Changes In Optical Rotatory Dispersion Research Articles

Chiral Crystallization of Ethylenediamine Sulfate

Optimized conditions for the chiral crystallization of ethylenediamine sulfate, which can serve as an ideal undergraduate experiment, are described. Large, flat, colorless crystals of ethylenediamine sulfate are obtained in an undisturbed evaporation dish within a period of approximately five to seven days. The crystals are ideal for polarimetry studies and observation using Polaroid sheets. Students become familiar with polarizing filters and how they can be used to distinguish between dextrorotatory and levorotatory crystals. Of 100 randomly sampled crystals, 47 crystals showed a (+)-rotation, while 53 crystals showed a (-)-rotation. The absolute value of the average optical rotation of the 100 crystals was found to be 158/mm ± 2 for the D line of sodium. The relative direction of rotation of polarized light was also determined by simply observing the change in optical rotatory dispersion of the crystals placed between two Polaroid sheets as one of the sheets was rotated clockwise or counter clockwise.

Interaction of metal ions with polynucleotides and related compounds. XX. Control of the conformation of polyriboadenylic acid by divalent metal ions

Divalent metal ions have profound effects on the conformation of poly A, as indicated by changes in optical rotatory dispersion and ultraviolet spectra, with different metal ions having distinctly different effects. At pH 6 and in the presence of 5 × 10 −2 M NaCl, two moles Ni(II) or Co(II) per mole of poly A residue stabilize the single helix; the same amount of Mg(II), Ca(II), Zn(II) or Mn(II) produce a mixture of double and single helix, while Cu(II) and Cd(II) induce the formation of a random coil structure. Under other conditions, these same metals have different effects, with the amount of added NaCl an important factor that can be used to control the way in which a particular divalent metal ion will induce the transition from a double helix to single helix or from helix to coil. The single helix is stabilized by metals binding to phosphate; the double helix is destabilized by metal ions binding to either phosphate or base, the former producing single helix and the latter random coil. The melting temperature of double helical poly A is diminished when metal ions bind to either type of site.

Ligand induced conformational changes in various normal and modified hemoglobins as indicated by changes in optical rotatory dispersion.

A decrease of the optical rotation in the wavelength region from 200 to 250 mμ is observed when ligands bind to hemoglobin. The change at 233 mμ amounts to about 8% of the total rotation at this wavelength, independently of the nature of the ligand (O2, CO, NO or ethyl‐isocyanide). The effect is interpreted as being caused by a small conformational change, which influences the electronic transitions of a number of peptide bonds or (and) a few aromatic side chains. The change is observed with αβ‐dimers, but not with the isolated single chains. The full effect is present however, in chemically modified hemoglobins which no longer show cooperative interactions in the ligand equilibrium curve. It is speculated that the conformational change takes place within the protomers but only when these are joined in αβ‐dimers (or tetramers). The chemical modifications might not prevent the initial ligand‐induced conformational change, but only abolish the transmission of the conformational change from one subunit to the others.

Studies on Myosin from Red and White Skeletal Muscles of the Rabbit: II. INACTIVATION OF MYOSIN FROM RED MUSCLES UNDER MILD ALKALINE CONDITIONS

The rapid loss of ATPase activity of myosin from the red, or slow, skeletal muscles of the rabbit under mild alkaline conditions has been studied in detail. The loss of ATPase activity is irreversible and consistent with a mechanism involving two first order reactions with rate constants of 0.3 and 0.007 min-1. No gross change in molecular structure accompanies inactivation, as judged by the lack of major change in optical rotatory dispersion or sedimentation. Slight increases in the rate of tryptic digestion and reactivity of sulfhydryl groups which may or may not be involved in the loss of enzymic activity are observed after inactivation. It appears that the molecular changes involved in loss of enzymic activity are relatively small and may be localized at or near the catalytic site. Myosin from rabbit cardiac muscle loses ATPase activity at pH 9.0 at the same rate as myosin from red skeletal muscles.

Changes in the optical rotatory dispersion induced by the dilution inactivation of dehydrogenases

Optical rotatory dispersion properties of three dehydrogenases of Bacillus subtilis were examined under various conditions. 1. 1. Alanine dehydrogenase ( l-alanine:NAD + oxidoreductase, deaminating, EC 1.4.1.1), which consisted of 6 subunits, and lactate dehydrogenase ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27), which consisted of 4 subunits, were partially inactivated by dilution at neutral pH. No significant changes in their optical rotatory dispersions and in their molecular weights accompanying the dilution inactivation were detected. 2. 2. Lactate dehydrogenase was completely inactivated by dilution at alkaline pH accompanied by dissociation into subunits. A change in optical rotatory dispersion, which indicated unfolding of the enzyme, was observed. 3. 3. Malate dehydrogenase ( l-malate:NAD + oxidoreductase, EC 1.1.1.37), which consisted of 4 subunits, was completely inactivated by dilution at alkaline pH in the absence of NADH accompanied by dissociation into subunits. A change in optical rotatory dispersion, suggesting unfolding and β-structure formation, was observed.

