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A simple method for measuring oxyhaemoglobin dissociation curves in a student practical class
34
BIOCHEMICAL EDUCATION
April 1979 Vol. 7 No. 2
A SIMPLE METHOD FOR MEASURING OXYHAEMOGLOBIN DISSOCIATION CURVES IN A STUDENT PRACTICAL CLASS
For some time we have been using a simple and rapid method for measuring oxyhaemoglobin dissociation curves in our student practical class. The method is based upon that published by Neville and Sasner (1964) which, unfortunately, is not readily available. Scott (1975) improved the method by using an oxygen electrode but does not explain clearly how to calculate the oxyhaemoglobin dissociation curve. We are using this opportunity to demonstrate our experiences with the method in class use, and to re-present the theory.
Principle of the method Yeast cells in an oxygenated buffer solution will consume and remove oxygen from the medium at a constant rate (Fig. la). When erythrocytes are present, the dissolved oxygen will be removed at a constant rate until the Po~ falls to the value at which oxyhaemoglobin begins to dissociate. After this time, part of the oxygen consumed by the yeast will come from oxyhaemoglobin so that the rate of decrease of oxygen concentration is retarded (Fig. lb). Consider Fig. lb. Extrapolation of the linear part of the trace (shown by line DB) to zero oxygen concentration gives a curve essentially the same as that obtained in the absence of blood. The extra time required to consume the oxygen released by the oxyhaemoglobin is thus the time represented by the line between points A and B. Point D on the trace, where the linear part merges with the sigmoidal part, is the m i n i m u m Po~ required for 100% saturation of the haemoglobin. The time represented by AB is the time required by the yeast to use the total oxygen present. T h u s point C, midway between A and B, corresponds to the time for half the combined oxygen to be consumed. The line drawn parallel to BD through C cuts the trace at E. This point gives the Po~ at which the haemoglobin is 50% saturated, i.e. the P50 value• This parameter is obtained directly, and therefore quickly, from the recorder trace• It may be sufficient for many purposes but, with a little more calculation the oxyhaemoglobin dissociation curve itself may be obtained.
GORDON L. ATKINS & JAMES DOYLE Department of Biochemistry University of Edinburgh Medical School Teviot Place Edinburgh, EH8 9AG, Scotland
Theory for the calculation of the dissociation curve The yeast consumes oxygen at a constant rate (R). Fig. 2a shows an experimental trace of o x y g e n concentration (Po, in m m Hg) versus time when only yeast was present. Fig. 2b shows the result of an experiment when blood was also present. (The final sigmoidal part of the curve is extrapolated to point A to correct for the small non-linear region of oxygen consumption by yeast at concentrations below about 2 m m Hg.) At Po2 above about 70 m m Hg the oxyhaemoglobin remains undissociated and the oxygen is derived solely from solution. Hence the trace is linear and can be used to calculate the constant rate of oxygen consumption by the yeast (R) in m m H g / m i n . R could be expressed in ml Oz/min or in/amole/min by multiplying by a suitable constant, but in calculating the final variable (percentage saturation of haemoglobin) the constant would be cancelled out. Below point D oxygen is obtained both from that in solution and that released by oxyhaemoglobin. Consider this section. During a time interval At: amount of oxygen consumed by the yeast = R At mm Hg amount of oxygen 9rovided by the solution = APo~ m m Hg If the amount released by the haemoglobin is AC m m Hg, then R.At= ~° o +AC or, A C = R . A t 2 A P o ~ m m H g R has been calculated, and the values of At and APo2 can be read from the trace. Thus AC can be calculated for many values of Po~. The amount of oxygen released up to time t is t
C(t) = ~, AC 0
and the total amount of oxygen released is C(°°) = ~ AC m m Hg 0 so that the percentage saturation at any given oxygen concentration (Po~) is
I C( °)- C(t) 1
x 100%
C(oo)
{b)
ta) 80
P02! \
(a)
\
i i =
'\X
\
\\ \
\ \
/
(bl
PC
\
\
60
/
/
/ -80
-60
\
,/ /' 40 ,\
-o
\
/
j /'
_
/
/ -20
20
\\
40
\
0
Time
Time
B
C
A
Figure 1. The retarding effect of erythrocytes on the linear consumption of oxygen by yeast cells: (a) yeast alone, (b) yeast + blood.
i
I
I Time
I
i
(rain)
A
C
B
Figure 2. Recorder traces obtained during an experiment: (a) yeast alone, (b) yeast + blood.
