THE REVERSIBLE INACTIVATION OF BACTERIOPHAGE BY BICHLORIDE OF MERCURY

1. The complete inactivation of antistaphylococcal phage by HgCl₂ (2.8 per cent for 216 hours) can be reversed by precipitation of Hg⁺⁺ with restoration of the phage to its original titre. 2. This behavior seems more compatible with the known properties of certain enzymes than with those of living protoplasm.     

THE PHOTODYNAMIC INACTIVATION OF PHAGE PRECURSOR BY METHYLENE BLUE

Methylene blue added to suspensions of activated staphylococci in amounts sufficient to furnish 1 x 10⁵ molecules of dye/bacterium inactivates the phage precursor content of the cells without causing cell death when the mixtures are exposed to strong light of 4000–8000 Å. There is a lag phase of approximately 15 minutes in the photodynamic inactivation of phage precursor by methylene blue. This delay seems to be due to a primary reaction between the cell and methylene blue after the completion of which exposure to light brings about the inactivation of precursor quite promptly.     

THE KINETICS OF INACTIVATION OF COMPLEMENT BY LIGHT

The photoinactivation of complement has been studied with a view to determining if possible how many kinds of molecules disappeared during the reaction. It was found that: 1. The apparent course of photoinactivation is that of a monomolecular reaction. 2. Diffusion is not the limiting factor responsible for this fact, because the temperature coefficient of diffusion is much higher than that of photoinactivation (Q ₁₀ = 1.22 to 1.28, and Q ₁₀ = 1.10 respectively). 3. There is no change in the transparency of serum solutions during photoinactivation, at least for light of the effective wave-length, which is in the ultra-violet region probably at about 2530 Ångström units. It is pointed out that under these conditions only one interpretation is possible; namely, that during photoinactivation a single disappearing molecular species governs the rate of reaction. This substance must be primarily responsible for the hemolytic power of serum when it is used as complement.     

THE REVERSIBLE INACTIVATION OF TOBACCO MOSAIC VIRUS BY CRYSTALLINE RIBONUCLEASE

The reversible inactivation of tobacco mosaic virus by crystalline ribonuclease is reported. Studies on the effect of time of standing on the amount of inactivation, and on the effect of dilution and repeated high speed centrifugation on the recovery of virus activity, and the preparation of an insoluble virus-enzyme complex show that the inactivation is brought about at least in part by a combination between virus and enzyme. The significance of the fact that ribonuclease has no detectable effect on the virus nucleic acid when the latter is in combination with protein in the form of virus is discussed with respect to the structure of the virus.     

THE INACTIVATION OF BACILLUS SUBTILIS SPORES IN PENICILLIN BY GAMMA RADIATION

SUMMARY: Sodium benzylpenicillin, contaminated with Bacillus subtilis spores by freeze-drying a suspension of spores in an aqueous penicillin solution ( c . 50% w/v), was exposed to gamma radiation and a 70-tube dilution method was used to determine the surviving spores after various doses. The correlation coefficient between log₁₀ percentage survival and dose was −0.9523. The regression of the former on the latter was calculated and the decimal reduction dose found to be 20.2 × 10⁴ rads. The regression and the decimal reduction dose were similar to those obtained when suspensions of spores in distilled water were irradiated.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

THE GROWTH OF BACTERIOPHAGE AND LYSIS OF THE HOST

1. A new strain of B. coli and of phage active against it is described, and the relation between phage growth and lysis has been studied. It has been found that the phage can lyse these bacteria in two distinct ways, which have been designated lysis from within and lysis from without. 2. Lysis from within is caused by infection of a bacterium by a single phage particle and multiplication of this particle up to a threshold value. The cell contents are then liberated into solution without deformation of the cell wall. 3. Lysis from without is caused by adsorption of phage above a threshold value. The cell contents are liberated by a distension and destruction of the cell wall. The adsorbed phage is not retrieved upon lysis. No new phage is formed. 4. The maximum yield of phage in a lysis from within is equal to the adsorption capacity. 5. Liberation of phage from a culture in which the bacteria have been singly infected proceeds at a constant rate, after the lapse of a minimum latent period, until all the infected bacteria are lysed. 6. If the bacteria are originally not highly in excess, this liberation is soon counterbalanced by multiple adsorption of the liberated phage to bacteria that are already infected. This leads to a reduction of the final yield.     

STUDIES ON THE PURIFICATION OF BACTERIOPHAGE

A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10^–14 mg. of nitrogen, or 6 x 10^–17 gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply this method of purification to a staphylococcus bacteriophage. Since this organism does not readily grow in synthetic medium, a diffusate of yeast extract medium was employed. The better of two preparations contained about 10^–12 mg. of nitrogen per unit of lytic activity. Although this is about one hundred times the amount of nitrogen found in an active unit of B. coli bacteriophage, nevertheless, the diffusion rate experiments gave results which paralleled those obtained with the coliphage. The diffusible particles of the crude staphylococcus bacteriophage had a radius of about 7 millimicra, and a calculated molecular weight of about 1,000,000, while the particles of the same phage which appeared in the ultrafiltrate through a thin collodion membrane had a radius of about 2.4 millimicra and a calculated molecular weight of about 45,000. It appears, therefore, that the active principle is distributed as particles of widely different sizes. However, since the smaller particles have all the properties of bacteriophage, the larger particles probably do not represent free molecules, but either are aggregates, or more likely, inactive colloids to which the active agent is adsorbed. The protein isolated, which bears the phage activity, is capable of stimulating the production of antilytic antibodies on parenteral injection into rabbits or guinea pigs. It retains its specific antigenicity when inactivated by formalin, but not when inactivated by drying.     

THE INFLUENCE OF PROTEINS ON THE REACTIVATION OF YEAST INVERTASE

1. Acid-inactivated yeast invertase could not be regenerated in the presence of the proteolytic enzymes trypsin, pepsin, and chymotrypsin. 2. Certain foreign proteins of non-enzymatic nature partially inhibited the reactivation of acid-inactivated invertase. 3. Certain proteins as gelatin, lacto-globulin, and carbohydrate-free horse crystalbumin did not prevent the reactivation of invertase at all. 4. Highly purified reactivated invertase was shown to exhibit an effect typical of original native invertase; that is, acceleration of its activity in presence of foreign protein at pH 3.0. 5. Native invertase was not digested by trypsin and chymotrypsin. 6. The addition of trypsin and chymotrypsin to reactivating invertase did not affect the invertase which had already reverted to the active form, but prevented further reactivation of inactive invertase.