Miss

In 1950 she travelled to North America and studied at the School of Social Service Administration of the University of Chicago, graduating with the degree of Master of Arts in Social Work. At this time, no postgraduate training in social work was available in Australia. Overseas travel was the only choice for those who sought further study. Few were able to do this and Miss McLelland joined the small group who fanned the basis of Australia's capacity to develop its own educational programme.

in all solutions, which is on the one hand the optimal H + -concentration for the activity of the enzyme and on the other hand the H + -concentration at which there is the lowest variation of enzyme activity as a result of a small random deviation from this concentration, since in the region of the optimal H + -concentration the dependence of the enzyme activity on the H + -concentration is extremely small.
At least as important in the work of Henri is the lack of consideration of the fact that on inversion of the sugar, glucose is formed initially in its birotational form and is only slowly converted to its normal rotational form. 11 ) Monitoring the progress of the inversion reaction by direct continuous observation of the polarization angle therefore leads to a falsification of the true rate of inversion, since this is superimposed on the change in polarization of the freshly formed glucose. This could be allowed for by including the rate of glucose equilibration in the calculations. However, this is not realistic, since highly complex functions are generated which can be easily avoided experimentally. A better approach is to take samples of the inversion reaction mixture at known time intervals, to stop the invertase reaction and to wait until the normal rotation of glucose is reached before measuring the polarization angle. Sörensen used sublimate (HgCl 2 ) while we used soda, which inactivates the invertase and removes the mutarotation of the sugar within a few minutes. 12 ) Incidentally, it should be noted that Hudson 13 ) already adopted the approach of removing mutarotation experimentally using alkali, but came to a quite different conclusion to ours concerning the course of the invertase reaction. Thus, he is of the opinion that after removing the problem of mutarotation, inversion by invertase follows a simple logarithmic function similar to that of inversion by acid, but this result is contrary to all earlier investigations and according to our own work is not even correct to a first approximation. Even if Henri's experiments need to be improved, their faults are not as grave as Hudson believes. (Sörensen also noticed that Hudson´s conclusions were incorrect). On the contrary, we are of the opinion that the basic considerations that started with Henri are indeed rational, and we will now attempt to use improved techniques to demonstrate this. It will become apparent that the basic tenets of Henri are, at least in principle, quite correct, and that the observations are now in better accord with them than are Henri's own experiments.
Henri has already shown that the cleavage products of sugar inversion, glucose and fructose, have an inhibitory effect on invertase action. Initially, we will not attempt to allow for this effect, but will choose experimental conditions which avoid this effect. Since the effect is not large, this is, in principle, simple. At varying starting concentrations of sucrose, we only need to follow the inversion reaction in a time range where the influence of the cleavage products is not noticeable. Thus, we will initially only measure the starting velocity of inversion at varying sucrose concentrations. The influence of the cleavage products can then be easily observed in separate experiments.

The initial reaction velocity of inversion at varying sucrose concentrations
The influence of the sucrose concentration on enzymatic inversion was examined by all authors already cited and led to the following general conclusions. At certain intermediate sucrose concentrations the rate is hardly dependent on the starting amount of sugar. The rate is constant at constant enzyme concentration but is reduced at lower and also at higher sugar concentration 14 ). Our own experiments were performed in the following manner. A varying quantity of a sucrose stock solution was mixed with 20 ccm of a mixture of equal parts of 1/5 M acetic acid, 1/5 M sodium acetate, a certain quantity of enzyme, and water to give a volume of 150 ccm. All solutions were prewarmed in a water bath at 25 ± <0.05° and held at this temperature during the reaction. The first sample was taken as soon as possible after mixing the solution, followed by further samples at appropriate intervals. Every sample of 25 ccm was transferred to a vessel containing 3 ccm of ½ M Soda to immediately stop the enzyme activity. The solution was examined polarimetrically after approximately ½ hour. The initial polarization angle was extrapolated from the first actual measurements. This extrapolation is certainly valid, since it was only over a few hundredths of a degree. Regular checks that the mutarotation was complete were made by repeated measurements ½ hour later. Every measurement recorded in the protocol is the average of 6 individual measurements, which only differed by a few hundredths of a degree. If we now plot the rotation as a function of time for a single experiment, we see that at the beginning of the process the rotation decreases linearly with time over a fairly long stretch. We define the initial velocity of the inversion as the decrease of rotation per unit time in the phase that can be regarded as linear. The experiments led to the following results: In Tables I through IV we give the rotation angle relative to the real zero  point of the polarimeter, corrected for the (very small) rotation of the enzyme  solution.  Table I Table III.
