Günter Barth

Magnetic circular dichroism studies: XIX. Determination of the tyrosine: Trytophan ratio in proteins

Abstract Recently (2), we reported the use of magnetic circular dichroism (MCD) for the determination of tryptophan in intact proteins (1). We pointed out that, in comparison to other methods currently in use, MCD has the unique advantage of giving unambiguous results even in cases in which the tyrosine: tryptophan ratio is high, that conformational and environmental effects do not interfere with the accuracy of the method, and that only small amounts of sample are required. Here, we wish to report an important refinement in the procedure that enables the technique to be extended to samples which, due to the presence of unknown quantities of water or inorganic salts, are not of analytical purity. This modification makes use of the fact that, in contradistinction to tryptophan, the quantitative determination of tyrosine by the usual chromatographic procedure subsequent to hydrolytic cleavage of the protein does not present serious difficulties. Consequently, the spectroscopic determination of the tyrosine: tryptophan ratio has the advantage that, given knowledge of the tyrosine content, accurate values for the tryptophan content can be obtained with only rough knowledge of the protein concentration. Thus, tyrosine serves as an internal standard in the determination of tryptophan by the MCD technique. In addition to enabling tryptophan determinations to be made with increased accuracy, this modification illustrates the advantages inherent in a spectroscopic method in which signals may occur with either positive or negative sign.

Magnetic circular dichroism spectroscopy: A definitive method for the determination of urinary porphyrins☆

Abstract The application of MCD for the determination of urinary porphyrins is assayed by specific application to the screening of workers exposed to lead arsenate. The definitive nature of this new spectroscopic method arises from: (1) its sensitivity to as little as 0.02 μg/ml of porphyrin dication; (2) its freedom from interference caused by other substances; (3) the highly characteristic shape of the MCD bands exhibited by porphyrin dications; and (4) the high information content of the MCD spectrum.

Partial reduction of aquomethemoglobin on a sephadex G-25 column as detected by magnetic circular dichroism spectroscopy and revised extinction coefficients for aquomethemoglobin

In a typical preparation of aquomethemoglobin, oxyhemoglobin is oxidized with potassium ferricyanide, and the resultant mixture of methemoglobin and potassium ferro- and ferricyanides is separated on a Sephadex G-25 column. We find that about 1% of the heme is reduced on the column and is eluted with the methemoglobin. Magnetic circular dichroism spectra show that the reduced species is oxyhemoglobin. Magnetic circular dichroism is more sensitive than is absorption spectroscopy to small amounts of oxyhemoglobin in such solutions; we can detect its presence at the 0.1% level. A redetermination of the extinction coefficients for methemoglobin gives a value of 0.80 for the absorbance ratio A570A630 at pH 6. This value lies clearly outside the currently accepted range of 0.83 to 0.87.

Magnetic circular dichroism studies XXXIV: Improved instrumentation for MCD measurements

Abstract The use of a 15-kG electromagnet in conjunction with a circular dichrometer of enhanced sensitivity affords MCD spectra of quality comparable to those obtained using a superconducting magnet and an instrument of lower sensitivity. To demonstrate this, the MCD spectra of zinc octaethylporphyrin, human serum albumin, a microsomal suspension containing cytochromes P -450 and b 5 , adenine, and benzene in the vapor phase are reported. The convenience, economy of operation, and availability of electromagnets should be a major incentive for the utilization of this spectroscopic technique in a variety of potentially interesting areas in chemistry and biochemistry.

Magnetic circular dichroism studies. XXXIII. The MCD spectrum of formaldehyde vapor

Abstract The MCD spectrum of formaldehyde in the vapor phase has been measured and analyzed. Triplet bands occur with unusually high ellipticity, allowing identification of several new singlet-triplet bands. Also, an additional singlet progression, based on two quanta of the out-of-plane band and a single quantum of the antisymmetric CH stretch, has been identified.

Magnetic circular dichroism studies—XXXI. Thioketones

Abstract The magnetic circular dichroism associated with the n -π* transition of several thioketones is reported and evidence is presented that the lowest energy band is of singlet-triplet origin.

Theoretical analysis of the magnetic circular dichroism spectrum of carbon disulfide

The upper level of the near‐ultraviolet absorption system of carbon disulfide (the R system) is the B2 spin‐multiplet component of a 3A2 state. A theoretical analysis of the magnetic circular dichroism spectrum of the R system shows that if, as has been assumed previously, the A1 and B1 spin‐multiplet components of the 3A2 state are degenerate, they lie above the B2 component. This result is at variance with earlier conclusions. The only ordering of the spin‐multiplet components which is consistent with all available data is one in which the A1 and B1 components are nondegenerate, with both of these components lying below the B2 component. The available data are insufficient to assign the relative ordering of the A1 and B1 components.

The magnetic circular dichroism spectrum of carbon disulfide

The magnetic circular dichroism spectrum of carbon disulfide vapor in the 300–380 nm region is reported and analyzed. The signs of the A terms associated with the P and R rotational branches of the triplet bands are negative and positive, respectively. These data are used in an accompanying theoretical paper (following paper in this journal) to calculate the zero field splittings in the 3A2 state of CS2.

The vapor phase magnetic circular dichroism spectrum of the 3Au ← 1Ag excitation of glyoxal

Abstract The vapor phase magnetic circular dichroism spectrum of the 520 nm band system of glyoxal is reported. The vibrational analysis of this spectrum, and its temperature dependence, extend and confirm the assignments made by Ramsay and his collaborators from magnetic rotation and emission data.

Magnetic circular dichroism of the 3B3μ ← 1 Ag excitation in pyrazine

Abstract The magnetic circular dichroism spectrum of the lowest lying singlet—triplet transition of pyrazine is reported. The experimental data are in accord with theoretical predictions based upon the assumption of Hunds case (b) coupling in the 3B3u state.