James A. Ryan

The structure of human acidic fibroblast growth factor and its interaction with heparin

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.

Rapid verification of identity and content of drug formulations using mid-infrared spectroscopy

A general method for the rapid verification of both identity and content of complete solid drug formulations has been devised. Infrared spectra for the samples were recorded using the diffuse reflectance technique, and specially written software was employed to identify the type of formulation and level of active ingredient. This software was devised to ensure reliable use when applied by those with minimal operator skills. Three differing drug tablet formulations containing simvastatin, enalapril maleate and lovastatin, as well as a capsule formulation containing finastride were studied. Adequate precision was obtained to reliably verify drug dosage levels. Near-infrared (NIR) and mid-infrared (MIR) spectrometers were evaluated for use with the method. The MIR instrument allowed sufficient resolution and spectral/structural selectivity to reliably verify correctness of either of two near derivative drugs necessarily present in the same clinical study. Drug tablet and capsule dosage levels tested ranged from 0.2 to 40 mg of drug. Approximately 1% (w/w) of the drug in the formulation was the minimum amount determined. Parameters affecting method ruggedness in routine use were optimized. Experimental addition of an extraneous material to a simvastatin formulation was easily detected and flagged by the routine test procedure. Subsequent data retrieval and searching against spectral libraries was used to demonstrate identification of the additive.

Stabilization of the FK506 binding protein by ligand binding

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.

The determination of dexamethasone and prednisolone esters by infrared spectroscopy with long-path cells.

Abstract An infrared method has been devised for the determination of the acetic and tertiary butylacetic esters of dexamethasone and the tertiary butylacetic ester of prednisolone. This method, an example of a more general application of the use of long-path infrared liquid cells to pharmaceutical analysis, is applicable with slight modification to other similar substances. Data obtained on various formulations were in good agreement with quantitative thin-layer chromatographic data for the steroids. The relative standard deviation is ± 1.07%. The method is selective for intact ester in the presence of hydrolysis products.

Sensitive infrared measurement of pilocarpine--isopilocarpine isomerization.

Abstract A sensitive and convenient method of measurement of the degree of isomerization of pilocarpine in 3-mg samples is described. The drug base is dissolved directly or is extracted from salts in basic buffer with chloroform. After evaporation the residue is dissolved in carbon disulfide and examined in a 3.0-mm pathlength infrared cell. The absorbance ratio A 1100cm -1 /A 1082cm -1 is used to calculate the isopilocarpine content from a standard curve. Drug formulation excipients do not interfere if they are insoluble in carbon disulfide, but non-polar substances which cannot be removed by prior extraction interfere. Results from the application of the method to pilocarpine salts are presented. The relative standard deviation of 50—50 isomer mixture is ±1.83 % for seven determinations.

Automatic quantitative infrared analysis of meprobamate. Biphasic method

Abstract The construction of a long-path, minimal-volume infrared cell has made possible the use of a research infrared spectrophotometer as a detector system for automatic analyzer system. Application to the automated assay of meprobamate is described. The method involves aqueous solutions of the drug, such as those resulting from dissolution studies of pharmaceutical dosage forms, and extraction into chloroform before measurement. A relative standard deviation of ± 1.08% was obtained. Sample measurement rate was about 20 per h.

Functional Group Spectroscopy Of Peptides - An Application Of Fourier Transform Infrared (FTIR) Absorbance Subtraction

In recent years peptide chemists have made available a wider variety of pure peptides whose structures were described by techniques such as amino acid sequencing and 13C and 1H nuclear magnetic resonance. With the advent of fourier transform infrared spectroscopy (FTIR), the natural course of events is to determine if FTIR data treatment can contribute significantly to enhance I.R. spectral applications to these compounds.