pletely covered the inside of the tube for the greater portion of its length. Fronds were chosen which were sufficiently stiff so that their own elasticity caused them to remain closely and evenly pressed against the inner surface of the glass tube even when liquid was poured in and out or shaken back and fourth in the tube.

The glass tube was sealed off at one end, while at the other it was furnished with a short piece of rubber covered with paraffin.® The covering of paraffin was continuous and care was taken to renew it each time the tube was used.

After placing the frond in the tube, the latter was filled with sea water (at the temperature of the bath) and the rubber tube was clamped shut. In some cases a small bubble of air was left in the tube to act as a stirrer: in other cases the tube was completely filled with sea water and the stirring was effected by a small piece of paraffin or by a glass bead covered with paraffin.

The tube was then placed in a large water bath in direct sunlight. The tube was slanted so as to receive the sunlight nearly at right angles. The light passed through a sufficient amount of water to filter out most of the heat rays. Some light was reflected from the surface of the water but this was practically constant during any one experiment. The temperature of the bath was kept constant within 1° in most of the experiments.

In order to determine the degree of alkalinity produced by photosynthesis two methods were used. In the first the indicator was added to the sea water containing Ulva after a definite exposure to sunlight; in the second the indicator was added to the sea water before the exposure began. In the latter case there was a possibility that the presence of the indicator might affect the amount of photosynthesis, but it was found by control experiments that

this was not the case with the concentrations employed in these experiments.

It was also necessary to ascertain whether the degree of alkalinity produced was a reliable measure of the amount of photosynthesis. This was done by making simultaneous determinations of the degree of alkalinity and the amount of oxygen evolved (by Winkler's method). The results show that the amount of photosynthesis, as indicated by the evolution of oxygen, is approximately a linear function (in this range) of the change in the PH value of the sea water. This being so we can measure the amount of photosynthesis by determining the change in PH value regardless of any possible complications such excretion of alkali by the plant.

Since the plants produce CO, by respiration this must be taken into consideration. Experiments conducted under precisely the same conditions except that light was excluded showed that the respiration was practically constant. It is, therefore, easy to make a correction for it.

In order to ascertain how much photosynthesis had taken place after a definite time the pink color produced by the Ulva was matched against the colors of a series of tubes (of the same size) containing the same concentration of indicator in a series of buffer solutions of known alkalinity." The matching was done under a "Daylight" lamp, which is invaluable for this purpose.

In this way the degree of alkalinity produced may be easily ascertained and since this corresponds to the amount of oxygen evolved it gives us a direct measure of photosynthesis, provided we know the amount of CO, or of O, corresponding to the observed changes in alkalinity. These may be determined in various ways which can not be discussed here.

7 For buffer solutions see: Sörensen, Biochem. Zeit., 21: 131, 1909; Ergeb. d. Physiol., 12: 392, 1912. Höber, R., Physik. Chem. d. Zelle u. d. Gewebe, 4te Aufl., 1914, S. 169. Bayliss, W. M., "Principles of General Physiology," 1915, p. 203.

For the PH values needed in these investigations mixtures of .05 M borax and 0.2 M boric acid (to each liter of boric acid 2.925 gm. NaCl is added) are useful. The following table gives the

In order to study the effects of temperature, light intensity, etc., it is not necessary to know the amount of CO, abstracted; it is sufficient to compare the time required to produce the same change in the color of the indicator under different conditions. This gives much more accurate results than comparison of the amounts of CO, abstracted in equal times. In case anything is added to the solution which changes its buffer value due allowance must be made for this.

It is evident that the method is accurate, simple, rapid and convenient, permitting us to measure minute amounts of photosynthesis at frequent intervals.

It may be added that aquatic plants are greatly to be preferred to land plants for quantitative studies of photosynthesis because in the latter the temperature can not be satisfactorily controlled while with the former the PH values of a series of mixtures (Palitzsch, S., Bioch. Zeit., 70: 333, 1915. Compt. rend. lab. Carlsberg, 11: 199, 1916). Cf. McClendon, J. F., Gault, C. C., Mulholland, S., Pub. 251 Carnegie Inst., 1917, pp. 21-69.

fluctuations can be confined within one degree, or less.

Similar experiments were made with a variety of fresh-water plants, including Spirogyra, Hydrodictyon and Potamogeton. The results were very satisfactory. The usual procedure was as follows: A gallon bottle was filled with the water in which the plants were growing, a little phenolphthalein was added and a solution of sodium bicarbonate was then added, drop by drop, until a pink color was produced. On pouring this into the tubes used in the experiments the pink color was not perceptible since the layer of liquid was not sufficiently thick.

