Search

A method for the determination of the composition of a conjugate between two different proteins by amino acid analysis followed by least-squares evaluation of the concentration ratio of the two components is presented. The method is based solely on calculations and avoids the use of labeled residues. A computer program, written in BASIC, is also presented to perform the calculations.

A semiautomated system for spectrophotometric measurement of enzyme activity is described. In comparison to a 1-ml reaction volume monitored continuously by a conventional spectrophotometer, this system requires 1/10 to 1/100 the volume of sample, and 1/8 to 1/4 the time for measurement and computation of 96 enzyme assays. The system hardware consists of a 96-well platereader interfaced to a personal computer. Absorbances of 96 reactions are measured at timed intervals. These data are transmitted electronically from the platereader to the computer through the modem port using a modem program. The reaction rates are computed from the timed absorbance readings using a spreadsheet program. Three enzyme assays are presented, but the method has been used for several other assays and is applicable to many spectrophotometric rate assays. Many laboratories currently possess one or both of the two major components of the relatively inexpensive system described.

Determination of isoenzymes by a method of differential substrate levels is fast and simple and usually does not require prior separations. It can often be adapted to existing total enzyme assays. Application has been made to mixtures of purified tissue fractions of human l -aspartate aminotransferase using a slightly modified commercial assay kit. Colorimetric rate measurements, accurate to 2–3%, allowed resolution of components to an accuracy of 6–9%. A generalized calibration scheme was devised which allowed standardization either by mixed or pure isoenzyme standards. The concept of an “error multiplier” has been developed for prediction of the most favorable operating conditions and for estimating the expected accuracy of the simultaneous determination. Curves are presented that provide a direct means of selecting optimum conditions for two-component isoenzyme systems. The computations are well suited to computer methods. However, in the event a manual data-reduction procedure is desired, a simple graphic scheme has been presented.

A reliability study was conducted to compare a new, computerized method of measuring the areas under chromatographic curves with the traditional hand method of determining the areas. Three runs of 30 separate injections of standard inorganic phosphate were made using the chromatographic and dry-ashing procedure developed in the laboratory of the Department of Pharmacology of the USC School of Medicine. Results indicate that there is no statistically significant difference between the traditional method and the new computer method for amounts greater than 10 nm, and the computer method is more precise for amounts less than 10 nm. Hence, great savings in time and an increase in precision can be made by using a computer for these analyses.

A microcomputer-controlled data acquisition system for spectrophotometric enzyme kinetics measurements has been assembled. The system uses an Apple IIe computer which is interfaced to the binary coded decimal output of a Gilford spectrophotometer. No analog-to-digital converter had to be purchased. A BASIC program which collects timed absorbance readings every 500 ms, plots the data in real time, performs a linear regression of the data to measure the reaction rate, and calculates the enzyme activity concentration is given in full. Details describing the interfacing of the computer to the spectrophotometer are presented which will permit other laboratories to readily assemble their own systems using this hardware. Kinetic data acquired by the system are highly reproducible and agree well with data processed much more slowly by manual techniques from strip chart recordings.

Preparation of protein-specific fluorescent probes with the desired degree of fluorochrome can be greatly facilitated by a technique that combines thin-layer chromatography (TLC) with quantitative image analysis (QIA). Using TLC QIA , the investigator can determine the fluorochrome/protein ratio on-line with only a few micrograms of protein as fluorochrome is conjugated to protein. In addition, this technique allows rapid quantitation of dye noncovalently adsorbed to fluorochrome-labeled protein.