an oxidation-reduction metabolic chain, the transfer of electrons and protons is catalysed by enzymes which facilitate the reduction of metabolic substrates. ~,2 This transfer is initiated by, for example, the luminous excitation energy of a sensitive receptor such as retinal in the bacteriorhodopsin membrane

RNction center

protein 3'4 or by the light excitation of the harvesting or antenna complex in photosynthetic b a c t e r i a : : The oxidation-reduction chains in these macromolecular membrane complexes are composed of numerous intermediates which alternatively pass from the oxidized to the reduced state. Each step of the metabolic chain may be kinetically defined by a specific transfer rate constant. In the case of the photosynthetic bacteria chromatophores of Rhodobacter capsulatus, it is possible schematically to present the different protein complexes involved in the transfer of electrons and protons from the light harvesting complex trapping the luminous energy, to the enzyme synthetizing ATP molecule, ATP synthase (ref 6 and Figure 1). In the light harvesting complex, a large number of intermediates are involved in the transfer of electrons and protons. Figure 2 presents these intermediates and their absorption maxima and kinetic constants for transferring electrons and protons. Spectroscopic and biochemical methods have shown that the light-harvesting antenna of Rh capsulatus consists of two major pigment-protein complexes, LH2 and LH 1.5'6 Each system is built of polypeptides that bind bacteriochlorophyll and carotenoid pigment molecules. This presentation analyses the pathways of energy transfer through the light-harvesting antenna pigments of the photosynthetic purple bacteria Rh capsulatus. The aim of the paper is to show students that when the resolution time of the spectrophotometer used is not sufficiently fast, a number of biological events are not discriminated by differential spectrophotometry. Increasing the time resolution of the apparatus give access to oxidation-reduction kinetics resolved in time for a large number of intermediates in a metabolic chain.

k-2

.

The first point to analyse is the Jablonski diagram, s which presents the time scales, for the phenomena of light absorption by a chromophore, for its internal energy conversion and for possible fluorescence emission. The time scale for ~hoton absorption by a biological molecule is approximately 10 - I s; the internal energy conversions occur in 10-ii_10-i_~ s; the lifetime of the ,possible emitted fluorescence photon can be of the order of 10-~s. When the photosynthetic bacteria chromatographores of Rh capsulatus are excited, they absorb the luminous energy hv and the outer electrons of the excited chromophore are transferred from degenerate initial states So to excited states St. Experimentally, the absorption spectrum of the chromophore may be recorded on a scale of seconds or minutes; the spectrum presents two characteristics: a light absorption peak at a given wavelength h r n a x and an absorption coefficient at this absorption peak ~Xmax. Using the Beer-Lambert law, quantitative measurements are possible. 9 Other spectrophotometric measurements can also be made. Using differential spectrophotometry, it is possible to study the appearance of the reduced states of the carotenoid chromophore

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Figure 1 Schematic presentation of the bacterial photophosphorylation machinery