The effect of methanol, urea and other solutes on the action of papain

The activity of papain (EC 3.4.4.10) decreases with increasing methanol concentration. At relatively low concentrations (2–4%), methanol, a few other solvents and urea (2 M) exhibit apparently competitive inhibition. However, the extent of inhibition by methanol and urea is not the same for the two substrates benzoylglycine ethyl ester and benzoylarginine ethyl ester. Partial methanolysis of benzoylarginine amide was observed in 30% methanol by a titration method. Methanol was a 5–6 times more effective nucleophile than water. In 50% methanol almost no activity remained. This loss of activity is believed not to be due to conformational change for the following reasons: (1) There was no change in optical rotatory dispersion up to 70% methanol. (2) There was no change in viscosity in 50% methanol. (3) Papain is an enzyme of unusual stability, since in 8 M urea it retains considerable activity and exhibits no essential change in optical rotatory dispersion. It is considered possible that the inhibitory effects are to a large extent due to interference with hydrophobic interactions between enzyme and substrates. Certain consequences for the X-ray analysis of papain crystals grown from water-methanol mixtures are pointed out.

Optical properties of ribonucleic acids predicted from oligomers

An empirical method has been developed for predicting the optical properties of single-strand polynucleotides from the corresponding properties of oligonucleotides. If only nearest-neighbor interactions are considered, the optical rotatory dispersion of polynucleotides can be predicted from that of dinucleoside phosphates and mononucleosides. These calculations are in good agreement with experimental results for poly A, poly C, poly U and tobacco mosaic virus RNA and other RNA's in the absence of salt at neutral pH. Thus in the limit of low salt concentration, the conformation of tobacco mosaic virus RNA is mostly a single strand with stacked bases. The nearest-neighbor formalism has been extended to take into account double-stranded conformations. At present there are not sufficient experimental data to include the contributions to the optical rotatory dispersion from all of the possible double-stranded dimers. However, the effects of double-strand formation can be estimated from the known optical rotatory dispersion of double- and single-strand homopolynucleotides. With these estimates it was possible to predict the change in optical rotatory dispersion expected when a single strand forms intramolecular base pairs. These calculations agree with the observed change in the optical rotatory dispersion of tobacco mosaic virus RNA when salt is added to a salt-free solution. A model has been constructed for the yeast alanine sRNA which includes the effects of base stacking as well as base pairing. The properties of this model are consistent with much of the experimental data on the conformation of sRNA's. The optical rotatory dispersion of partially double-stranded alanine sRNA is in good agreement with nearest-neighbor predictions.

Acid Denaturation of Horse Carbonylhemoglobin in the Absence of Oxygen

Abstract Unlike ferrihemoglobin (Hb+), denatured carbonylhemoglobin (COHb) has almost the same absorption spectrum in the visible and near ultraviolet region as the native form; the very slight difference appears only in the Soret band. Kinetic and equilibrium experiments under anaerobic conditions show that COHb is considerably more resistant to acid denaturation at 25° than is Hb+ although the rates of denaturation of both proteins depend on pH in much the same way. These conclusions are based on assays of denatured protein by precipitation at the isoionic point, by the measurements of unmasking of imidazole groups inaccessible in the native protein, and by changes in optical rotatory dispersion. Air, when present, oxidizes the denatured protein practically instantaneously, but it has no discernible effect on the denaturation rate. It does, however, predictably shift the equilibria by removing the initial product, unoxidized acid-denatured protein. COHb, unlike Hb+, is not stabilized by formate, but another stabilizer of Hb+, azide, produces complex effects. Bromphenol blue greatly accelerates the rate of denaturation and shifts the denaturation equilibrium to higher pH. The possible significance of the absence of a marked spectral change is discussed in terms of a possible nonbonded clathrate structure for the prosthetic group in COHb. Changes in the Cotton effects in the Soret band differ in acid-denatured COHb and Hb+, and there are differences in the regenerability of the denatured proteins. These facts add support to the evidence that conformation differences exist in the native forms; thus the difference in stability between COHb and Hb+ need not be solely the direct consequence of the positive charge on the prosthetic group of the latter as previously postulated.

Citrate-condensing Enzyme-Oxalacetate Binary Complex: STUDIES ON ITS PHYSICAL AND CHEMICAL PROPERTIES

Abstract A binary complex is formed between citrate-condensing enzyme and oxalacetate. Complex formation is accompanied by a change in protein conformation, as judged by changes in ultraviolet absorption, a small change in optical rotatory dispersion, and increased stability to denaturation. The stability of the complex in urea has made it possible to estimate that there is 1 oxalacetate molecule per active site on the enzyme, and that the dissociation constant of this complex is about 6 x 10-7 m. Urea treatment of the enzyme results in the exposure of sulfhydryl groups, changes in the ultraviolet absorption, changes in the optical rotatary dispersion spectrum indicative of unfolding, and changes in the sedimentation behavior. Urea treatment of the complex, however, shows essentially none of these changes. A number of analogues were tested as to their ability to bind to the oxalacetate site. On the basis of these studies it was concluded that oxalacetate was bound through both carboxyl groups, as well as through the carbonyl oxygen. It seems that once the oxalacetate site on the enzyme is occupied, then urea (or other denaturants) cannot react with the enzyme.

Physicochemical studies on denaturation of myosin—Adenosinetriphosphatase. II. Changes in chromatographic profile and optical rotation

The inactivation of myosin A-ATPase proceeds according to the first-order law. The rate constants of the inactivation were proportional to the 1.3, 0, and −3.3 powers of [H +], respectively, in the ranges of pH 5.2–6.0, 7.5–8.5, and 10.0–10.5. In these three ranges of pH, Δ H‡ values were 31.6, 52.6, and 42.1 kcal./mole, respectively. During the incubation at 36 °C. the α component in chromatographic profiles on diethylaminoethylcellulose decreased, the β component increased, and finally the β component decomposed. However, specific ATPase activities of both the α and the β components were not constant and decreased with time. Under three conditions (pH 7.0 and 30 °C., pH 5.7 and 20 °C., and pH 10.3 and 20 °C.), the change in optical rotatory dispersion with time was measured and compared with that of ATPase activity. The helical content decreased very slowly and only by a few per cent, even after ATPase activity disappeared.

ANOMALOUS ROTATORY DISPERSION OF ENZYME COMPLEXES, II. THE ASYMMETRIC BINDING OF COENZYMES AND INHIBITORS TO LIVER ALCOHOL DEHYDROGENASE

The inhibition of the yeast and liver alcohol dehydrogenases' by chelating agents is accompanied by progressive changes in optical rotation.2' 3 Such alterations of the optical rotatory properties of yeast alcohol dehydrogenase are due to the binding of chelating' agents to the asymmetric zinc site of the enyme resulting in a characteristic Cotton effect. The rotatory power of this Cotton effect, when superimposed upon the background dispersion curve of the native protein, accounts for the rotational changes which appear during the course of enzymatic inhibition, and these changes can be differentiated from those due to alterations in protein conformation.4 Such observations suggested that changes in optical rotatory dispersion might be a frequent concomitant of the binding of small molecules to proteins and might, thereby, be used to identify the interactions with enzymes of optically active, chromophoric substrates, coenzymes, inhibitors, and other site specific reagents.4 To this end we have investigated the alcohol dehydrogenase of horse liver. This zinc containing enzyme5' 6 interacts stoichiometrically with two moles of coenzyme, DPN(H), to form the enzymatically holoenzyme7 and is inhibited by a number of chelating agents.' Zinc is not removed during chelation nor does the enzyme dissociate into subunits by electrophoresis or ultracentrifugation. 1,10phenanthroline, OP, reversibly inhibits the enzyme by chelation of the zinc atom forming a mixed complex: LADH-Zn OP.9 The coenzyme binds in a specific though as yet unknown manner at or near the zinc site of the enzyme. A variety of approaches indicate that zinc is a component of the active center of liver alcohol dehydrogenase.'I The present report demonstrates that the binding of reduced diphosphopyridine nucleotide to liver alcohol dehydrogenase is accompanied by the appearance of a pronounced Cotton effect at 327 m,. The magnitude of this Cotton effect is a direct function of the molar ratio of the interactants, thereby permitting a quantitative assessment of coenzyme binding. Moreover, characteristic changes in the optical rotation of the enzyme-coenzyme complex identify its interaction with substrate and inhibitors. Similar effects are observed when the analogues of DPNH bind to liver alcohol dehydrogenase. The considerations pertinent to the choice of metalloenzymes for the exploration of the molecular basis of enzyme action have been discussed3 and preliminary accounts of the present studies have been reported.2' 13 Methods and Materials.-Crystalline alcohol dehydrogenase of horse liver was obtained from C. F. Boehringer and Soehne, Mannheim, W. Germany. Prior to use, the enzyme was dialyzed for 5 days against 0.1M phosphate buffer, pH 7.5 at 4? to remove low molecular weight impurities which absorb radiation at 280