David Hawke

Microsequence analysis of peptides and proteins. II. Separation of amino acid phenylthiohydantoin derivatives by high-performance liquid chromatography on octadecylsilane supports.

A comparison of the separation of the common phenylthiohydantoin derivatives of amino acids on DuPont octadecylsilane with that obtained with Ultrasphere octadecylsilane supports is given together with the effect of acetate, phosphate, and trifluoroacetate buffers in the elution solvents. An important change in performance for two different batches of DuPont Zorbax octadecylsilane was noted. The use of combined trifluoroacetate/acetate buffer with Ultrasphere octadecylsilane gives optimal separations and peak sharpness. Practical examples of the performance of this system in low-nanomole NH2-terminal sequence analysis are discussed with emphasis on identification of unusual amino acid derivatives and interfering background peaks.

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.

Microsequence analysis of peptides and proteins: III. Artifacts and the effects of impurities on analysis

Levels of contaminants in the parts-per-billion range can adversely affect amino acid microsequence analysis (low-nanomole to subnanomole range) in two ways; (a) contaminants in solvents used in the purification of proteins and peptides can derivatize reactive amino acids to form unusual products or react with free α-NH2 groups to effectively prevent sequence analysis, and (b) contaminants in the reagents and solvents used in Edman chemistry can give spurious peaks on HPLC analysis of amino acid phenythiohydantoin derivatives or react with the phenylthiocarbamylpeptidyl derivatives to give lower initial and repetitive yields of the subsequent phenylthiohydantoin derivatives. Practical examples of these problems and their solutions are described. With proper care in the preparation of solvents and reagents for sample purification and Edman chemistry, microsequence analysis in the low-nanomole to subnanomole range can be made routine.

Unique amino terminal structure of rat little gastrin

The heptadecapeptide form of rat gastrin was purified by a combination of DEAE cellulose, Sephadex G50 affinity, and high performance liquid chromatography. An amino terminal pyroglutamyl blocking group was removed by incubation with PCA peptidase. Amino acid analysis before and after the unblocking reaction revealed the presence of one additional residue of arginine and proline compared with porcine gastrin. Microsequencing analysis of the unblocked peptide revealed that the sequence of the remaining hexadecapeptide was RPPMEEEEEAYGWMDF. The corresponding sequence of porcine gastrin is GPWMEEEEEAYGWMDF amide. The presence of carboxyl-terminal amide group in rat gastrin is strongly supported by complete immunoreactivity with antibodies specific for amidated C-terminal sequences of mammalian gastrins. The Arg and Pro substitutions in the amino terminal region can explain poor crossreactivity of rat gastrin with antibodies specific for the amino-terminal portion of porcine or human gastrin and its more basic chromatography pattern on ion exchange resins.

Purification of Somatostatin from Frog Brain: Coisolation with Retinal Somatostatin-Like Immunoreactivity

Abstract: Somatostatin‐like immunoreactivity (SLI) was purified from frog brain and retina, and the structure of the brain peptide was determined. Frog brain (101 g) and retinal (45 g) tissues were extracted with 3% acetic acid, yielding 9.6 and 0.44 nmol of SLI, respectively. SLI was further purified by chromatography on a somatostatin immunoaffinity column followed by sequential application to reverse‐phase C‐18 HPLC columns. The brain and retinal peptides, purified roughly 100,000‐fold with net yields of 7.5 and 2.3%, respectively, appeared identical in the final steps of purification. The amino acid sequence of brain SLI, as determined by a gas‐phase automated Edman degradation technique, was as follows: Ala‐Gly (Cys)‐Lys‐Asn‐Phe‐Phe‐Trp‐Lys‐Thr‐Phe‐Thr‐Ser‐ (Cys). Our data indicate that despite structural variations in somatostatins of other lower vertebrates, the amino acid sequence of frog brain and, by deduction, retinal SLI is identical to that of somatostatin tetradecapeptide. These findings support the physiological relevance of studies directed at elucidating the neurotransmitter function of somatostatin using the well‐established models of frog brain and retina.

Amino terminal fragments of human progastrin from gastrinoma

Two peptides which copurified from a human gastrinoma were found to correspond to the amino acid sequence deduced for the amino terminal portion of human and porcine progastrin. The sequence of peptide A is Ser-Trp-Lys-Pro-Arg-Ser-Gln-Gln-Pro-Asp-Ala-Pro-Leu-Gly-Thr-Gly-Ala-Asn- Arg-Asp-Leu-Glu-Leu which is identical to an amino terminal portion of human progastrin. The sequence of peptide. B is identical to that of peptide A except it is missing the first five amino acids. If peptide A corresponds to the amino terminus of progastrin, the signal peptidase cleaves at an Ala-Ser bond.

Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R3-R14 of Aplysia

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R3-R14, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R3-R11 have been postulated to play a role in cardiovascular control, peptide II was without effect at less than or equal to 10(-4) M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.

Routine fast atom bombardment mass spectral analysis of high molecular weight peptides--atrial gland peptides from Aplysia californica.

Three peptides isolated from the atrial glands of Aplysia californica were analysed by Fast Atom Bombardment Mass Spectrometry. Survey scans over the mass range 1650 to 7500 at 500 resolution were used to locate signals for the protonated molecular ion and two subunits which result from cleavage of a single disulfide bond. A more accurate mass determination was made by accumulating scans over a narrow mass range. The amounts of sample used for each measurement ranged between 10 and 30 pmoles. Measured mass values are within 0.5 amu of calculated average molecular weights. Results illustrate the utility of the technique for accurate molecular weight determinations on limited quantities of high molecular weight peptides.

Microsequence analysis of peptides and proteins: IV. Structural studies on human leukocyte interferons

Abstract The sequence of the tryptic peptides of three major species of human leukocyte interferon was determined by microsequencing procedures. The peptides were aligned by comparison with the amino acid sequences predicted by the DNA sequences of recombinants containing leukocyte interferon-coding inserts. In addition, extended NH 2 -terminal amino acid sequences of two human leukocyte interferons produced in Escherichia coli by recombinant DNA methodology are also reported. This report demonstrates application of microsequencing methodology to low nanomole and subnanomole amounts of proteins and peptides of biological interest.

Preparation of Peptides and Proteins for Sequence Analysis at the Low Nanomole to Subnanomole Level by Reverse-Phase High-Performance Liquid Chromatography: Results for Cytochromes P450 and Fibronectin

A number of key developments have led to the routine microsequence analysis (low nanomole to subnanomole range) of peptides and proteins in a number of laboratories. In spinning cup microsequence analysis, the developments were improved instrument design and performance (1–4), better methods for solvent and reagent purification (2, 5), automated conversion of amino acid anilinothiozolinone to phenylthiohydantoin (PTH) derivatives (6), high sensitivity separation and quantitation of PTH derivatives by reverse-phase HPLC (2, 7–9), and retention of µg amounts of proteins and peptides in the spinning cup with polybrene (2, 10). The ability to perform routine microsequence analysis on µg amounts of peptides and proteins has led to structural studies on relative rare substances which possess important biological properties. In our laboratory we have used this methodology to obtain structural information on human leukocyte and fibroblast interferons (11–15) and on bovine adrenal opioid peptides (16–18). Over the past two years these studies have led us to consider another critical aspect of microsequence analysis, namely the compatibility of sample preparation with microsequence analysis.