A new fluorescent microassay method for pepsin using succinyl-albumin.

A method was developed for fluorescent microassay of pepsin with a fluorescent reagent, fluorescamine, and a nonquenching substrate, succinyl-albumin. In this method hydrolysis of succinyl-albumin by pepsin at pH 2,0 was stopped by adding phosphate buffer, pH 6.1, and newly liberated amino groups in the reaction mixture were determined quantitatively by fluorescence after adding fluorescamine. Fluorescence increased linearly with 1.0 to 18 ng of hog pepsin. The assay was 200 times more sensitive than the modified micromethod of Anson [(1939) J. Gen. Phys.22, 79–89].     

A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol

An alternative primary amine assay utilizing 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione (fluorescamine) is compared with the frequently applied trinitrobenzenesulfonic acid assay as a means of estimating protein primary amine modification with cyanuric chloride attachment of polyethylene glycol (PEG). The results of the two assays are compared for three different proteins and several advantages of the fluorescamine assay are emphasized. In particular, the fluorescamine assay was found to be unaffected by free PEG up to 0.08% in the assay mixture. The fluorescamine assay can be used to measure the partition coefficients of native or PEG-modified protein in two-phase aqueous polymer systems.     

A sensitive,fluorometric method for the assay of microsomal hydroxylase: N-demethylation of p-chloro-N-methylaniline

A fluorometric method for the assay of microsomal hydroxylase activity is described. N-Demethylation of p-chloro-N-methylaniline yields p-chloroaniline, which is coupled with fluorescamine, extracted with ethylacetate, and measured fluorometrically. This method can determine low levels of N-demethylase activity.     

A sensitive fluorometric assay for reducing sugars.

A simple and rapid fluorometric assay for reducing sugars that is sensitive to the nanomolar range has been developed. The assay involves the derivatization of a given sugar with hydrazine at pH 3 to form a hydrazone, which is reacted with fluorescamine following adjustment of pH to first 9.4 and then 7.4. The amount of sugar in a sample is quantitated by measuring the fluorescence intensity at an excitation wavelength of 400 nm and an emission wavelength of 490 nm. The assay is precise and reproducible, as indicated by intra- and inter-run variations of at most 3% and 4%, respectively. In addition to reducing sugars, the assay can also be used to measure aliphatic and aromatic aldehydes, but not acetone. Compared with an existing fluorometric sugar assay, the assay reported here does not require chromatographic separation of the fluorescent derivative from unreacted fluorescamine. The assay can, however, be potentially adapted for postcolumn detection of aldehydes, reducing sugars, and hydrazones in HPLC.     

Protamine, a substrate for thrombolytic agents and enzymes of similar specificity.

Protamine, a highly purified basic polypeptide of 4000 molecular weight containing 80–85% arginine, is a useful substrate for the assay of plasmin, activated plasminogen, and enzymes of similar specificity, e.g., urokinase, coagulation factor Xa, trypsin, and thrombin, and is also an excellent secondary substrate for activator assays of urokinase and streptokinase. The assays were performed manually, and also automated procedures for continuous multiple sample analyses were used. The relative sensitivities for various plasmin-like enzymes were: trypsin > plasmin > urokinase > factor Xa > thrombin. Using protamine with manual assay procedures, the amino-terminal groups of the enzyme-degraded protamine digestion products were detected and quantitated by the colorimetric ninhydrin or the fluorometric fluorescamine procedures, and using protamine with an automated system the ninhydrin method was used. Assigning the CTA casein assay for plasmin a nominal sensitivity of 0.1 (for 0.1 CTA unit of plasmin), the sensitivities of the various assay methods were casein, automated protamine, and manual protamine with ninhydrin, 0.1; manual protamine-trichloroacetic acid with fluorescamine, 0.005; and manual protamine direct fluorescamine, 0.0005. A unit of plasmin, based on the uptake of 1 μequiv base/min during hydrolysis of 0.4% protamine sulfate under standard conditions, is equal to approximately 1.7 × 10³ RFI units or 2.9 CTA units; or, 1 CTA unit of plasmin resulted in an average uptake of 0.346 μmol of base or equivalent bonds split per minute.     

Use of fluorescamine-labeled casein as a substrate for assay of proteinases.

Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.     

