spect has suggested the substitution of a method that is less laborious and time consuming, and on which we are now working. The fact that the product is not completely soluble shows that denaturation occurred. For this reason we are now endeavoring to perfect a method of extraction which will prevent such a result.

Results of treatment with the vaccine. Eleven cases were treated in 1914, before and during the season for autumnal catarrh. Six cases were treated in advance of the attack. One of these was cured for the season, four had very mild symptoms, and one was not improved. Five cases were treated during the attack. The symptoms of four subsided after one to four injections, whereas one patient received no benefit. Altogether, there were five cures for the season. In four cases there was marked improvement. In two cases there was no improvement. Of the two cases that were not improved, one had a polypoidal degeneration of the middle turbinate with underlying bone necrosis. The patient had distinct asthmatic attacks every night and it was impossible to say whether the attacks were due to his hay fever or his local nasal condition. The other was a physician who reacted to both ragweed and golden-rod pollen. He received in all thirty-three injections, alternating the ragweed extract and the golden-rod extract. He came very irregularly. It is possible that at times the treatment was too intensive. His physical condition was so poor that possibly he could not develop a tolerance.

DISCUSSION. Nine of our cases reacted to ragweed pollen and two reacted to that of both ragweed and golden-rod. Both of these latter cases received both golden-rod and ragweed antigen hypodermically. One was cured but the other was not improved. When a patient is sensitive to more than one pollen, individual doses of each extract should be administered, in order to determine when the tolerance is sufficiently raised for each. Mixing the antigen is too empirical.

There are two ways of determining when a patient has become sufficiently immune to warrant discontinuance of the treatment. 1. With the complement-fixation test.

2. From the size, intensity and duration of the wheal produced by skin scarification, at different times, namely, before and during the treatment.

Clowes (20) was one of the first investigators in this country to

immunize hay fever patients with pollen extracts. He performed complement-fixation tests, before and during the treatment, and showed that an increase in antibodies is produced in a few weeks.

The scarification method is the one we have generally used to diagnose and determine the degree of immunity induced. The wheal produced by the initial vaccination is measured, its time of appearance and its duration noted. After five or six treatments the patient is revaccinated and the wheal is observed again as before, and compared with the former results. When the wheal is very small or does not appear, the patient is sufficiently immune and probably will go through the season with very mild symptoms or none at all.

Naturally the question arises whether such immunization is permanent. We believe it is safe to say that, while immunity may not be successfully carried over to the succeeding year, recurrences are much milder at least and require less re-immunization. An attack the following year can probably be overcome by a few injections.

The best time to begin treatment is probably about ten weeks before the attack may be expected to occur. Regularity of attendance at about weekly intervals is important.

We feel that cures were not accomplished in two cases because treatment was begun too early; and in two other cases, because the patients were treated too irregularly. Furthermore, it is probable that some of these cases were susceptible to pollen other than that from ragweed and golden-rod. At the time of our initial work, we were not prepared with as large a variety of pollens as we now possess for the continuance of this work.

Our heartiest thanks are due to Dr. Wm. J. Gies, for assistance in the conduct of the work done at the College of Physicians and Surgeons; also to Dr. E. P. Bernstein, for many valuable suggestions.

BIBLIOGRAPHY

1. WOLFF-EISNER: Das Heufieber, sein Wesen und seine Behandlung, 1906.

2. DUNBAR: Berl. klin. Woch., 1905, Nos. 26, 28, 30; Zeitschr. f. Immunitätsforsch., 1907, 7; Deutsche med. Woch., 1911, 37, p. 578.

3. RICHÉT and HERICOURT: Compt. rend. de la Soc. biol., 1898.

4. VAUGHAN and WHEELER: Jour. Inf. Dis., 1907, 4.

5. SLEESWIJK: Zeitschr. f. Immunitätsforsch., 1909, 2.

6. FRIEDBERGER and HARTOCH: Ibid., 1909, 3.

7. FRIEDMAN: Ibid., 1909, 2.

8. WOLFF-EISNER: Berl. klin. Woch., 1904, Nos. 42 and 44.

9. CALMETTE: Compt. rend., de l'acad. des sciences, 1907, June.

10. Rosenau and ANDERSON : U. S. Pub. Health and Marine Hospital

Service, Hyg. Lab. Bull., 36, 1907; Bull. 64, 1910.

11. Отто: Мünch. med. Woch., 1907, No. 34.

12. NEUFELD and DOLD: Berl. klin. Woch., 1911, Nos. 2, 24; Arb. aus

d. Kais. Gesundheitsamt, 1911, 38.