Amount of enzyme about half as large as in Fig. 1.  To analyze these experiments, we assume with Henri that invertase forms a complex with sucrose that is very labile and decays to free enzyme, glucose and fructose. We will test whether such an assumption is valid on the basis of our experiments. If this assumption is correct, the rate of inversion must be proportional to the prevailing concentration of the sucrose-enzyme complex. 18 ) If 1 mole of enzyme and 1 mole of sucrose form I mole of sugar-enzyme complex, the law of mass action requires that where [S] is the concentration of free sucrose, or since only a vanishingly small fraction of it is bound by enzyme, the total concentration of sucrose; Φ is the total molar enzyme concentration, φ is the concentration of the complexed enzyme, [Φ-ϕ] is the concentration of free enzyme, and k is the dissociation constant.  Table IV. Enzyme amount approximately the same as in the experiment of Fig. 1. 18 The authors use the word "Verbindung", which is normally used these days for compound. English texts of the period use the expression "molecular compound" for the invertase:sucrose complex (A.J. Brown, J. Chem. Soc. Vol. 81, pp. 373-388, 1902). This quantity must be proportional to the starting velocity, v, of the inversion reaction, therefore This function is formally the same as the association curve 21 ) of an acid 22 ) ρ = [H + ] [H + ]+ k and in order to achieve a better graphical representation we will plot the logarithm of the independent variable on the abscissa. We can therefore plot V as a function of log[S] and should obtain the well known association curve. At this point, we do not know the true scale of the ordinate. We only know that the maximal value V =1 should be reached asymptotically and that the foot of the ordinate of value ½ should give the value of k. In order to find the scale, we use the following graphical procedure.
Let us assume that we have a number of points from the experiment that we assume should give an association curve. Since the scale of the points on the ordinate is arbitrary, we have to assume that it will be different from that of the abscissa. Setting s = log[S], the function that we wish to display graphically is V = 10 s 10 s + k or, if we substitute 10 = e p , where p (= 2.303) is the modulus of the decadic logarithm system, Differentiating, we obtain dV ds = p ⋅ k ⋅e ps (e ps + k) 2 This differential quotient defines the tangent of the slope of the specified part of the curve. The association curve has a region whose slope is especially easy to determine, since it is practically linear over an extended stretch. This is the middle of the curve, in particular around the region where the ordinate has a value of ½. We know (cf. the work just referenced) that this ordinate corresponds to the point log k on the abscissa. If we now substitute the value ½ for V and log(k) for s, i.e. k for e ps , in the differential equation, we obtain This means that the middle, almost linear part of the curve has a slope relative to the abscissa whose tangent is 0.576 (i.e. a slope of almost exactly 30°). This obviously only applies if the ordinate and the abscissa have the same scales. We now join the experimental points of the middle part of the curve by a straight line and find that the tangent of its slope has the value ν. 23 ) From this we can conclude that that the units of the abscissa are related to those of the ordinate in the ratio of 0.576:ν, i.e. that the units of the ordinate are the ν/0.576 of those of the abscissa. We can now calculate the proper scale of the ordinate. (cf. Fig. 1a, 2a, 3a, 4a; "rational scale"). We now determine the position of the point 0.5 on this new scale. The ordinate of the curve, which corresponds to this point, gives the value of log k at its foot on the abscissa. We now know the value of k and can construct the whole association curve point for point. We will do this to test whether all the observed points fit well to this curve, and in particular that the value of 1 is not exceeded. Doing this for our experiments, we determine a value for ν for each curve; we then construct the curve according to this and find, with one exception to be discussed, a good agreement of the observed and calculated points.
A second method to determine the scale of the ordinate is the following. If several points at the right hand end of the curve are well determined, and if it is clear that the maximal value has been reached, we can rescale the ordinate to make this value equal to 1. Then we again construct the sloping middle part of the curve by joining the points with a straight line and determine which point corresponds to the ordinate 0.5 on the new scale. We now have all data to construct the curve.
