A. D. McLAREN

MECHANISM OF ENZYME INACTIVATION BY ULTRAVIOLET LIGHT AND THE PHOTOCHEMISTRY OF AMINO ACIDS (AT 2537 Å)

Abstract— >The inactivation of the enzymes chymotrypsin, lysozyme, ribonuclease, and trypsin by ultraviolet light can be accounted for quantitatively by summing the products of (1) the probability that light is absorbed by a given amino acid residue, the molecular extinc tion coefficient, and (2) the probability that absorbed light induces a chemical change in the residue, the quantum yield for the residue. The principal residues involved are cystyl and tryptophanyl. Peptide bond rupture is not important. Energy transfer among chromophores within molecules of enzymes need not be invoked in order to account for photochemical inactivation.

QUANTUM YIELDS FOR ENZYME INACTIVATION AND THE AMINO ACID COMPOSITION OF PROTEINS

Summary A formula has been derived which as a first approximation, predicts the quantum yields for the inactivation of enzymes. The enzymes can be divided into a group containing cystine and the subtilisins which do not. The formula applied to subtilisin gives good agreement. For some of the enzymes, only cystine needs to be considered in the calculation; for others good agreement is found if contributions from aromatic groups are included. On this basis a quantum yield for tobacco mosaic virus protein is computed as 0.0047 at 2537 Å.

PHOTOBIOLOGY OF RNA BACTERIOPHAGES—I. ULTRAVIOLET INACTIVATION AND PHOTOREACTIVATION STUDIES*

Abstract— Biologically active f2‐RNA, Obtained from bacteriophage f2, was inactivated by ultraviolet (u.v) light (2537 Å) with a quantum yield of 3.3 ± 0.3 times 10‐3 when assayed in the dark with protoplasts of an F‐ strain of E. coli k12. Assay under “black light” gave a quantum yield of 2.7 ± 0.5 times 10‐3 which was just enough lower to suggest that 17 per cent photorecovery of the u.v. lesions has taken place.

PHOTOCHEMISTRY OF PROTEINS XXIII. PROTEIN‐PROTElN INTERACTION BETWEEN TRYPSIN, SOYBEAN TRYPSIN INHIBITOR, AND OTHER PROTEINS

Compound formation between trypsin and inhibitor and derivatives of these proteins has been studied by means of light scattering and differential spectrophotometry. it has been fouiid that a non‐specific attraction between oppositely charged molecules is not sufficient for compound formation and that the makeup of the bonding configuration of functional groups involved in compound formation is not constant over a wide range of pH.

In vitro photoreactivation of ultraviolet-inactivated ribonucleic acid from tobacco mosaic virus.

Abstract— Treatment of ultraviolet‐inactivated tobacco mosaic virus ribonucleic acid (TMV–RNA) with extracts obtained from the local lesion host, Nicotiana tabacum var. Xanthi, n.c., and simultaneous illumination at 365 nm results in up to a four‐fold increase in infectivity over non‐illuminated controls. The active material in the extract appears to be associated with protein, based on its inactivation by boiling, precipitation with ammonium sulfate, and exclusion from Bio‐Rad P100 polyacrylamide. Partially purified DNA photoreactivating enzyme from yeast or pinto bean has no activity on ultraviolet‐irradiated TMV–RNA.

A KINETIC ISOTOPE EFFECT IN THE PHOTOREACTIVATION OF U.V.‐INACTIVATED TOBACCO MOSAIC VIRUS RNA*

Abstract— Inactivation of tobacco mosaic virus RNA (TMV‐RNA) by u.v. radiation is slower in D2O than in H2O, and TMV‐RNA which has been inactivated in D2O is photoreactivated faster (on Pinto bean) than TMV‐RNA which has been inactivated in H2O. The maximum amount of photoreactivation is unaffected by the solvent, H2O or D2O, present during irradiation. These deuterium isotope effects for inactivation and photoreactivation suggest that pyrimidine hydrates are photoreactivable lesions on Pinto bean.

PHOTOBIOLOGY OF RNA BACTERIOPHAGES–II U.V.–IRRADIATION OF f2: EFFECTS ON EXTRACELLULAR STAGES OF INFECTION AND ON EARLY REPLICATION*

Abstract— The effect of u.v. irradiation (2537 Å) on the RNA bacteriophage f2 has been studied with respect to the adsorption of f2 to E. coli K12 (male strain), the penetration of f2‐RN A into the host cell and the conversion of the phage nucleic acid to the double‐stranded replicative intermediate. The biological parameter most sensitive to u.v. was the plaque‐forming ability of the phage. Its loss could be attributed to several factors. (1). A binding of capsid protein to phage nucleic acid interfering with host penetration by the f2‐RNA. (2). Desorption of some irradiated phage at 37° from their attachment sites on the host. (3). Molecular alterations in the RNA preventing formation of the replicative intermediate within the host.

The effect of high pressure on the rates of proteolytic hydrolysis. I. Chymotrypsin

Abstract A convenient method which requires only small amounts of enzyme is described for the measurement of the relative rate of hydrolysis of casein by crystalline chymotrypsin. This method is applicable to other proteolytic enzyme-substrate systems provided split products are liberated which absorb light in the ultraviolet region. From the change of the specific rate constant of an enzymatic reaction with pressure it is possible to calculate the volume of activation, Δ V . If this rate constant refers to the rate of disappearance of [ES], then the calculated Δ V may be interpreted as the difference in volume between [ES] and the activated complex [ES]. A relative value of −13.8 ml. for Δ V has been experimentally estimated for the chymotrypsin hydrolysis of casein at 14.8 °C. and pH 7.60. The rate of hydrolysis of 0.02 M l -tyrosine ethyl ester at 25.1 °C. and pH 7.80 has been found to follow zero-order kinetics. A volume of activation of −13.5 ml. was calculated for this hydrolysis.

PHOTOREACTIVATION ACTION SPECTRUM OF TOBACCO MOSAIC VIRUS-RIBONUCLEIC ACID INACTIVATED BY U.V. RADIATION†

Abstract— Initially photoreactivation of irradiated (2537 Å) nucleic acid on pinto bean increases linearly with time of illumination with white light of 250 ft‐c. Maximum amounts of photo‐reactivation depend on the quality of light used. The action spectrum shows a peak in the ‘black light’ region, where the greater amount of photoreactivation is found, and a shoulder in the blue light region. Maximum repair is obtained with ‘black light.’ Photoreactivation does not occur at wavelengths above 550 nm. Photoprotection by illumination of leaves prior to inoculation by irradiated RNA was not found. The action spectrum for photoreactivation does not resemble the action spectrum for photosynthesis.

PHOTOCHEMISTRY OF A MACROMOLECULE: TOBACCO MOSAIC VIRUS

Abstract— A history of the photochemistry of the first known virus is presented. Hollaender and Duggar recognized that the virus contains nucleic acid because of the shape of their action spectrum for photoinactivation. An analysis of this spectrum in detail presents a challenge to photochemists because of the combined role of protein and nucleic acid in determining rates of inactivation. At present these rates can not be explained simply on the basis of the photochemistry of each of the constituents; that is, the photochemistry of free infectious nucleic acid differs in several respects from that exhibited by nucleic acid irradiated in intact virus. At present it is believed that pyrimidine hydrate and dimer formations are important, but an explicit solution to the problem is not in sight.