Andrew M. Tometsko

Fluorometric method for quantitative determination of free amine groups in peptide-containing Merrifield resins.

Abstract A highly sensitive fluorometric method for determining free amine groups on the Merrifield peptide resin is presented. The method, as described, is capable of quantitatively analyzing 1.5 nmoles of free amine on 7 mg of resin, and is currently being used to follow the kinetics of amino acid incorporation into resin bound peptides. In addition the method should be applicable to monitoring the free amine content of resins during solid-phase synthesis and/or solid-phase Edman degradation of peptides.

Determining the availability of activated amino acids for solid phase peptide synthesis

Abstract Data concerning the reaction characteristics of each amino acid are of fundamental importance for providing optimum reaction conditions and high yields during polypeptide synthesis. Loss of activated amino acid as a function of time would have a pronounced effect on the efficiency of synthesis. An assay procedure is described which quantitatively determines the availability of DCC activated amino acids as a function of preincubation time. The assay was employed to follow the inactivation of t-BOC.Leu(DCC) and indicated a significant loss of activated amino acid.

Quality control during peptide synthesis. I. Quantitative monitoring of resin bound amines

Abstract A fluorescent assay procedure, capable of determining 99.9% coupling (free amine remaining) during solid phase synthesis is described. The maximum free amine content of completely deblocked resin usually ranges from 1000 to 5000 nmoles 10 mg sample, and at 99.9% coupling, the resin would contain 1–5 nmoles of free amine. This assay procedure is able to quantitatively analyze 0.1 nmole of free amine.

EVALUATING THE STABILITY AND REACTIVITY OF A LIGHT-SENSITIVE PROBE BY ENZYME ANALYSIS

Abstract— Enzymes can provide well defined functional targets for determining the availability and reaction characteristics of light sensitive probes. Trypsin and chymotrypsin are inhibited by flash photolysis with a photoprobe, 4‐fluoro‐3‐nitrophenylazide (FNPA). Inhibition is competitive in the dark, and non‐competitive (irreversible) following photolysis of the FNPA enzyme solution. Photoinactivation is dependent upon the concentration of FNPA, the flash rate, and the time of photolysis. The enzymes can be protected from photolytic inactivation with FNPA if photolysis is carried out in the presence of 2,4‐dinitrophenol or 4‐fluoro‐3‐nitroaniline which compete with FNPA for binding sites. The photo‐probe is effective over a wide pH range (i.e. pH 2–11), and provides a sensitive tool for probing conformational and charge adjustments which increase or decrease the affinity of the binding site. Chymotrypsinogen was also sensitive to photolysis, indicating that FNPA binding sites are present in the zymogen structure.

Resin probe analysis: II. Amino acid coupling reactions

Abstract Resin probe analysis has been employed to evaluate the availability of dicyclohexylcarbodiimide (DCC)-activated amino acids, the relationship between coupling time and reaction yield, and the influence of triethylamine (TEA) concentration on peptide bond formation. Results are presented for five amino acids which indicate that the coupling reactions plateau within 5 min, and no significant increase in yield is observed for longer incubation times. Large decreases in coupling yield (70–90%) were observed at concentrations of TEA above 0.01 m . Inactivation appears to be dependent in part upon amino acid structural features. In the absence of TEA, DCC-activated t-butyloxycarbonyl (Boc)-glycine was stable in the activated state for hours. peptide bond formation showed little or no amino acid concentration-dependence in the range of 0.01–0.04 m . Resin probe experiments provide quantitative data on reaction progress and factors that influence the availability and reactivity of activated amino acids.

Computer approaches to protein structure III. Transformation of atomic coordinates

Abstract The study of polypeptide and protein structure with computer-generated molecular models provides numerous advantages over conventional framework molecular models. One notable advantage is the dynamic nature of the computer model. Computer programs, discussed in this report, transform the coordinates of atoms of a peptide or protein, resulting in the rotation of the molecule about a fixed reference point in the x - y or y - z planes. The angle of rotation is increased by 5° after each structure is drawn on the cathode ray tube. As a result, for one complete revolution of the molecule, the computer draws 72 different images and the cathode ray tube is photographed to provide a permanent record of each structure.

Computer approaches to protein structure. IV. Control tapes for polypeptide synthesis

Abstract The use of tape reading instruments for the solid phase synthesis and/or solid phase Edman degradation of polypeptides and proteins has created a need for fast and reliable methods of generating control tapes. A computer program is described which generates a single control tape for the simultaneous synthesis of two proteins. With this program, the investigator specifies the desired amino acid sequences and the computer, through the program, generates a command array for the complete synthesis of the proteins. As a result, the control tape for weeks of instrument operation, and therefore weeks of chemical synthesis, is available in minutes. Knowledge of computer programming is not essential to generate control tapes with the described program.

Computer approaches to protein structure. VI. A multiple option model building program

Abstract Conformation analysis of proteins is complicated by the presence of thousands of atoms which often are involved in multiple interactions. As a result, complete molecular models of proteins contain a relatively high information density. Computer graphics provides a means of controlling the flow of information by permitting the construction of large numbers of models containing different amounts of structural detail. The multiple option FORTRAN IV program described in this report permits the observer to focus on specific interactions and/or regions of a protein model with all or only specified amino acid side chains present on the backbone. The models can be readily translated, rotated, and adjusted in size, and constructed with specified visibility limits. Minor adjustments in the basic program provides complete families of stereoscopic pairs for three dimensional analysis of the models.

Computer approaches to protein structure: V. viewing models of proteins from the inside☆

Abstract The dynamic nature of computer models is discussed. In addition to varying the three dimensional position of atoms and the amount of detail presented in a given model, the computer is also able to change the position of the camera relative to the atoms of a protein model. Thus, the camera could be positioned outside of the model (the usual case) or it could be moved inside. From inside the protein model, internal atoms are immediately before the camera and interactions can be studied at close range. Atoms behind the camera are out of sight but could be brought into view by rotating the structure. The computer program discussed in this report constructs the peptide backbone of a protein, amino acids that contain ring systems, and the heme prosthetic group (myoglobin). In addition, the program varies the position of the camera and the positions of the atoms to provide unique views of protein models. Methods also are described for preparing animated and stereoscopic three dimensional presentations from the CRT models.