Difference Spectrophotometry Research Articles

Spectrophotometric Measurements of Phytochrome in vivo and Their Correlation with Photomorphogenic Responses of Phaseolus

Direct in vivo measurements of phytochrome have been made in Phaseolus vulgaris by 2-filter difference spectrophotometry (Ratiospect). All measurements were made at 730 versus 800 nm and it is assumed that the Delta (DeltaOD) is directly proportional to the PFR concentration of phytochrome present. Dose response curves were determined for both physiological and spectrophotometric responses for red induction and far-red photoinactivation. For induction, saturation occurs at 100 mj/cm(2) and for inactivation at 30 mj/cm(2). The rate of hook opening and the physiological response measured 20 hours after induction are both shown to be directly proportional to the initial amount of PFR present spectrophotometrically. The sensitivity of the tissue correlates well with the absolute amount of phytochrome present, the inner portion of the hook having the maximum concentration of 0.042 Delta (DeltaOD)/g fresh weight. If the total reversible phytochrome concentration is reduced by exposure to red light and allowing PFR to decay out of the system the remaining sensitivity of the tissue is shown to be directly correlated with the amount of PR remaining in the tissue. PFR disappears rapidly in the dark at 25 degrees , and is not detectable after 6 hours. There is no indication that PFR reverts in the system to PR. At 4 degrees , PFR does not disappear measurably up to 1 hour and is nearly totally reversible to PR.

Spectroscopic changes accompanying some reactions of trypsin and α-chymotrypsin

Difference spectrophotometry is used to measure changes in the ultraviolet spectra of trypsin and α-chymotrypsin caused by complexation with some of their substrates. The sign of the trypsin/ligancl difference spectra is opposite to the sign of the α-chymotrypsin/ligand spectra. The detailed appearance of the spectra depends upon the enzyme/ligand combination. The trypsin vs trypsinogen difference spectrum has the same sign and shares some of the peak locations found in the trypsin/substrate spectra; the chymotrypsin vs chymotrypsinogen difference spectrum shares several peak locations with the α-chymotrypsin/substrate spectra. The concentration dependence of the α-chymotrypsin/p-toltienesulfonyl-L-arginine methyl ester difference spectrum fits the assumption that the species responsible for the difference spectrum is in equilibrium with free enzyme and substrate; the dissociation constant obtained is the same as the Michaelis-Menten constant obtained by kinetic analysis.

Preparation and Some Properties of Yeast Mitochondria

Abstract Well preserved mitochondria were isolated from Saccharomyces carlsbergensis by a procedure which involves digestion of the cell wall with snail gut juice. The yeast mitochondria thus prepared oxidized members of the tricarboxylic acid cycle, reduced nicotinamide adenine dinucleotide, d- and l-lactate, and tetramethyl-p-phenylenediamine reduced by ascorbate. Respiratory control in response to the addition of adenosine diphosphate was observed with all of these substrates. The highest respiratory control ratio (5 to 6) was obtained with α-ketoglutarate as substrate. The yeast mitochondria actively oxidized externally added NADH, and this oxidation likewise showed respiratory control. The ADP to oxygen or phosphorus to oxygen ratios observed in this system were about 0.9 with tetramethyl-p-phenylenediamine and lactate; 1.7 with succinate, NADH, citrate, and pyruvate plus catalytic amount of malate; and 2.5 with α-ketoglutarate. These respirations were completely insensitive to Amytal and rotenone. It is suggested that phosphorylation Site I is absent from these yeast mitochondria. Effects of uncouplers and inhibitors of oxidative phosphorylation on yeast mitochondria were very similar to those observed with mammalian mitochondria. The P:O ratios in the respiration with NADH as a substrate showed constant values when measured between pH 5.4 and 7.5, and at a range of tonicity between 0.3 and 1.0 m sorbitol. The yeast mitochondria could be stored for a long period of time without appreciable loss of phosphorylation activity. The components of electron transport of isolated yeast mitochondria were studied by means of difference spectrophotometry. Electron micrographs of the isolated yeast mitochondria and mitochondria in situ were also presented.

Identification and determination of phenols and chloro-phenols in very dilute aqueous solutions by gas-liquid chromatography, paper chromatography and spectrophotometry

After concentration of the organic substances from water samples of 700 to 20,000 l by adsorption on active carbon and desorption with chloroform in a large Soxhlet apparatus, tlie organic compounds were separated by extraction into 5 different groups: acids, phenols, bases, neutral and amphoteric substances. The phenol group was investigated by gas and paper chromatography, ultraviolet difference spectrophotometry and infrared spectroscopy. Phenol, 2,4,6-trichlorophcnol, 2- and 3-cresol, 2,4-xylenol and 2,4-dichlorophenol were identified in samples of raw and treated water. Quantitative measurements proved to be possible with gas chromatography. The conditions for quantitative desorption and separation were studied.