The first method will be chosen if the middle part of the curve is well determined, the second if the points at the right hand end of the curve are determined more reliably. If possible, both methods are used to confirm the agreement of the values obtained; in case of slight disagreements, the average value is taken. Using a combination of these methods we were able to obtain all of the curves shown. In all 4 cases (curve 1a, 2a, 3a, 4a), a family of dissociation curves was constructed for all possible combinations of likely scales for the ordinate and the best fitting curve was selected by shifting to the right or the left until the observed experimental points gave the best fit. It is indeed possible to find curves in all cases that fit within the limits of the allowed tolerances, even though the 4 experimental series were performed with quite different amounts of enzyme.
The dissociation constant for the invertase-sucrose complex found in the individual experiments were: 24 ) in good agreement, although experiments were carried out with different amounts of enzyme. We have here, for the first time, a picture of the magnitude of the affinity of an enzyme for its substrate and we measure the size of a "specific" affinity according to the van´t Hoff definition of chemical affinity. The meaning of this affinity constant is the following. If we could prepare the enzyme-sucrose complex in a pure form and were to dissolve it in water at a concentration such that the undissociated fraction was present at a concentration of 1 mol in 1 liter, there would be √0.0167 mol or 0.133 mol of free enzyme and the same amount of free sucrose in the solution.
The accuracy with which k can be determined is different in the 4 different experiments (Fig. 1a, 2a, 3a, 4a). To an inexperienced observer, the unavoidable arbitrariness in plotting the observed points will appear questionable. But in fact this has little influence. For example, the worst of our curves is arguably Fig. 3a.
Here we find log k = 1.8. Perhaps we could draw an acceptable curve for log k = -1.7 or -1.9. But assuming log k = -2.0 would not be compatible with the shape of a dissociation curve, and the same applies for log k = -1.5. 25 ) Thus, the variance of the true value of k is not large, even for a curve as poor as in Fig. 3a, as long as we have shown in a number of better experiments that the curve can be regarded as an "association curve".
The agreement of the theoretical curve with the observed points is satisfactory from the lowest useable sucrose concentrations up to ca. 0.4 M (corresponding to a logarithmic value of ca. -0.4). However, at higher concentrations there is a deviation such that the rate becomes slower rather than remaining constant. 26 ) However, we are not concerned with this deviation, since in this situation we are not confronted with the pure properties of a dilute solution. It is to be expected that the developed quantitative relationships are only valid over a limited range. The reasons for the failure of the law at high sugar concentrations can be attributed to factors whose influence we cannot express quantitatively. The most important influence can be summarized as "change of the nature of the solvent". We cannot regard a 1 molar solution of sucrose, containing 34% sugar, simply as an aqueous solution, since the sugar itself changes the character of the solvent. This could lead to a change in the affinity constant between enzyme and sugar as well as the rate constant for the decay of the complex. As an example of the manner in which an affinity constant can change when the nature of the solvent changes on addition of an organic solvent, we can consider the investigation of Löwenherz 27 ) on the change in the dissociation constant of water on addition of alcohol. There is no change in the affinity up to 7% alcohol, but there is a progressive decrease as the concentration is increased further. 25 Theoretical dissociation curves can obviously be generated with log k = -2.0 or -1.5 ; they mean the points are not well explained assuming these values of k. 26 The quantities of enzyme in the experimental series I, II, III, IV are calculated from the initial velocities to be almost exactly 1:2:0.5:1. 27 R. Löwenherz, Zeitschr. f. physikal. Chem. 20, 283 (1896) Biochemische Zeitschrift Band 42.

The influence of the cleavage products and other substances.
The cited authors, especially Henri, have already shown that the cleavage products glucose and fructose have an influence on the hydrolysis of sucrose. Henri found that the influence of fructose is greater than that of glucose. We now have the task of determining this influence in a quantitative manner. Like Henri, we assume that invertase has affinity not only for sucrose, but also for fructose and glucose, and we attempt to determine the values of the affinity constants. We did this in the following manner: As before, the initial rate of hydrolysis of sucrose at a certain enzyme concentration is determined. In a second experiment, a known concentration of fructose or glucose is added and the initial rate of hydrolysis of sucrose is determined and compared. It is found that this is reduced. We can conclude from this that the concentration of the sucrose-enzyme complex is reduced in the second case, under the assumption that the initial rate is always an indicator of the complex. If v 0 and v are the initial velocities and φ 0 and φ the corresponding sucrose-enzyme complex concentrations, then ν 0 : ν = ϕ 0 : ϕ If the concentration of enzyme, Ф, partitions between the sucrose concentration S and the fructose concentration F, and if φ is the concentration of the sucrose-enzyme complex and ψ that of the fructose-enzyme complex, it follows from the law of mass action that where k and k 1 are the respective affinity constants. From these 2 equations, elimination of ψ leads to can be determined as follows: In a parallel experiment without fructose, the initial rate is v 0 and the concentration of the sucrose-enzyme complex is φ 0 ; in the main experiment, these two are equal to v and φ, respectively; therefore ν : ν 0 = ϕ : ϕ 0 and ϕ = ν ν 0 ⋅ϕ 0 In the fructose-free experiment, according to equation (2) on p. 11 and finally by substitution in (1)   Fig 5. Graphical representation of the experiment in Table 5.
