Quantitation of collagen in polyacrylamide gels by fluorescent scanning of MDPF1-labeled proteins: An improvement over densitometric scanning of gels stained by coomassie blue

Purified α chains of collagen were made fluorescent by coupling with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) and then electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. The migration of MDPF-labeled collagen was similar to unlabeled collagen α chains except that MDPF-α1(I) migrated closer to MDPF-α2. The area under the peaks recorded from fluorometric scanning of MDPF-labeled α1(I), α2, and α1(III) was linear from 10^?5 to 10^?8 g. The standard curves for the three α chains were similar. The results from nonreplicate determinations had ±6% SE. This method is an improvement over staining with Coomassie blue. It has a greater sensitivity, peak area is independent of migration distance, has a wider range of linearity, and permits observation of bands during electrophoresis with quantitation immediately after electrophoresis.     

Gel electrophoresis of fluorescent labeled cyanogen bromide cleavage products at the submicrogram level.

A simple method is described for visualizing submicrogram amounts of the cyanogen bromide cleavage products of proteins on sodium dodecyl sulfate-polyacrylamide gels. The peptide fragment mixture is conjugated with the fluorogenic reagent 2-methoxy-2,4,-diphenyl-3-(2H)-furanone prior to electrophoresis. The fluorescent peptide bands are visible under ultraviolet light, thus avoiding the need for fixation and staining. The determination of the structural homology of two immunologically related proteins is presented to illustrate this methodology.     

Fluorescent labeling of proteins. A new methodology

A new reagent, 2-methoxy-2,4-diphenyl-3(2$\text{H}$)-furanone (MDPF) has been utilized for the fluorescent labeling of proteins. MDPF, which is nonfluorescent, reacts with primary amino groups to form fluorescent $\text{N}$-substituted 3,5-diphenyl-5-hydroxy-2-pyrrolin-4-ones. Antibodies labeled with MDPF afford intense immunofluorescent staining.     

Estimation of molecular weight by polyacrylamide gel electrophoresis using heat stable fluorophors

The utilization of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) as a fluorescent label for proteins in SDS polyacrylamide gel electrophresis is described. The procedure is very sensitive and detects disks containing as little as 1 ng of protein. The fluorescence signal is linear with concentration of protein to 500 ng. Emphasis is placed on the use of MDPF-labeled proteins as standards for the estimation of molecular weights of fluorescamine-labeled proteins. Data are provided which demonstrate the need for only three standard proteins in the molecular weight curve. Finally, the relative mobilities of proteins from nonreplicate determinations exhibited an average variation of only 3.9%.     

Comparative study of different fluorescent dyes for the detection of proteins on membranes using the peroxyoxalate chemiluminescent reaction

We have previously shown that the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2) chemiluminescent reaction in acetone can be used for the detection of proteins labeled with the fluorescent reagent 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. To improve this method, in this work we have designed and constructed a cell that allows us to perform this chemiluminescent reaction on PVDF membranes with a homogeneous distribution of the reagents. Using this cell we have examined the analytical properties of several recently developed fluorescent protein dyes chemically different from MDPF. We have found that the metal chelate dye SYPRO Ruby can also be excited by the high-energy intermediate produced in the TCPO-H(2)O(2) reaction.     

A new assay for proteases using fluorescent labeling of proteins

A simple assay for proteases based on the fluorescent labeling of insoluble proteins (fibrin) or of soluble casein by 2-methoxy-2,4-diphenyl-3(2H)furanone has been developed. Fluorescence of the liberated peptide-fluorophors resulting from enzymatic hydrolysis is easily measured in the supernatant after separation of the unreacted fluorescent fibrin by centrifugation or from unreacted casein-fluorophor by acid precipitation. Nanogram quantities of trypsin, chymotrypsin, and elastase can be measured.     

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.     

The possible involvement of 3-methoxy-2(5H)-furanone in the etiology of Narthecium asiaticum maxim. associated nephrotoxicity in cattle

Two samples of Narthecium asiaticum Maxim leaves collected in Japan were found to contain 103 microg(-1) and 160 microg g(-1) dry matter of 3-methoxy-2(5H)-furanone respectively. 3-Methoxy-2(5h)-furanone was suggested to be the toxic principle of N. asiaticum causing nephrotoxicity in cattle in Japan. Two other furanones, which are thought to be non-toxic, were also isolated from the two samples. These were 4-methoxy-2(5H)-furanone and 5-hydroxy-4-methoxy-2(5H)-furanone.     

Isolation and characterization of 3-methoxy-2(5H)-furanone as the principal nephrotoxin from Narthecium ossifragum (L.) Huds

The principal substance in Narthecium ossifragum (L.) Huds, responsible for the nephrotoxic effects on cattle, moose, goats and other ruminants has been isolated and identified by X-ray crystallography as 3-methoxy-2(5H)-furanone. The Fourier-transform infra-red, 1H and 13C nuclear magnetic resonance, and mass spectra are also given. The concentration in four different batches of plant material varied from 113 to 344 microg g(-1) (wet weight). Extracts of N. ossifragum and fractions derived from them, including purified 3-methoxy-2(5H)-furanone, were each dosed intraruminally, to young goats. 3-Methoxy-2(5H)-furanone of 99.9% purity (15 mg kg(-1) live weight) caused increased concentration of creatinine in serum within 2-3 days, typical of kidney damage caused by N. ossifragum, while toxic effect was obtained down to 4 mg kg(-1) live weight with less purified material (> or = 95%). Toxic effect was also obtained with synthesized 3-methoxy-2(5H)-furanone (30 mg kg(-1) live weight). The isomer 4-methoxy-2(5H)-furanone, detected in some of the batches of the plant material, was not toxic when dosed at 60 mg kg(-1) live weight.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

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.     

Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting

Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.     

New automated fluorometric peptide microassay for carnosine in mouse olfactory bulb

A procedure for assaying peptides at the picomole level in tissue extracts has been developed and used to measure the dipeptide carnosine in mouse olfactory bulb. In this procedure the tissue extract is reacted with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), and the resultant fluorophors are resolved on a high performance reverse-phase column. Quantitation is performed in a filter fluorometer equipped with a flow cell. Carnosine was found to be present at a level of 1.93 ± 0.44 nmol/mg of tissue (mean + SD of 11 samples), in agreement with previous findings by other methods.     

Separation of collagenase and a metal-dependent endopeptidase of rat uterus that hydrolyzes a heptapeptide related to collagen.

1. The synthetic peptide, 2,4-dinitrophenyl-L-Pro-L-Leu-Gly-L-Ile-L-Ala-Gly-L-Arg-amide (DNP-peptide) was tested as a potential substrate for uterine collagenase. Rat uteri were homogenized and the insoluble fraction was extracted at 60 degrees C to obtain collagenase. The extracts were chromatographed on Sephadex G-150 to yield two peaks of DNP-peptide hydrolyzing activity. Peak I was completely inhibited by EDTA and had a molecular weight greater than 100 000. Peak II was inhibited about 90% by EDTA and had an apparent molecular weight of about 70 000. 2. Peak II coincided closely, but not exactly, with the peak of collagenase activity. It differed from collagenase in heat stability, binding properties on CM-Sephadex and failure to display latency. 3. Peak II represents a new endopeptidase activity. It has a pH optimum of 7 and it cleaves the DNP-peptide at the Gly-Ile and, possibly, the Leu-Gly bond. 4. The DNP-peptide is not a satisfactory substrate for the assay of impure collagenase preparations nor does it inhibit the action of collagenase on collagen substrate when added in 30-fold molar excess.     

A convenient fluorescent assay for vertebrate collagenases

A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors.     

Spectrophotometric determination of fluorophor, protein, and fluorophor/protein ratios in fluorescamine and MDPF fluorescent antibody conjugates.

Goat and rabbit gammaglobulin were labeled with the recently introduced fluorogenic reagents fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (IV), or MDPF, and the molar absorption coefficient at 385 nm of the bound fluorogens was determined indirectly via unbound, hydrolyzed reagent. Based on absorption measurements at 280 and 385 nm, the content of bound fluorogen (F) and protein (P) and the $\text{F}\text{P}$ ratio in dilute solutions of antibody conjugates may conveniently be determined with a nomograph or calculated with individual formulas. The molar $\text{F}\text{P}$ ratio of our preparations was normally between 4 and 14, but it may rise to a maximum of about 20 without loss of antibody specificity. Fluorescence is proportional to absorption (fluorophor concentration).MDPF and fluorescamine offer a number of advantages over conventional fluorophors for antibody labeling, and since the method may be adapted to other proteins, it is to be expected that the method described here will be widely applicable.     

Chemical modification of gastric microsomal potassium-stimulated ATPase

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K⁺-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K⁺-stimulated ATPase and H⁺-transporting activities of the gastric microsomes, while the Mg²⁺-ATPase was not affected. Half-maximal inhibition occurred at about 3 μg 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent K_m for ATP protection was about 50 μM. Nucleotide selectivity for protection was ATP > ADP > ITP > GTP > CTP > AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the M_r 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the M_r 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg²⁺-ATPase and K⁺-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K⁺-ATPase and may be actually at the ATP binding site.     

A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge-coupled device after staining with silver or labeling with fluorophore.

A multiple mini two-dimensional electrophoretic method which results in three two-dimensional protein spot patterns being positioned side by side in an individual gel has been developed. Preparation time has been minimized by employing disposable capillary tubes for the isoelectric focusing gels and reducing the number of second-dimensional gels required. Commercially available vertical slab units were used for the second-dimensional electrophoresis. The protein spot patterns were visualized either by staining the second-dimensional gel with silver or fluorescently labeling the focused proteins while present in the isoelectric focusing gel and subsequently electrophoresing them into the second-dimensional gel. The fluorescently labeled second-dimensional gel was imaged while still present in the glass mold immediately following electrophoresis. Two fluorophores were compared: 2-methoxy-2,4-diphenyl-3(2H)-furanone and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein hydrochloride. A rapid imaging system based on a cooled charge-coupled device was used to view both the silver-stained and fluorescently labeled two-dimensional spot patterns. The sensitivity of detection of protein spots in the mini two-dimensional gels was similar for the two types of fluorescently labeled gels and the silver-stained gels.     

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryh?nen et al. (L. Ryh?nen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.