When the algae were placed in these tubes in sunlight a pink color appeared in a short time. If the tubes were placed in the dark the color disappeared as the result of respiration. In many cases the algae lived for several days in these tubes and made considerable growth, showing that they were not injured.

The method is well adapted to class work. For ordinary laboratory demonstrations Pyrex glass is not necessary since any good glass® will answer. It will be found that some algæ (particularly blue-green and unicellular green algae) will operate satisfactorily in diffused daylight. It is important, however, that the plants be in active condition. Aquatics are apt to prove unsatisfactory in fall and winter while in spring and summer the same species may be very active.

SUMMARY

Minute amounts of photosynthesis can be accurately measured by placing aquatic plants in solutions containing bicarbonates, with a little phenolphthalein, and observing changes in the color of the indicator.

The convenience, simplicity and rapidity of the method make it as useful for class-room demonstration as for quantitative investigations. W. J. V. OSTERHOUT, A. R. C. HAAS

LABORATORY OF PLANT PHYSIOLOGY,

HARVARD UNIVERSITY

8 This solution should be freshly made each day. Open bottles, test-tubes, beakers or tumblers may be employed.

FRIDAY, MAY 3, 1918

CONTENTS

A Chemical Study of Enzyme Action: Dr. K. GEORGE FALK

The Conservation of Wheat: DR. HARRY SNYDER

Scientific Events:

Theodore Caldwell Janeway; Medical Terminology; Lectures on Public Health; Research Grants of the American Association for the Advancement of Science; The National Academy of Sciences

Scientific Notes and News

University and Educational News

Discussion and Correspondence:

Note on a Reverse Concentration Cell: PROFESSOR FERNANDO SANFORD. Hering's Contributions to Physiological Optics: DR. CARL HERING. Reform of the World's Calendar: T. G. DABNEY

Scientific Books:

423

429

433

438

A CHEMICAL STUDY OF ENZYME

ACTION1

IN making up the list of papers to be presented at this meeting to-day, it was stated that the intention was to "get at the fundamental things in enzyme activity." Since the chemical nature of an enzyme is as fundamental for the understanding of an enzyme action as any other factor, I shall present some results obtained during the last six years bearing on this question.2 It will not be necessary to give a definition of enzymes here or to present a classification of enzyme actions. This has been done 436 repeatedly and it would appear that at present nothing essential can be added in this respect. The question will be taken up as a chemical problem. Certain definite chemical changes may be accelerated under definite conditions; certain products obtained from living organisms have the property of accelerating these changes; these accelerations can be controlled within limits by altering the conditions. The problem in its simplest terms is the study of the chemical nature of these products of animal or plant origin which accelerate the changes. At the same time, influences physical in nature, such as the solvent and the colloidal properties of the materials must not be lost sight of, as they undoubtedly play a part in modifying the velocities of the reactions.

438

Stejneger and Barbour's Check-list of North American Amphibians: PROFESSOR ALEX

ANDER G. RUTHVEN

440

Since enzymes manifest their actions by increasing the velocities of chemical reactions, a large amount of work has been done in studying the kinetics of such reactions. The actual results obtained from such studies in so far as light has been thrown on the chemical nature of enzymes has been disappointingly meager. In fact the results which might be expected from such studies have been in large measure unsatisfactory. This may be shown by a brief survey of some of the work on the kinetics of invertase action, to which, from this point of view, more attention has been paid than to any other enzyme action. Invertase, as is well known, hydrolyzes cane sugar to form glucose and levulose. O'Sullivan and Tompson3 in 1890, as a result of the study of the kinetics of this reaction, concluded that the reaction was of the first order, the velocity being proportional to the concentration of the cane sugar. Duclaux in 1898, Brown" and also Henri in 1902, found that the velocity of this reaction was not of the first order as shown by the lack of constancy of the velocity coefficients. Henri' suggested in 1905 that because of the colloidal nature of enzymes, the reaction belongs to a two-phase system to which the simple mass law is not applicable in the given manner. Hudson in 1908 as a result of some new work in which the mutarotation of the invert sugar was taken into account, found that the hydrolysis of cane sugar in the presence of invertase gave velocity coefficients that were constant when calculated by the unimolecu