Influence of glucose and fructose.    The protocol given describes the design of the experiment. As seen, the progress of cleavage is compared at optimal acidity and identical temperature in mixtures that are identical in terms of sucrose and enzyme but which differ in their content of fructose or glucose or in the absence of these substances. The nature of such experiments leads to certain limitations.
The total concentration of sugars should not be so high that the character of the solvent is changed. In general, it is not advisable to use total concentrations of more than 0.3 M. This necessitates the use of relatively low concentrations of sucrose. This means that the rate of conversion does not stay constant for long periods, so that the progress curve deviates from linearity after small changes in optical rotation, which leads to difficulties in estimating the initial rate unless graphical extrapolation procedures are used that are not free of arbitrariness. These deviations from linearity are often more pronounced with pure sucrose (e.g. Fig. 8, I) than in experiments with mixed sugars (Fig. 8, II), since the concentration of the inhibitory cleavage products changes relatively more strongly in the pure sucrose experiments than in experiments in which a certain amount of the inhibitory substance is present from the beginning of the experiment. The initial velocities needed for the calculations can only be obtained by graphical extrapolation: the actual curve is constructed by eye from the observed points and a tangent is estimated by eye to give the initial rate. This procedure cannot be regarded as highly accurate, but will suffice to give us a good idea of the size of the value we are interested in. The (geometrical) tangents are shown as dotted lines in Fig. 5. The value of the ratio of the trigonometrical tangents Tan I Tan II is calculated from Fig. 5  Applying the same procedure to experiment (Fig. 7), we obtain   Table 8. Influence of fructose.
From the experiment (Fig. 9) we obtain the following. Note, there is no deviation from a straight line in these experiments.  Fig. 9. Graphical representation of the experiment in Table 9. Influence of glucose and fructose.
For experiment (Fig. 6) we obtain Tang I Tang II = 1.133 so that k glucose k sucrose = 6.7 For experiment (Fig. 8)  The inhibitory influence of other substances was measured in the same manner. Before doing this, as a test for the correctness of the procedure described above, we had to show that foreign substances that were expected to have no affinity to invertase did not inhibit the cleavage of cane sugar as long as their concentration did not change the character of the solvent. We therefore convinced ourselves again that a 0.1 normal concentration of potassium chloride had absolutely no inhibitory effect and that even a normal concentration had no significant effect (Tables 10 and 13).  At a concentration of 0.2 M, ethanol does not show the slightest inhibitory effect (Table 10). In contrast, there is a slight inhibition at normal concentration, which is without doubt due to a change in the character of the solvent and does not 28 There was a discrepancy between the numbers in Table 10 and Fig. 10. In order to reproduce Fig. 10  arise from an affinity of the enzyme to alcohol. If one wished to calculate the effect in terms of an affinity as done previously, graphical estimation of the ratio k alcohol k sucrose would give a value of 36. Such a weak affinity can be equated to 0 within error limits (i.e. k alcohol = ∞), especially when we bear in mind that another inhibitory factor, namely the change in character of the solvent, certainly plays a role. The investigation of other carbohydrates or of poly-alcoholic substances was now of particular interest. Table 11.  Table 11.
Effect of lactose. The behavior of milk sugar was of special interest (Tables 11 and Fig . 11). Its inhibitory influence was so slight, that it was hardly detectable inside the error limits. If we evaluated the very slight signal changes, we would find Experiment 1 Experiment 2 k lactose k sucrose = at least 30 36 Since we cannot say whether the small effects can be used reliably, we have to be satisfied with the statement that an affinity of milk sugar to invertase is not measurable with certainty. This is in agreement with our expectations, since binding of a disaccharide such as lactose to invertase would lead to hydrolysis, as is the case for sucrose, whereas lactose is not cleaved. Mannose.