Fluorometric assay of secondary amino acids

A method is described for the conversion of secondary amino acids to primary amines which can be assayed with fluorescamine (I). Secondary amino acids undergo oxidative decar?ylation when reacted with halogenating agents. The resulting imines are hydrolyzed to primary amines, which are subsequently allowed to react with fluorescamine (I) to yield fluorescent pyrrolinones (II). This reaction sequence provides an efficient fluorometric assay for secondary amino acids. Thus, the fluorescamine procedure is now applicable to the full array of natural amino acids.     

A fluorescence-based homogeneous assay for measuring activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogeneous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K(m) of 367 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.     

KDAC8 substrate specificity quantified by a biologically relevant,label‐free deacetylation assay

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme‐substrate pairs of label‐free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine‐based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady‐state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry‐based experiments and cell‐based experiments will allow the identification of specific KDAC‐substrate pairs and lead to a better understanding of the biological consequences of these interactions.     

Sensitive fluorescent determination of trypsin-like proteases

A new sensitive assay for trypsin-like proteases has been developed using as substrate protamine sulfate from herring with the amino terminal group blocked with dinitrofluorobenzene. Enzymatic hydrolysis liberated amino groups which were quantitated by measuring fluorescence after reaction with fluorescamine. Thrombin was capable of hydrolyzing this substrate at a concentration as low as 8.3 NIH units per ml. Amino acid analysis of the protamine suggests that thrombin is capable of hydrolyzing a peptide bond other than an arginyl-glycine bond. Inhibition of thrombin by n-acetylimidazole suggests a relationship between the clotting and proteolytic activities of thrombin.     

An evaluation of fluorometric proteinase assays which employ fluorescamine

The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37 degrees C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.     

Application of fluorescamine to the study of protein-DNA interactions.

The reactivity of alpha-amino groups of basic proteins towards fluorescamine is essentially abolished if salt linkages with DNA phosphate groups are formed. This observation prompted the elaboration of a very general assay which allows the determination of binding parameters for the interaction of proteins with DNA and chromatin. Protamines, labeled with fluorescamine prior to their binding by DNA appear to be useful probes to monitor the formation and nature of DNA-protein complexes.     

A fluorescamine assay for submicrogram quantities of protein in the presence of Triton X-100

The sensitivity of the fluorescamine assay is increased by hydrolysis of the protein sample in 2 n NaOH. The assay is linear from 0.125 to 10 μg of protein and can measure concentrations as low as 0.5 μg/ml. The presence of several detergents including Triton X-100 in both the protein sample (up to 10%) and in the fluorescamine assay itself (up to 0.33%) does not interfere.     

A fluorescent assay for bacterial cell wall lytic enzymes

A sensitive fluorimetric assay is described for the measurement of N-acetylmuramic acid l-alanine amidase as well as lysozyme. The method uses Bacillus subtilis cell walls labeled with fluorescamine on the free amino group of diaminopimelic acid. The method can easily detect the lytic activity of 0.02 μg of pure N-acetylmuramic acid l-alanine amidase in 30 min and of 1 μg of hen egg-white lysozyme in the same period. The method is particularly suitable for measurement of competition between various cell wall preparations for the same enzyme.     

Ribonucleotide reductase activity during the cell cycle of Saccharomyces cerevisiae

A fluorometric amino acid analyzer using fluorescamine for the assay of the full array of natural amino acids including proline on a single column is reported. The proline determination was carried out by specific introduction of a solution of N-chlorosuccinimide into the flow system. Single column fluorometric amino acid analysis was carried out in a significantly shorter time and with a sensitivity almost two orders of magnitude greater than that obtained with a commerical colorimetric ninhydrin amino acid analyzer.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine–threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [³²P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and V_max of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R ² = 0.956.     

Asymmetric labeling of amino lipids in liposomes

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H⁺ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin K⁺ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents.When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching.These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surfaces, monitor vesicular fusion, and assess liposomal structure.     

A recording viscometer for assaying mammalian collagenase.

A recording viscometer for monitoring the action of mammalian collagenase on soluble collagen is described. For this system, where only one peptide bond is cleaved per subunit, it is shown theoretically that the decrease in viscosity is proportional to the fraction of molecules cleaved. Experimental confirmation was obtained by parallel monitoring of hydrolysis by using the fluorescamine assay of liberated amino groups. The initial velocity of reaction is proportional to substrate concentration and enzyme concentration.