3 O'Sullivan and Tompson, J. Chem. Soc., 57, 834 (1890).

4 Duclaux, Ann. Inst. Pasteur, 12, 96 (1898).

5 Brown, J. Chem. Soc., 81, 375 (1902).

6 Henri, Z. Physik. Chem., 39, 215 (1902).

7 Henri, Z. Physik. Chem., 51, 19 (1905).

8 Hudson, J. Am. Chem. Soc., 30, 1160, 1564 (1908).

lar formula. He therefore claimed to have confirmed the conclusions of O'Sullivan and Tompson. Michaelis and Menton' in 1913 disagreed with Hudson in attempting to express the velocity of the reaction as a simple logarithmic function of the sugar concentration and elaborated the view of Henri of the two-phase system and formation of an intermediate compound. Bayliss 10 in 1911 developed the view of such intermediate compounds as adsorption compounds and concluded that the rate of enzyme action was a function of the amount of adsorption compound in existence at any particular time. Nelson and Griffin" in 1916 developed the two-phase system view of invertase action and in 1917, as a result of an extended series of experiments, Nelson and Vosburgh12 summarized and stated clearly the present status of the problem of the kinetics of invertase action. Their conclusions may be stated briefly as follows:

I. The velocity of inversion is directly proportional to the concentration of the invertase.

II. The velocity is nearly independent of the concentration of the cane sugar in the more concentrated sugar solutions, while in very dilute sugar solutions the velocity increases with increase in concentration of the substrate and finally reaches a maximum.

III. The results obtained agree with the heterogeneous reaction view and contradict the claim that the kinetics of invertase action conform to the unimolecular law for homogeneous reactions.

› Michaelis and Menton, Biochem. Z., 49, 333 (1913).

10 Bayliss, Proc. Roy. Soc. London (B), 84, 90 (1911).

11 Nelson and Griffin, J. Am. Chem. Soc., 38, 1109 (1916).

12 Nelson and Vosburgh, J. Am. Chem. Soc., 39, 790 (1917).

IV. Adsorption is one of the controlling factors in the kinetics of invertase action, and the velocity of inversion curve has the same general shape as adsorption curves, as suggested by Henri.

This brief review will show the uncertainty of the conclusions from the results obtained in the study of the kinetics of one of the most carefully measured of enzyme actions. The factors controlling the velocity of this reaction are just beginning to be cleared up, the simple earlier views being incomplete.

An unsuccessful attempt to formulate the kinetics of enzyme action in a comparatively simple way may be mentioned. The hydrolysis of urea to form ammonia and carbon dioxide was used by D. D. van Slyke1s to develop a general theory of the kinetics of such enzyme actions based upon the assumption of an intermediate compound between enzyme and substrate. Unfortunately, in the development of the equations a further assumption was introduced which limits their validity and applicability to definite conditions which are realized only in special cases.14

The study of the kinetics of enzyme action has not, therefore, led to any results with regard to the chemical nature of enzymes, even in the simplest cases of chemical changes. Practically all enzymes are colloids, and when the substrate also is a colloid, as in the action of a protease on a protein, it is obvious that the conditions are complicated to such an extent that a quantitative study of the kinetics of such a reaction appears to be almost hopeless, although valuable qualitative results may be obtained.

The study of the chemical nature of enzymes is complicated in most cases by

13 D. D. Van Slyke and G. E. Cullen, J. Biol. Chem., 19, 146 (1914).

14 J. Biol. Chem., 28, 389 (1917) .

reason of the complexity of the substances whose changes they accelerate. This difficulty can be obviated for a few of the enzymes. For example, the lipases and esterases accelerate the hydrolysis of fats and esters. While the mechanism of the hydrolysis of an ester to form acid and alcohol in the absence of lipase is not known definitely, still the compositions and properties of the initial and final products undergoing the enzymatic change are known. This eliminates, partly at any rate, one of the unknown factors of the enzyme problem, and is the main reason for studying lipase in connection with the question of the chemical nature of the active catalyst, the enzyme.

Practically all enzymes are colloids or are intimately associated with substances having colloidal properties. Furthermore, in a large number of cases, it seems that the enzyme is associated with protein matter, either as an essential part of the protein molecule, or accompanying it in such a way that separation has not yet been effected. Among the enzymes which chemically show the characteristics of proteins may be mentioned the amylase obtained by Sherman,15 proteases and lipases. On the other hand, the invertase described by Nelson16 is a carbohydrate phosphoric acid complex containing about one per cent. of nitrogen in the form of protein.

These facts make it evident that for the case of lipase, to use a specific example, the isolation of the enzyme in a pure state is a phase or part of the problem of the isolation of a pure protein, since in the separation of the active lipase from inactive material present with it, the resulting bodies approach more and more nearly in proper

15 Sherman and coworkers, J. Am. Chem. Soc., 1912-1917.

16 Nelson and Born, J. Am. Chem. Soc., 36, 393 (1914).