Mannitol
The inhibitory effect was low. This example was used to determine a weak affinity quantitatively by adequate variation of experimental conditions. Considering the small signals, the agreement is not bad, and the average value of k mannitol k sucrose = 13 should give a reasonable impression of the relative affinities. Glycerin.
We have obtained the experimental series Fig. 15, Table 15 and an individual experiment (Fig. 10) To help understand these values, it should be noted that an increase in the dissociation constant corresponds to a decrease of the affinity of the enzyme to the respective substance. Thus, the affinity of sucrose is by far the largest.  On the basis of these data, we are now able to solve the old problem of the reaction equation of invertase in a real manner without resorting to the use of more than one arbitrary constant. Of all authors, V. Henri was closest to this solution, and we can regard our derivation as an extended modification of Henri's derivation on the basis of the newly gained knowledge.
The basic assumption in this derivation is that the decay rate at any instant is proportional to the concentration of the sucrose-invertase complex and that the concentration of this complex at any instant is determined by the concentration of enzyme, of sucrose and of reaction products that are able to bind to the enzyme. Whereas Henri introduced an "affinity constant for the cleavage products", we operate with the dissociation constant of the sucrose-enzyme complex, k = 1/60, with that of the fructose-enzyme complex, k= 1/17, and with that of the glucoseenzyme complex, k= 1/11. We also use the following designations: Φ = the total enzyme concentration ϕ = the concentration of the enzyme-sucrose complex Ψ 1 = the concentration of the enzyme-fructose complex Ψ 2 = the concentration of the enzyme-glucose complex S = the concentration of sucrose F = the concentration of fructose G = the concentration of glucose } i.e. the concentration of the respective sugar in the free state, which is practically equal to the total concentration.
Since the cleavage yields equal amounts of fructose and glucose, G is always equal to F.
According to the law of mass action, at any instant We can eliminate ψ 1 and ψ 2 by first dividing (2) by (3) to give and further by dividing (1) by (3) to give For abbreviation we substitute 1 k 1 + 1 k 2 = q so that Substituting in (4), this gives We can now proceed to the differential equation. If a is the starting amount of sucrose t is the time x is the amount of fructose or glucose, so that a-x is the remaining amount of sucrose at time t, the decay velocity at time t is defined by v t = dx dt According to our assumptions, this is proportional to φ, so that the differential equation derived using equation (4) is: . where C is the only arbitrary constant, which is proportional to the amount of enzyme. 31 ) The general integral of the equation can be calculated without difficulty: To eliminate the integration constant, we substitute the values of x=0 and t=0 for the start of the process to give 32 ) 0 = −k ⋅(1 + a ⋅ q)⋅ ln a + const and find by subtraction of the last two equations the definite integral  6) or on substituting the value for q: We can now incorporate k into the constant on the right hand side of the equation and obtain (7) Like the Henri function, this is characterized by a superposition of a linear and a logarithmic function of the type where the meaning m and n can be seen by inspection of the previous equation: they are factors whose magnitude is dependent on the respective dissociation constants and starting quantity of the sugar.
Substituting the determined values of k, k 1 and k 2 at 25° we obtain  (10) is strictly followed. The hitherto unknown function of the right hand side of the equation finds its definitive form in our equation (8). Otherwise nothing is changed and it can be easily seen that the constant in equation (8) must be proportional to the enzyme concentration.
While it is not necessary to test the correctness of equation (9) for varying amounts of enzyme, it still has to be tested whether the constant has the same value if the amount of enzyme is kept constant and the amount of sugar is varied, and whether the constant in a single experiment is independent of the time.
For these calculations, we use the data from experimental series I, and must first convert the values for x, for which we have so far used arbitrary polarimetric units, into concentration units. To do this we use the observation that the theoretical rotation of a sucrose solution which originally shows a rotation of m° is -0.313 x m° after complete cleavage of the sugar (cf. Sörensen, l.c., p. 262). The value of the constant is very similar in all experiments and despite small variation shows no tendency for systematic deviation neither with time nor with sugar concentration, so that we can conclude that we can conclude that the value is reliably constant.