Structural investigations on dehydrogenases by means of difference spectrophotometry

The distribution and localization of tyrosine in glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde 3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) and in lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by difference spectrophotometry through spectrophotometric titration, solvent perturbation and iodination. In both dehydrogenases the alkaline dissociation of some phenolic hydroxyl groups is anomalous. The apparent pK′s are 11.3 and 11.5, and the titrations are irreversible. As measured by the solvent perturbation method, maxima of 45 and 50% of the tyrosyl residues may be located on the surfaces of the glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenases respectively. About 65 and 80% of the tyrosyl groups are accessible to iodine in glycine buffer (pH 9.2). The remainder becomes available to iodine only after the proteins have been denatured with concentrated urea. On the basis of the above findings the structural composition of these dehydrogenases is discussed.

THE INFLUENCE OF IONIC STRENGTH AND ORGANIC SOLVENTS ON ACID TRANSITIONS OF PROTEINS.

1. 1. The influence of ionic strength, dioxane, and ethylene glycol on the acid transition of ribonuclease has been studied by difference spectrophotometry. In this transition two of the three buried tyrosyl residues of the molecule can be normalized if the temperature is high enough, but an increase in the ionic strength will repress the normalization of one of them. Dioxane makes the normalization of the second one easier, and ethylene glycol has very little effect. 2. 2. Dioxane also raises the p K of the transition. Ribonuclease, serum albumin, and acetic acid all show the same dependence of Δ p K on dioxane concentration, suggesting that the increase is caused by a change in the bulk dielectric constant of the medium. Chymotrypsinogen is much more strongly affected, and lysozyme is less strongly affected. 3. 3. The denaturation of ribonuclease at pH 2.3 by dioxane shows some very unusual features. The difference spectra are anomalous. The denaturation is very strongly affected by the ionic strength, and in an opposite direction to what is usually observed: denaturation occurs at lower dioxane concentrations as the ionic strength is raised. A possible explanation for these unusual features is based on Weber 's observation that dioxane first denatures ribonuclease, and at higher concentrations promotes the formation of helical regions in the molecule.

Observations on spectrophotometrically assayable phytochromein vivo in etiolatedPisum seedlings

Using 2-filter difference spectrophotometry, thein vivo phytochrome of etiolated pea seedlings was measured. On a fresh weight basis, the reversible optical density difference, Δ(Δ O.D.), at 660 and 730 mμ was highest in growing regions such as the epicotyl hook or the root tip, and decreased along the axis of shoot and root. The aging of the seedling did not affect the pattern of phytochrome distribution in the epicotyl, but Δ(Δ O.D.) per unit weight declined slowly in root-tips of intact seedlings and much more rapidly in those of excised root cultures. Although apparent amounts of phytochrome vary in each tissue, per cent conversion of PR to PFR was simply dependent upon total irradiation energy; that is, any tissue obtained from etiolated peas showed approximately 50% conversion of PR to PFR by 3 minutes exposure to red light of about 1.8 kiloergs x cm−2 x min−1. After conversion of PR to PFR by a brief red irradiation, dark transformation was observed in intact and excised tissues of stems and roots; after 4 hours at 26°C, most of the PFR disappeared optically, and total Δ(Δ O.D.) decreased to 1/2∼1/3 of the original level. No significant difference in rate was observed between intact and excised stem samples, or intact root samples, but the rate in excised roots was slower than in the intact. Contrary to previous reports, dark transformation occurs at 0°C, though at roughly 1/60th the rate observed at 26°C.

States of amino acid residues in proteins: IV. Bound tyrosine and tryptophan residues in pepsin as observed by difference spectrophotometry

The alkali denaturation of pepsin was studied between pH 1 and 13 by difference spectroscopy and by activity measurements at pH 1.8, and the tryptophan and tyrosine residues were differentiated into two and three groups, respectively that differed in state. As the pH value of a pepsin solution is raised progressively from 1.8, the denaturation proceeds in several steps. The proteolytic activity drops first with p K = 3.1, and the band shift of two presumed tryptophan residues follows the inactivation (p K = 4.0). The band shift of the remaining four tryptophan residues proceeds between pH 7 and 8 and in parallel with an irreversible inactivation. At higher pH values, the ionization of tyrosine residues in various states occurs successively; 11 rapidly ionizing residues with p K = 10.0, 5 rapidly ionizing residues with p K = 11.5, and 1 slowly ionizing residue with a higher p K value. These results are discussed with reference to data reported previously.

THE PROPERTIES OF THYROGLOBULIN. X. THE EFFECT OF UREA.

Abstract The properties of bovine thyroglobulin in solutions of urea were examined by the techniques of velocity sedimentation, viscosity, optical rotation and ultraviolet difference spectrophotometry. Below 2 M urea, a limited dissociation into two equal subunits took place without change in molecular asymmetry. At higher concentrations of urea, progressive destruction of the secondary and tertiary properties of the protein occurred. In 9 M urea, two sedimenting components were present in approximately equal amounts. These two molecules appeared to be highly unfolded forms of the native molecule and its subunit. No time effects were found in any of the transitions observed. The properties of thyroglobulin in urea solutions can be understood if there is (a) a distribution of energy states amongst the protein molecules which governs the susceptibility to unfolding by urea, and (b) a large number of unfolded states in which each molecule of thyroglobulin can exist.

Use of Protein Difference Spectrophotometry to Determine Enzyme-Cofactor Dissociation Constants

Use of Protein Difference Spectrophotometry to Determine Enzyme-Cofactor Dissociation Constants