BIOCHEMICAL EDUCATION
April 1979 Vol. 7 No. 2
35
Table 1. Stages in the manual calculation of a oxyhaemoglobin dissociation curve
Po2
70.50 60.00 52.50 45.00 40. S0 36.00 31.50 27.00 22.50 18.00 13.50 9.00 4.50 0.00
t
At
APoz
R " At
AC
Ct = ~,/XC
C~ - Ct
% saturation
2.92 3.65 4.22 4.94 5.45 6.07 6.78 7.66 8.73 9.76 10.90 11.80 12.50 12.90
0.73 0.57 0.72 0. S 1 0.62 0.71 0.88 1.07 1.03 1.14 0.90 0.70 0.40
10.50 7.80 7.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50
13.27 10.36 13.09 9.27 11.27 12.91 16.00 19.45 18.73 20.73 16.36 12.73 7.27
2.77 2.86 S.59 4.77 6.77 8.41 11.50 14.95 14.23 16.23 11.86 8.23 2.77
2.77 S.64 11.23 16.00 22.77 31.18 42.68 57.64 71.86 88.09 99.95 108.18 110.95
108.18 105.32 99.73 94.95 88.18 79.77 68.27 53.32 39.09 22.86 11.00 2.77 0.00
97.50 94.92 89.88 85.58 79.48 71.90 61.53 48.05 35.23 20.01 9.91 2.50 0.00
Table 1 shows the steps of the calculation for the trace of Fig. 2b together with the values for plotting the dissociation curve. We find that about twelve points is a suitable number for a plot. but the accuracy of the results is not affected by using more or less. The calculations can be performed in a short time with a hand calculator. If the calculations are done frequently, the method can be programmed for any computer with sufficient storage space, fifteen data points requires about 90 storage registers. We have written a version in FORTRAN (obtainable from G.L.A on request) which also has a subroutine for plotting the results on a teletype or line printer (see Fig. 3).
lOOT 0
Calibration of the cell We adjusted the volume to about 4.0 ml. and did not vary this afterwards. The cell was calibrated using buffer from a flask open to the air and kept in the water bath used to control the cell temperature. The flask was swirled from time to time to keep the buffer oxygenated. I. With this buffer in the cell, the recorder was set to 100% by using the potentiometer control on the oxygen electrode circuit. 2. The cell was then calibrated for zero oxygen concentration by adding a pinch of sodium dithionite (reducing agent) to the buffer in the cell. The recorder zero was adjusted when the pen came to a halt. 3. The cell was then washed out with water × 3 and buffer × 2 using a 20ml syringe. A piece of plastic tubing was attached in order to suck out the wash fluid. This washing routine was repeated between each experiment.
0 0
Experimental procedure
0 ©
sot
%l!
0fo o
0
rnm H 9
35
70
Figure 3. Tracing of an oxyhaemoglobin dissociation curve plotted directly by a line printer.
1. Buffer (5 ml) was pipetted into a small container. 2. Using a plastic tipped automatic pipette. 10()~1 yeast suspension was added to and mixed with the buffer. 3. The recorder (set to 6 0 0 m m / h r , or some other speed in the light of experience) was switched on so that the chart paper was moving. 4. The buffer/yeast mixture was quickly placed in the cell and the cell cap inserted allowing excess fluid to be displaced through the injection port. A tissue held over the cap absorbed the excess. When human blood was used, a polythene glove was worn for protection. 5. The oxygen consumption rate of the yeast was recorded. 6. If the trace was suitable, the procedure was repeated using the same volume of yeast. This time an equal volume of blood (100~1) was added quickly by automatic pipette. The mixture was placed in the cell and a trace recorded as before.
