Methods that use FDNB* for the determination of available lysine need a correction for loss. The correction procedure has been studied, especially for vegetable materials.To minimise loss of DNP-L, hydrolysates were filtered hot and residues washed thoroughly. Hydrolysis of dinitrophenylated protein was incomplete after 16 h reflitx in 5 4 M-HCI, the yield of DNP-L being improved by a second hydrolysis of residues. The effect of obtaining higher values after hydrolysis in a large volume of acid (dilute hydrolysis) has been largely eliniinated. Although DNP-L added to dinitrophenylated materials (Carpenter's procedure)' was partly destroyed during acid digestion, DNP-L in protein (indigenous DNP-L) was diminished less. DNP-protein was therefore tried as recovery agent instead of DNP-L.The estimated coeficient of variation was 3.8%. Various materials were assayed by this method and by TLMIT (the Silcock rnethodP. The two values were closer for animaI than for vegetable materials, and for undamaged than for heatdamaged materials. The loss of indigenous DNP-L during an assay (the criterion being comparison with TLMI) was about half that predicted from the recovery of added DNP-L, but about equal to that predicted from recovery from DNP-protein. Less DNP-L was lost during acid digestion in presence of animal than of vegetable protein. DNP-Lt was adsorbed by residues.A modiJed Carpenter method is described in the Appendix.The use of the new, smaller, correction factor is discussed.

calculating the extra rate in this unit at a series of instants during the course of the manometric experiment. These extra rates are then extrapolated to zero time so as to eliminate changes in the activity of the enzyme during the actual experiment owing to (i) progressive alterations in pH, substrate and buffer concentrations as C02 is taken up or evolved, (ii) destruction brought about bytheviolent shaking. The extrapolated rates are finally corrected for the limiting effect of diffusion by means of extensions of the equations previously developed by Roughton, on the basis of the application of the stationary film theory to gas-enzymic reactions. 3. Detailed examples of the computation of enzymic activity are given for CO2 uptake experiments both below and above pH 8-0 and also for C02 output experiments. 4. Applications of the methods to the study of the kinetics of carbonic anhydrase under varied conditions are given in the following paper.

The velocity constant of the reaction C02 + OH--HC03is more than a million times greater than the velocity constant of the reaction C02 + H20 -+ H2C03, the loss of the proton from the H20 molecule apparently causing, as Faurholt has emphasized, an enormous increase in the affinity for C02. In consequence of this, it is only below pH 8 that the rate of (2) becomes negligible in comparison with the rate of (1): in the range pH 9-10 the two rates are of the same order, whilst above pH 10 the rate of (2) becomes predominant.Similar studies on the rate of combination of C02 with very weakly acidic organic hydroxides, such as methyl alcohol, ethyl alcohol and glucose, have shown [Faurholt, 1927] that C02 reacts slowly and in an analogous way with the organic hydroxide molecule: C02 + HOX=HCO3X. ...... (3)The inhibitory effect of the proton is again shown by the fact that the reaction with the organic anion-CO2+OXI=CO3X-...... (4) -is also very much more rapid and complete than the reaction with the molecule.In the case of stronger oxy-acids the affnity of the anion for H+ is correspondingly lower, and Faurholt tacitly assumed that the affinity of the anion for C02 would fall pari passu and hence that acids with pK of the order of 7 (e.g. cacodylic acid) could be safely used as buffers in the measurements of the velocity constants of the reactions C02 + H20#H2C03 and C02 + OH-HC0O3-, without fear of the buffer taking any part other than the "instantaneous" supply or removal of the H+ ions involved in the ionization of carbonic acid, i.e. H20O3=H+ + HC03-. The possibility of any direct reaction of the buffer with C02, H2CO3 or HC03-(except in the case of the borate ion, which Faurholt suggested might form some special complex with C02) was thus implicitly excluded, not only by Faurholt but by all others who have worked in this field.Preliminary experiments on the activity of carbonic anhydrase in phosphate solutions of varying concentration and pH have, however, prompted us to examine more critically the role of the buffer in reactions (1) and (2). To this end we have measured manometrically both the rate of C02 uptake by buffer solutions shaken violently with C02, and the rate of C02 output from bicarbonate buffer mixtures, in the presence of a much wider range of buffer concentration George Henry Lewes Student.

No abstract

AN active inhibitor of carbonic anhydrase found in the sera of pig and other mammals, was briefly described in a recent note [Booth, 1936]. Carbonic anhydrase is normally found only in corpuscles, and its absence from the plasma has distinct advantages in the regulation of the blood pH in vivo [Ro ughton, 1935]. The function of the inhibitor was thought to be a protective one in that it provides a temporary mechanism for ensuring that any carbonic anhydrase which may be set free into the circulating plasma as a result of corpuscle destruction does not annul these advantages. Much of the force of this suggestion was lost on finding that the inhibitor is not present in the blood of man. However, even though the inhibitory activity of serum may be accidental it was considered of interest to study (a) the kinetic relations of the inhibitor to the enzyme, (b) its own chemical nature, and (c) its distribution. METHODSThe dehydration rates of carbonic acid were determined with the apparatus of Meldrum & Roughton [1933 a] in which bicarbonate solution is suddenly mixed with phosphate buffer and the C02 evolution is observed manometrically. The hydration rates (CO2 uptake by buffer) were determined in a similar apparatus, as described by Meldrum & Roughton [1933 b], but with certain modifications. The technique is essentially the same as that used for measuring C02 output rates except that C02 was added to the manometer vessel (or "boat") and the rate of change of pressure due to its uptake by buffer solution observed manometrically. The boat was first evacuated to 0.1 atm.: then 2 ml. C02 at 1 atm. were introduced into the 60 ml. gas space, making the final concentration of C02 25 p.c. at 0-133 atm.1 George Henry Lewes Student.