BIOCHEMICAL EDUCATION 23(2) 1995

102

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Figure 2 A kinetic model with transfer kinetic constants o f electrons and protons for energy migration through the light harvesting antenna o f Rh capsulatus at 77°K when an imposed potential is applied to the chromatophore preparation. ~(j Varying the potential imposed on the biological preparation and measuring the reduced absorption at a given wavelength, the characteristic midpoint potential of the entity studied is obtained. This permits ordering of the different intermediates in an oxidation-reduction chain. The time scale of events is, in the example given, about a few milliseconds. The total or the partial reduction of the compound and then its reoxidation during time has been shown but time resolution of the apparatus was not short enough to measure the real time kinetics of reduction and reoxidation. By increasing the apparatus time resolution, it is possible to record the reduction and the re-oxidation of the chromophore studied directly on a microcomputer. Two types of recording were analysed: (a) the absorbance variations &A = f(h) of the intermediate induced by its flash reduction. Laser flash excitation is very brief (~ps), and a time lag is observed before detection of absorbance variations. The data &A = f(h) are then recorded. The difference spectra presented are the average of x experiments (Fig 3); (b) the reduced absorbance variations AA = f(t) is measured at a selected wavelength (Fig 4). Each point in the figure represents one experiment and the line is the best fit for the data. Figure 3 shows the rise in reduction of the BChl896nm chromophore varying the lag time between the laser flash excitation and the ~A variations. If the lag time between the excitation flash and the measured &,4 is increased, more BCh1896nm chromophore is reduced (A in Fig 3). Analyzing the decrease in reduction or the reoxidation of the intermediate (B in Fig 3), it is seen that increasing the time lag can induce a decrease of the reduced state of the chromophore. In these experiments, the difference spectra were recorded on a microcomputer and were the average of x data acquisitions. Quantitative analysis was then possible by students: in Fig 3, A, the amplitude maximum of the spectrum recorded, with a 150 p.s time lag between flash excitation and absorbance variation, represents 100% of the intermediate reduction. Spectrum 3 in Fig 3A shows only 78.5% reduction, spectrum 2, 67.8% and spectrum 1 53.5%. Figure 4 presents the rise-time kinetics of Rh capsulatus BChls96n m recorded at 910 nm and 77°K after excitation of the antenna pigment and 800 nm.ll The experiments were carried out at 77°K to permit transformations in one direction only (Fig 2). In addition, spectral resolution was better at this temperature than at room temperature. The figure shows the reduction and reoxidation of the excited chromophore versus time. From the kinetics, it is possible to define a rise time of reduction (%) which represents the time needed for 50% of total chromophore reduction and a decay time of reduction ('rd) which represents the time need for 50% of chromophore reoxidation. In ref 11 the values calculated for -r~ and To are respectively of 12 and 200 ps. These two parameters are constant characteristics for biological oxidation-reduction events which are of the order of the picosecond in the experiments presented.

BIOCHEMICAL EDUCATION 23(2) 1995

Discussion Classical spectrophotometry enables the characterization of a biological chromophore by its maximum absorption wavelength (hm,×) and its molecular absorption coefficient (~<,.,,). These two parameters are macroscopic and simply indicate that the chromophore absorbs light energy over a range of wavelengths. These two parameters characterizing the chromophore studied are very important when recording the differential spectrum between an oxidized and a reduced state of a biological intermediate. Quantitative studies are feasible because the amplitude AA of the phenomenon observed can be varied by changing the physico-chemical conditions imposed on the chromatophore preparation. "~ Two possibilities are encountered depending on the type of spectrophotometer used: (a) if the resolution of the apparatus is limited to the ms or the ~s scale, only biological events taking place on this scale can be observed. The apparatus used is the limiting element in this case and a large number of intermediates in the oxidation-reduction chain could be missed. Biological events can only be seen when +10

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Wavelength(nm) Figure 3 Time-resolved difference spectra of BChln9~..... chromophore in the bacterial photophosphorylation process, A: Spectral changes during the rise in the reduced form o f the BChls~ ..... chromophore. Difference spectra were measured at 300 ns, 750 ns, 35 pts, 150 p~ after the laser flash excitation, respectively. B: Spectral changes during the reduction decay o f the same chromophore. Difference spectra were measured at 20 pos, 600 Its, 50 ms, 120 ms after the laser flash excitation, respectively. Each difference spectrum is the average o f x acquisitions o f data

103 I

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Letters to the Editor

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From M Carroll Quality monitoring l

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Figure 4 Rise-time kinetics o f Rh capsulatus BChls~ .... recorded at 910 nm and 77~K after antenna pigment excitation at 800 nm. The dots represent measured kinetics and the line is the best fit to data. The curve on the left is the laser response function taking place in the order of resolution of the spectrophotometer; (b) if the spectrophotometer used has a resolution time of 10-12 s, a large number of intermediates in an oxidationreduction metabolic chain may be discriminated. The limiting step is now not the apparatus but the biological event observed. It is possible to characterize these oxidation-reduction events by their reduction rise (rr) and reduction decay (Td) which are two microscopic constants characteristic of the compound studied and measured on the biological time scale. With the evolving technology of spectrophotometers and using microcomputers for data acquisition, it is now possible to analyze on a biological time scale a large number of oxidationreduction intermediates in a metabolic chain. However, metastables or oscillating intermediates can exist in biological oxidation-reduction chains ~2 having lifetimes <10-~2s: at present, it is very difficult to analyse the variations in their oxidation-reduction in real time.