13. KRAUS: Zeitschr. f. Immunitätsforsch., 1911, 8.

14. RITZ and SACHS: Berl. klin. Woch., 1911, No. 22.

15. IZAR: Zeitschr. f. Immunitätsforsch., 1911, 10.

16. FRIEDBERGER and MITA: Ibid.

17. ZINSSER: Jour. Exp. Med., 1913, 17.

18. BORDET: Ann. de l'Inst. Past., 1903, 17.

19. ZINSSER and DwYER: Proc. Soc. Exp. Biol. and Med., 1914, 11, p.

74; Jour. Exp. Med., 1914, 20, pp. 387, 582.

20. CLOWES: Soc. for Exp. Biol. and Med., 1913, 10, p. 48.

THE INFLUENCE OF LOW TEMPERATURES UPON ENZYMES

A review

JOSEPH SAMUEL HEPBURN

(University Fellow in Biological Chemistry, Columbia University, 1912-1913)

Introduction. The influence of low temperatures on enzymes is a subject of growing importance to the chemist, the biologist, and the bromatologist. Problems in this field may be studied from either the potential or the kinetic side; for either the resistance of an enzyme to, or its activity at, low temp. may be investigated. Various researches, conducted during the last half century, have demonstrated that enzymes survive exposure to low temp. and also act as catalysts at such temp. The reports of these researches are widely scattered in the literature; and frequently the original papers may be obtained for consultation only with difficulty. It is the purpose of this paper, which is based on primary sources, to give a résumé of our present knowledge of this subject. One section is devoted to the resistance of enzymes to low temp., and one to their activity at such temp. In the first section, the following data for each enzyme are given, so far as they have been recorded in the original literature: source of enzyme; temp., time and mode of exposure. In the second section, the data given for each enzyme, so far as recorded by the various observers, are: source of enzyme, temp. and time of incubation, substratum, degree of progress of reaction, and results of comparative experiments carried out at higher temp.

2. Resistance of enzymes to low temperatures. The researches reviewed below demonstrate that the following enzymes survive exposure to low temp. and again exert their usual catalytic power when brought into a suitable environment:-lipase, protease of plants, pepsin, trypsin, rennin, thrombin, zymase, invertase, maltase, diastase, inulinase, oxidase, peroxidase, catalase, and simple and aldehyde reductase. This order will be followed in presenting the data.

LIPASE. According to Kastle and Loevenhart (1) the lipase of a pig pancreas, which had been held in cold storage at 4° C. for 7 days, retained 40 percent of its power to produce hydrolysis of ethyl butyrate. A 10 percent aqueous extract of pig pancreas was held at 1° C. for 72 hr., and a 10 percent aqueous extract of pig liver was kept on ice for 48 hr. During holding at these temp., both extracts gained in power to hydrolyze butyric ester, a zymogen having become activated.

Pennington and Hepburn (2) demonstrated the presence of active lipase in the crude abdominal fat of chickens of known history, held hard frozen for periods of 122, 13, 16, 28, 29 and 42 months at a temp. of -9.4° to - 12.2° C., and of chickens, whose history prior to freezing was unknown, kept at that temp. for periods of 54 and 89 months. The chickens held for 28 or more months were not marketable and are of scientific interest only. As the period of holding hard frozen grew longer, the activity of the lipase toward esters usually became greater, and the acidity of the crude fat increased. Apparently a zymogen became converted into its active form, thus giving rise to increased activity of the lipase. The increase in activity of the lipase, and in the acidity of the crude fat, occurred less rapidly in hard frozen chickens than in birds held at higher temp., e. g., room temp. Active lipase was also found in the crude abdominal fat of a chicken kept at o° C. for 24 hr. after death. Pennington and Robertson (3) detected lipase in eggs which had been held at a temp. of o° C. for 66 days.

PROTEASES OF PLANTS. Kovchoff (4) demonstrated the power of these enzymes to survive freezing. Wheat seedlings which had germinated for 17 days, excoriated peas, peas excoriated after germination for 5 days, and certain tissues of the bean, Vicia fabaetiolated caulis tops, etiolated leaves and green leaves were studied separately. Each sample was frozen for 24 hr., then permitted to undergo autolysis at room temp., in the presence of toluene as a bactericide, for a period varying from 2 days to 5 weeks. The amounts of protein and non-protein nitrogen were then determined. Almost invariably the former decreased and the latter increased during the autolysis. Therefore, these proteases had survived freezing and had