Determination of the P50 value Methods l. Blood This may be fresh or up to two days old. We have used human and chicken blood, but rat blood would do because each experiment requires no more than 100~l. The blood is heparinised. Before pipetting, it is oxygenated by blowing air on its surface in a small vessel with swirling for a few seconds. 2. B u f f e r s All the buffers used, pH 7.0, 7.25. 7.45, 7.65 and 7.80. were 20mM phosphate in isotonic NaCl (0.9% w/v). 3. Yeast Dried bakers yeast (10% w/v in 0.9% NaC1) was prepared at least 30 min beforehand. 4. A p p a r a t u s We used thermostatted oxygen electrodes (Rank Brothers, Cambridge) coupled to linear recorders (Servoscribe).
For class work we calibrated the oxygen electrode with air. Assuming the atmospheric pressure to be 760mm Hg, that air contains 20% oxygen, and given that the water vapour pressure is 31.8 mm at 30°C, 47.1 mm at 37°C and 55.3 mm at 40°C. Then, for 37°C 2O Po~= ~ x ( 7 6 0 - 47)= 143 mm Hg i.e. at full saturation Po~ is approximately 150 mm Hg. There were 100 divisions across the chart paper, therefore each division corresponded approximately to 1 . 5 m m H g . P50 was then calculated by counting the number of divisions from the baseline to point E (Fig. 2b) and multiplying by this factor. If SI units are preferred the conversion factor is I mm Hg = 0.133 kPa.
36
BIOCHEMICAL EDUCATION
Table 2. Results of a class experiment on one batch of fresh human blood. PS0values are in mm Hg. Temp. °C 30 ° 37 °
pH 7.0
33.7 36.0
7.25
24.7 25.5 25.5
7.45
15.0 15.7 15.25 17.2 16.5
21.0 19.5 21.0 19.S 22.5
7.65
19.5
7.80
18.0 17.0
Table 3. Measurements of P50 (ram Hg) on chicken blood at pH 7.45. Temp. °C
PS0
35
49.5 51.8
40
54.0 54.0
42 °
saponin-treated: 40
30.0 30.0
31.5
30.0 30.0
saponin-treated: 7.45
April 1979 Vol. 7 No. 2
12.7 9.7
Secondly. the experiment is simple and quick to perform, and the P50 value can be read straight from the charl using pencil and ruler. Thirdly, if the full dissociation curve is required it can be calculated manually in a few minutes using a hand calculator. We have used the technique to demonstrate the effects of temperature, pH and plasmolysis on the oxyhaemoglobin dissociation curve. Other teaching uses might include a comparative study of different animals' blood, and the behaviour of red cell populations of different ages.
Results and Discussion PS0 values measured on human blood and chicken blood are presented in Tables 2 and 3. The advantages of this method are many. First, none of lhe solution volumes needs to be measured accurately, The amounts of yeast and blood used are governed only by practical considerations, such as the time required for a complete experiment and the necessity of obtaining a large enough displacement from the linear portion of the trace in order to allow accurate measurement.
tNeville, J. R. and Sasner, J. J. (1964) A new method for the measurement and expression of oxyhaemoglobin dissociation curves. S.A.M.-TR-64-75. 2Scott, G. E. (1975) A rapid method for obtaining the half saturation tension of small blood samples using a standard Clark electrode system. Brit. J. Haematol. 30, 39-45.
Biological Sciences. A Subject Index of AudioVisual Materials
The Donor-Acceptor Approach to Molecular Interactions
E d i t o r , D. M a s l i n . I n s t i t u t e of Biology, 41 Q u e e n ' s G a t e , L o n d o n S W 7 S H U , E n g l a n d . £6.00 or $12.00.
By Viktov G u t m a n n , Pp 279. P l e n u m Press, Ne~ Y o r k a n d L o n d o n , 1978. £17.32 or $27.50.
The Index includes all audio-visual materials relevant to lhc biological sciences thai are available in the U.K. The Index is comprehensive rather than selective. The lille of each item is entered under the one or two n:osl appropriate subject headings. Out" readers will be interested in Biochemistry, Cell Biology and/or Molecular Biology, Genetics. Cancer, hnmunolog 3. The entries are grouped according to the nature ot the medium, e.g. sound films, videotapes, silent fihns. fihn loops, fihn strips, projectm slides, audiotapes, biosets, overhead projector transparencies. Under each entry the supplier's code is given and the details concerning the supplier can be found at the back of the index. At a rough count there seem to be aboul 500 suppliers listed. In order to use the index one naturall3 has to work hard and a good deal ol "research" is required. Thus under biochemistry one finds the Open Universi b films entered twice since there are lxso suppliers but there are some intriguing entries and by wriling to the supplier for a catalogue I am sure one could unearth some unexpected treasures. There are for example sets of overhead projector transparencies on metabolism and a long lisl of selflearning material. The entries for cell biology are even more extensive. Anyone who is interested in the application of audio-visual aids will find this a most useful compendimn. 1 do nol believe that its use is merely confined to the U.K. for man 5 of the entries originalc from other countries so that it is probably a useful enlr6e world wide. P, N, Camphell
In this book Viktor Gutmann draws together the threads of many of his lectures and papers of the last decade or so and presents his Donor-Acceptor approach as a simple means ot explaining the behaviour and structure ot molecules and ions in both the crystalline and the solution stale. The firsl inlroductory chapter presents the reader ,aith the simple basic rules of charge distribution and associated bond lengtl; varialion. Nol surprisingly the second chapter introduces the concepts ol Donor and Aceeptm numbers as applied to solvents one ol the author's main areas ol research interesi. Using the foundations provided by: these chapters ~e are led through a ~idc area o| chenristry: bond lengths in cr)stals, inleriacial i~henomcmt molecular association in the liquid slate, ion solvation, redox properties, ionization equilibria, complex stabilities, kinetics ~,i subslilulion reaclions, and calM>sis. Finally, in a shor~ chaptel of! biochemical considerations, the author outlines the applical~ilit 3 <,1 his approach to the oxidation ot haemoglobin, carbonic anh},drase, corrinoids and the activalion of phosphate transfer. While lifts book is not written with biochemisls in mind those who wish lo explore a philosophy of chemical thinking and its resultant lisl of explanations would be advised lo read it. A simple qualitative approach which uses no quanlum mechanical methods bill which at the same time is not contradk:torx to these oilers an ahernalive x~a3 ot explaining chemical heha~ tour
REFERENCES
J B (Ell l)eparlnlenl ot Inorganic and Slruclnral Chemist D Uni~ersit3 oI l,eeds, U.K.
BIOCHEMICAL EDUCATION
April 1979 Vol. 7 No. 2
A SIMPLE METHOD FOR MEASURING OXYHAEMOGLOBIN DISSOCIATION CURVES IN A STUDENT PRACTICAL CLASS
For some time we have been using a simple and rapid method for measuring oxyhaemoglobin dissociation curves in our student practical class. The method is based upon that published by Neville and Sasner (1964) which, unfortunately, is not readily available. Scott (1975) improved the method by using an oxygen electrode but does not explain clearly how to calculate the oxyhaemoglobin dissociation curve. We are using this opportunity to demonstrate our experiences with the method in class use, and to re-present the theory.
Principle of the method Yeast cells in an oxygenated buffer solution will consume and remove oxygen from the medium at a constant rate (Fig. la). When erythrocytes are present, the dissolved oxygen will be removed at a constant rate until the Po~ falls to the value at which oxyhaemoglobin begins to dissociate. After this time, part of the oxygen consumed by the yeast will come from oxyhaemoglobin so that the rate of decrease of oxygen concentration is retarded (Fig. lb). Consider Fig. lb. Extrapolation of the linear part of the trace (shown by line DB) to zero oxygen concentration gives a curve essentially the same as that obtained in the absence of blood. The extra time required to consume the oxygen released by the oxyhaemoglobin is thus the time represented by the line between points A and B. Point D on the trace, where the linear part merges with the sigmoidal part, is the m i n i m u m Po~ required for 100% saturation of the haemoglobin. The time represented by AB is the time required by the yeast to use the total oxygen present. T h u s point C, midway between A and B, corresponds to the time for half the combined oxygen to be consumed. The line drawn parallel to BD through C cuts the trace at E. This point gives the Po~ at which the haemoglobin is 50% saturated, i.e. the P50 value• This parameter is obtained directly, and therefore quickly, from the recorder trace• It may be sufficient for many purposes but, with a little more calculation the oxyhaemoglobin dissociation curve itself may be obtained.