Acknowledgements

Dear Sir, I was interested to read the article ~ on student evaluation of biochemistry teaching as applied to large classes. We have experienced a similar situation with our first-year intake of 285 medical, dental and science students, 2 and have devised a similar solution to that used at Leeds University. We make extensive use of computer-marked questions of the multiple-choice type (MCQ). It is a simple matter to convert the computer output to a Likert-style scale of 1-5, where 1 = Strongly Disagree, 3 = Neutral/Don't Know, 5 = Strongly Agree. It takes me about one hour to analyse 55 such questions, a pleasingly small investment of my time. However, the value of the information derived from such analysis depends critically on the nature of the questions asked: a teaching matter of concern to the students may not appear as an item on the MCQ-based questionnaire. Hence I always add several free-response questions, including the final open-ended one: "What would have helped you to learn better the material in this module?" However, analysing over 250 responses to 7 such questions takes a considerable time, typically one hour per question! The issue of monitoring the quality of our biochemistry teaching is likely to play an increasingly im?ertant role in our professional lives over the next few years (in the UK at least; it probably already has that significance in the USA and elsewhere). Obviously we do not want to commit a disproportionate amount of resources to quality control, and MCQ-based questionnaires can make a useful contribution here. How do other colleagues cope with this problem? And just as importantly, how do they use the information derived from students" evaluation of biochemistry teaching?

References l Houghton J D and Booth A G (1994) Biochem Educ 22,201-203 2McCrorie P (1993) Trans Biochem Soc 21,379-383

We thank P Winterton for checking the English

Mark Carroll Department o f Biochemistry Queen Mary and Westfield College University o f London London E1 4NS, UK

References ~Pelmont, J (1989) Enzymes, Presses Universitaires de Grenoble, Grenoble ZAudigi6, CI and Zonszain,F (1993) Biochimie M~tabolique-Biosc&nces & Techniques, Doin, Paris 3Dunach, M, Berkowitz, S, Marti, T, Wu Hr, Y, Subramaniam, S, Khorana, H G and Rothschild, K J (1990) J Biol Chem 265, 1697816984 4Subramaniam, S, Greenhalgh, D A, Rath, P, Rothschild, K J and Khorana, H G (1991) Proc Nat! Acad Sci USA 88, 6873-6877 5Cramer, W A and Crofts, A R (1982) in Photosynthesis: Energy Conservation by Plants and Bacteria, Vol I (edited by Govindjee), pp 387-467, Academic Press, New York ~'Jackson, J B (1982) in Photosynthesis: Energy Conservation by Plants and Bacteria, Vol I (edited by Govindjee), p 317-375, Academic Press, New York 7Crofts, A R and Wraight, C A (1983) Biochim Biophys Acta 726, 149185 XLakowicz, J R (1992) Topics in Fluorescence Spectroscopy -- Biochemical Applications, Vol 3, Plenum Press, New York ~Prats, M (1989) Biochem Educ 17, 151-153 mRodriguez, F and Prats, M (1991) Biochem Educ 19, 88-911 ~ Zhang, F G, Van Grondelle, R and Sundstr6m, V (1992) Biophys J 61, 911-920 ~2Vos, M H, Lambry, J C, Robles, S J, Youvan, D G, Breton, J and Martin, J L (1991) Proc Nail Acad Sci USA 88, 8885-8889

B I O C H E M I C A L E D U C A T I O N 23(2) 1995

From F Vella Biochemistry without a ficence Dear Sir In the editorial which appeared in the October 1994 issue of Biochemical Education, the definition of molecular biology as "biochemistry without a licence" was attributed to Severo Ochoa. I think that the source was not Severo Ochoa but Erwin Chargaff, who wrote: "My definition, incidentally, would be that molecular biology is essentially the practice of biochemistry without a licence." This appeared in an essay entitled Amphisbaena which was published in his book Essays on Nucleic Acids (Elsevier, 1963, p 174-199) and later reprinted in his Voices in the Labyrinth (Seabury Press, New York, 1977, p 92-113). Chargaff described the genesis of this definition in his autobiography Heraclitean Fire (The Rockefeller University Press, New York, 1978, p 140). The paragraph in which this occurs runs as follows: