Abstract Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.

In contrast to DNA chips, serial analysis of gene expression (SAGE) is not dependent on genes having been previously identified for their monitoring. Although useful, the method can be technically challenging, and particularly the last steps including concatenation and cloning may result in less than optimal results. We propose that many of the encountered problems can be attributed to the purification of the 26-bp ditags by polyacrylamide gel electrophoresis. Low yields, gel contaminants, potential exposure to degrading enzymes during handling and lengthy separation all disfavor the method. We introduce purification of 26-bp ditags by reverse-phase high-performance liquid chromatography (HPLC) using polystyrene/divinylbenzene columns and tetraethylammonium acetate buffer with acetonitrile as mobile phase. The method is fast and gives excellent results. Ditags purified by HPLC readily ligate to high-molecular-weight concatemers leading to their efficient cloning. The method should substantially facilitate the construction of SAGE libraries.

A family of rigid macroporous HPLC materials, reversed phase and anion exchange, has been evaluated for the analysis and purification of a range of de-protected, dimethoxytrityl-off, oligonucleotides. A 25-base pair (bp) double-stranded DNA ladder was used to determine the resolving range for the four pore sizes of reversed-phase media. The 100 Å pore size resolves up to 50–75 bp, the 300 Å up to 250–300 bp, the 1000 Å up to 400–450 bp and the 4000 Å pore size is capable of resolving in excess of 500 bp. The dynamic capacity of these four pore sizes was also determined using a synthetic oligonucleotide with two ion-pairing agents at ambient and 60 °C. The dynamic capacity was shown to decrease with increasing pore size and that with the triethylammonium acetate ion-pairing agent there was negligible temperature dependency. The dynamic capacity was higher when tetrabutylammonium bromide was used at elevated temperature. A strong anion-exchange functionality on a pH-stable polymeric particle was used to investigate the selectivity and resolution of the technique. Using a poly-T-oligonucleotide size standard, resolution of full length oligonucleotide (n) from the truncated species due to coupling failure (n−1, n−2, etc.) was demonstrated up to at least the 30mer. Resolution of a phospho diester contaminant from a phospho thioate oligonucleotide and a truncated sequence was demonstrated using anion-exchange HPLC at high pH.

INTRODUCTION • The use of Biophysical techniques in research and development is increasing rapidly • ICH Guideline Q6B: ‘The higher-order structure of the product is examined using procedures such as circular dichroism, nuclear magnetic resonance (NMR), or other suitable techniques, as appropriate’ • SGS M-Scan offers established GxP compliant absorbance, CD, DSC, AUC and SEC-MALS • The company has recently extended its range of biophysical techniques, and now offers fluorescence, FTIR and DLS services • We demonstrate here the application of SGS M-Scan’s orthogonal analysis capabilities to the characterisation of biological material

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

An M(r) 22 500 protein was purified from isolated oil bodies from sunflower cotyledons. N-terminal amino acid sequencing showed that this protein belonged to the 2S albumin group of storage proteins. A corresponding cDNA clone encoded a preproprotein, comprising two mature 2S albumin proteins one of which corresponded to the oil-body associated albumin. In contrast, the second albumin encoded by the cDNA was related to components purified from a total albumin fraction by RP-HPLC.

Two distinct cDNAs encoding oleosins (oil body proteins) have been identified by degenerate PCR as transcripts present in the developing seeds of sunflower (Helianthus annuus L. cv. Dwarf Sunbred). One (pSOM) of these is closely related to a reported sunflower oleosin, whilst the other (pSO5) has not been previously described. Different expression patterns were observed for the two cDNAs, pSO5 being expressed earlier than pSOM in seed maturation and oil deposition. The results support the contention that oleosin proteins are synthesised either during or closely after the formation of the oil body. Translation in vitro of synthetic oleosin transcripts was enhanced by the addition of microsomes but suppressed by the addition of purified signal recognition particle (SRP) complex. Deletion of 62 amino acid residues at the C-terminus of the oleosin did not alter the in vitro targeting of the protein to the microsomal membrane. Taken together these data support the idea that oleosins are targeted to the ER membrane as part of oil body biogenesis.