GORDON L. ATKINS & JAMES DOYLE Department of Biochemistry University of Edinburgh Medical School Teviot Place Edinburgh, EH8 9AG, Scotland
Theory for the calculation of the dissociation curve The yeast consumes oxygen at a constant rate (R). Fig. 2a shows an experimental trace of o x y g e n concentration (Po, in m m Hg) versus time when only yeast was present. Fig. 2b shows the result of an experiment when blood was also present. (The final sigmoidal part of the curve is extrapolated to point A to correct for the small non-linear region of oxygen consumption by yeast at concentrations below about 2 m m Hg.) At Po2 above about 70 m m Hg the oxyhaemoglobin remains undissociated and the oxygen is derived solely from solution. Hence the trace is linear and can be used to calculate the constant rate of oxygen consumption by the yeast (R) in m m H g / m i n . R could be expressed in ml Oz/min or in/amole/min by multiplying by a suitable constant, but in calculating the final variable (percentage saturation of haemoglobin) the constant would be cancelled out. Below point D oxygen is obtained both from that in solution and that released by oxyhaemoglobin. Consider this section. During a time interval At: amount of oxygen consumed by the yeast = R At mm Hg amount of oxygen 9rovided by the solution = APo~ m m Hg If the amount released by the haemoglobin is AC m m Hg, then R.At= ~° o +AC or, A C = R . A t 2 A P o ~ m m H g R has been calculated, and the values of At and APo2 can be read from the trace. Thus AC can be calculated for many values of Po~. The amount of oxygen released up to time t is t
C(t) = ~, AC 0
and the total amount of oxygen released is C(°°) = ~ AC m m Hg 0 so that the percentage saturation at any given oxygen concentration (Po~) is
I C( °)- C(t) 1
x 100%
C(oo)
{b)
ta) 80
P02! \
(a)
\
i i =
'\X
\
\\ \
\ \
/
(bl
PC
\
\
60
/
/
/ -80
-60
\
,/ /' 40 ,\
-o
\
/
j /'
_
/
/ -20
20
\\
40
\
0
Time
Time
B
C
A
Figure 1. The retarding effect of erythrocytes on the linear consumption of oxygen by yeast cells: (a) yeast alone, (b) yeast + blood.
i
I
I Time
I
i
(rain)
A
C
B
Figure 2. Recorder traces obtained during an experiment: (a) yeast alone, (b) yeast + blood.
BIOCHEMICAL EDUCATION
April 1979 Vol. 7 No. 2
35
Table 1. Stages in the manual calculation of a oxyhaemoglobin dissociation curve
Po2
70.50 60.00 52.50 45.00 40. S0 36.00 31.50 27.00 22.50 18.00 13.50 9.00 4.50 0.00
t
At
APoz
R " At
AC
Ct = ~,/XC
C~ - Ct
% saturation
2.92 3.65 4.22 4.94 5.45 6.07 6.78 7.66 8.73 9.76 10.90 11.80 12.50 12.90
0.73 0.57 0.72 0. S 1 0.62 0.71 0.88 1.07 1.03 1.14 0.90 0.70 0.40
10.50 7.80 7.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50
13.27 10.36 13.09 9.27 11.27 12.91 16.00 19.45 18.73 20.73 16.36 12.73 7.27
2.77 2.86 S.59 4.77 6.77 8.41 11.50 14.95 14.23 16.23 11.86 8.23 2.77
2.77 S.64 11.23 16.00 22.77 31.18 42.68 57.64 71.86 88.09 99.95 108.18 110.95
108.18 105.32 99.73 94.95 88.18 79.77 68.27 53.32 39.09 22.86 11.00 2.77 0.00
97.50 94.92 89.88 85.58 79.48 71.90 61.53 48.05 35.23 20.01 9.91 2.50 0.00
Table 1 shows the steps of the calculation for the trace of Fig. 2b together with the values for plotting the dissociation curve. We find that about twelve points is a suitable number for a plot. but the accuracy of the results is not affected by using more or less. The calculations can be performed in a short time with a hand calculator. If the calculations are done frequently, the method can be programmed for any computer with sufficient storage space, fifteen data points requires about 90 storage registers. We have written a version in FORTRAN (obtainable from G.L.A on request) which also has a subroutine for plotting the results on a teletype or line printer (see Fig. 3).
lOOT 0
Calibration of the cell We adjusted the volume to about 4.0 ml. and did not vary this afterwards. The cell was calibrated using buffer from a flask open to the air and kept in the water bath used to control the cell temperature. The flask was swirled from time to time to keep the buffer oxygenated. I. With this buffer in the cell, the recorder was set to 100% by using the potentiometer control on the oxygen electrode circuit. 2. The cell was then calibrated for zero oxygen concentration by adding a pinch of sodium dithionite (reducing agent) to the buffer in the cell. The recorder zero was adjusted when the pen came to a halt. 3. The cell was then washed out with water × 3 and buffer × 2 using a 20ml syringe. A piece of plastic tubing was attached in order to suck out the wash fluid. This washing routine was repeated between each experiment.
0 0
Experimental procedure
0 ©
sot
%l!
0fo o
0
rnm H 9
35
70
Figure 3. Tracing of an oxyhaemoglobin dissociation curve plotted directly by a line printer.
1. Buffer (5 ml) was pipetted into a small container. 2. Using a plastic tipped automatic pipette. 10()~1 yeast suspension was added to and mixed with the buffer. 3. The recorder (set to 6 0 0 m m / h r , or some other speed in the light of experience) was switched on so that the chart paper was moving. 4. The buffer/yeast mixture was quickly placed in the cell and the cell cap inserted allowing excess fluid to be displaced through the injection port. A tissue held over the cap absorbed the excess. When human blood was used, a polythene glove was worn for protection. 5. The oxygen consumption rate of the yeast was recorded. 6. If the trace was suitable, the procedure was repeated using the same volume of yeast. This time an equal volume of blood (100~1) was added quickly by automatic pipette. The mixture was placed in the cell and a trace recorded as before.
Determination of the P50 value Methods l. Blood This may be fresh or up to two days old. We have used human and chicken blood, but rat blood would do because each experiment requires no more than 100~l. The blood is heparinised. Before pipetting, it is oxygenated by blowing air on its surface in a small vessel with swirling for a few seconds. 2. B u f f e r s All the buffers used, pH 7.0, 7.25. 7.45, 7.65 and 7.80. were 20mM phosphate in isotonic NaCl (0.9% w/v). 3. Yeast Dried bakers yeast (10% w/v in 0.9% NaC1) was prepared at least 30 min beforehand. 4. A p p a r a t u s We used thermostatted oxygen electrodes (Rank Brothers, Cambridge) coupled to linear recorders (Servoscribe).
For class work we calibrated the oxygen electrode with air. Assuming the atmospheric pressure to be 760mm Hg, that air contains 20% oxygen, and given that the water vapour pressure is 31.8 mm at 30°C, 47.1 mm at 37°C and 55.3 mm at 40°C. Then, for 37°C 2O Po~= ~ x ( 7 6 0 - 47)= 143 mm Hg i.e. at full saturation Po~ is approximately 150 mm Hg. There were 100 divisions across the chart paper, therefore each division corresponded approximately to 1 . 5 m m H g . P50 was then calculated by counting the number of divisions from the baseline to point E (Fig. 2b) and multiplying by this factor. If SI units are preferred the conversion factor is I mm Hg = 0.133 kPa.
36
BIOCHEMICAL EDUCATION
Table 2. Results of a class experiment on one batch of fresh human blood. PS0values are in mm Hg. Temp. °C 30 ° 37 °
pH 7.0
33.7 36.0
7.25
24.7 25.5 25.5
7.45
15.0 15.7 15.25 17.2 16.5
21.0 19.5 21.0 19.S 22.5
7.65
19.5
7.80
18.0 17.0
Table 3. Measurements of P50 (ram Hg) on chicken blood at pH 7.45. Temp. °C
PS0
35
49.5 51.8
40
54.0 54.0
42 °
saponin-treated: 40
30.0 30.0
31.5
30.0 30.0
saponin-treated: 7.45
April 1979 Vol. 7 No. 2
12.7 9.7
Secondly. the experiment is simple and quick to perform, and the P50 value can be read straight from the charl using pencil and ruler. Thirdly, if the full dissociation curve is required it can be calculated manually in a few minutes using a hand calculator. We have used the technique to demonstrate the effects of temperature, pH and plasmolysis on the oxyhaemoglobin dissociation curve. Other teaching uses might include a comparative study of different animals' blood, and the behaviour of red cell populations of different ages.
Results and Discussion PS0 values measured on human blood and chicken blood are presented in Tables 2 and 3. The advantages of this method are many. First, none of lhe solution volumes needs to be measured accurately, The amounts of yeast and blood used are governed only by practical considerations, such as the time required for a complete experiment and the necessity of obtaining a large enough displacement from the linear portion of the trace in order to allow accurate measurement.
tNeville, J. R. and Sasner, J. J. (1964) A new method for the measurement and expression of oxyhaemoglobin dissociation curves. S.A.M.-TR-64-75. 2Scott, G. E. (1975) A rapid method for obtaining the half saturation tension of small blood samples using a standard Clark electrode system. Brit. J. Haematol. 30, 39-45.
Biological Sciences. A Subject Index of AudioVisual Materials
The Donor-Acceptor Approach to Molecular Interactions
E d i t o r , D. M a s l i n . I n s t i t u t e of Biology, 41 Q u e e n ' s G a t e , L o n d o n S W 7 S H U , E n g l a n d . £6.00 or $12.00.
By Viktov G u t m a n n , Pp 279. P l e n u m Press, Ne~ Y o r k a n d L o n d o n , 1978. £17.32 or $27.50.
The Index includes all audio-visual materials relevant to lhc biological sciences thai are available in the U.K. The Index is comprehensive rather than selective. The lille of each item is entered under the one or two n:osl appropriate subject headings. Out" readers will be interested in Biochemistry, Cell Biology and/or Molecular Biology, Genetics. Cancer, hnmunolog 3. The entries are grouped according to the nature ot the medium, e.g. sound films, videotapes, silent fihns. fihn loops, fihn strips, projectm slides, audiotapes, biosets, overhead projector transparencies. Under each entry the supplier's code is given and the details concerning the supplier can be found at the back of the index. At a rough count there seem to be aboul 500 suppliers listed. In order to use the index one naturall3 has to work hard and a good deal ol "research" is required. Thus under biochemistry one finds the Open Universi b films entered twice since there are lxso suppliers but there are some intriguing entries and by wriling to the supplier for a catalogue I am sure one could unearth some unexpected treasures. There are for example sets of overhead projector transparencies on metabolism and a long lisl of selflearning material. The entries for cell biology are even more extensive. Anyone who is interested in the application of audio-visual aids will find this a most useful compendimn. 1 do nol believe that its use is merely confined to the U.K. for man 5 of the entries originalc from other countries so that it is probably a useful enlr6e world wide. P, N, Camphell
In this book Viktor Gutmann draws together the threads of many of his lectures and papers of the last decade or so and presents his Donor-Acceptor approach as a simple means ot explaining the behaviour and structure ot molecules and ions in both the crystalline and the solution stale. The firsl inlroductory chapter presents the reader ,aith the simple basic rules of charge distribution and associated bond lengtl; varialion. Nol surprisingly the second chapter introduces the concepts ol Donor and Aceeptm numbers as applied to solvents one ol the author's main areas ol research interesi. Using the foundations provided by: these chapters ~e are led through a ~idc area o| chenristry: bond lengths in cr)stals, inleriacial i~henomcmt molecular association in the liquid slate, ion solvation, redox properties, ionization equilibria, complex stabilities, kinetics ~,i subslilulion reaclions, and calM>sis. Finally, in a shor~ chaptel of! biochemical considerations, the author outlines the applical~ilit 3 <,1 his approach to the oxidation ot haemoglobin, carbonic anh},drase, corrinoids and the activalion of phosphate transfer. While lifts book is not written with biochemisls in mind those who wish lo explore a philosophy of chemical thinking and its resultant lisl of explanations would be advised lo read it. A simple qualitative approach which uses no quanlum mechanical methods bill which at the same time is not contradk:torx to these oilers an ahernalive x~a3 ot explaining chemical heha~ tour
REFERENCES
J B (Ell l)eparlnlenl ot Inorganic and Slruclnral Chemist D Uni~ersit3 oI l,eeds, U.K.