Search

Peptidases, including chymotrypsin, thermolysin, trypsin, V8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7-8 and 40-50 degrees C. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic alpha-, beta-, and gamma-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides' identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current 10-40 times higher than that of the [M + 2H]2+ ion for unhydrolyzed beta-endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol.

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.

A rapid method using orthophthaldialdehyde and mercaptoethanol to form a fluorescent adduct with released α-amino groups was developed and characterized as a means for assaying peptidase activity on physiological peptide substrates. A typical assay consisted of withdrawing a 10-μl aliquot of the substrate-enzyme solution at various time intervals, which was then added to 3 ml of reagent; the fluorescence was measured relative to 0.1 μg/ml quinine sulfate in 0.1 n H 2 SO 4 following a standardized 2-min reaction time. The assay can be performed either in the presence or absence of sodium dodecyl sulfate; addition of the detergent can be a convenient means for stopping the reaction. Contrary to previous suggestions, addition of detergent was found to slightly decrease fluorescence of dipeptide adducts with this reagent rather than causing a substantial enhancement. However, enhancement was observed for cases where the spacing between isoindoles or between isoindole and indole was similar to that for the disubstituted derivative of lysine. This method was characterized by assaying porcine intestinal peptidase on the peptides Leu-Gly, Gly-Gly, Ala-Gly, and Gly-Leu-Tyr. Using an immobilized form of this enzyme, excellent agreement was found between amino acid determinations and fluorescence-derived calculated values for the fraction of bonds hydrolyzed for dipeptide substrates. It was shown that the fluorescence ratio (F t − F 0 ) (F c − F 0 ) corresponds to the fractional hydrolysis of dipeptides where F t , F 0 , and F c are the relative fluorescences at time t , time zero, and complete hydrolysis, respectively.

A rapid, sensitive fluorimetric assay has been devised for available (nonconjugated) lysine residues of proteins. The ϵ-amino groups of a protein (1- to 40-μg sample) react with o-phthalaldehyde to produce a fluorescent adduct of the protein, whose fluorescence intensity, corrected for the contribution of N-terminal amino groups, is a linear function of lysine content (r = 0.98 for linear regression of 13 protein and peptide standards with lysine content ranging from 0 to 92 mol per 105 g protein). The reaction, which is specific for free amino groups, approaches completion within 1 min at 21°C ( t 1 2 = 6–12 s ) and requires no preliminary heating or hydrolysis of the sample. Triplicate analyses of samples as small as 1 μg gave an accuracy of 10% or better. The method can also be adapted to analysis of unknown protein samples if conditions are used to eliminate from analyses those samples for which the contribution of N-terminal amino groups to the total fluorescence is not insignificant. Consequently, this method by virtue of its simplicity and sensitivity can be used for routine nutritional analyses for available lysine.