METHODS FOR ASSAYING PROTEINASES AND PROTEINASE INHIBITORS

Abstract: Methods are provided for determining proteinases and proteinase inhibitors based on the ability of phenanthradine coumpounds to intercalate between the bases of DNA with a resulting increase in their fluorescence. The DNA is complexed with a protein to block binding of the phenanthradine. Proteinase activity degrades the complexed protein, exposing DNA to phenanthradine binding accompanied by increased fluorescence.

Description: METHODS FOR ASSAYING PROTEINASES AND PROTEINASE INHIBITORS This invention relates to methods for measuring proteinase enzymes and to methods for measuring proteinase inhibitors. Proteinases and their inhibitors have become the subject of explosive research interest recently, since they control very important processes; involvement of proteinases and their inhibitors has been shown in the regulation of many fundamental biological processes, including regulation of virus infectivity, λ phage development, pathogenicity of bacteria, healing in plants and tumour invasiveness. Their importance in human diseases is well established, as proteinases or their inhibitors have been implicated in hypertension, Alzheimer disease and lung disease. Proteinases and/or their inhibitors also play a role in blood clotting (antithrombin III deficiency) , hereditary angioedema (CjQ inhibitor deficiency) , acute lymphocytic leukemia (neutral endopeptidase which cleaves endorphins shows elevated levels) and in metastatic tumours. Proteinases are also implicated in parasitic diseases such as malaria and in AIDS. o^-antiproteinase (or α,-antitrypsin) is one of the major an iproteinase inhibitors in human serum. It inhibits all serine proteinases, to which it binds with very high affinity, and its major function is probably the inhibition of neutrophil elastase in the lung and in the liver, to prevent destruction of the matrix proteins of connective tissue. Genetic deficiency of α j -antiproteinase is a proven cause of early onset emphysema and possibly liver diseases. Inactivation of ct j -antiproteinase by cigarette smoking has been proposed as a causal factor of emphysema in smokers. There is therefore a need for convenient, reliable and sensitive methods for measuring proteinases and their inhibitors in many clinical situations. The assays for proteinases available to date can be divided into three groups. (i) Activity can be measured by hydrolysis of synthetic substrates that release chromogenic compounds e.g. nitroanilides, or fluorogenic molecules e.g. 6-aminoguinolone (Tuppy et al. (1962) Hoppe-Seylers 2. Physiol. Che ., vol. 329, pp. 278 - 288) . These assays are rapid and can be very specific. Sensitivity is usually around 1 μg of proteinase. (ii) Insoluble proteins, as substrates, give rise to hydrolysis products in soluble forms. Substrates are azo-proteins and radiolabelled insoluble proteins, e.g. collagen or elastin (Phillips et al. (1989) J. Clin. path. Vol. 37, pp. 329 - 331; Truden et al. (1985) A er. J. Path., vol. 121, pp. 165 - 175; Takahashi et al. (1973) Biochim. Biophys. Acta, vol. 327, pp. 138 - 145). Sensitivity can be in the nanogram range, but substrates are usually difficult to prepare, and reaction times can be very long. (iii) The concentrations of proteinases (and their inhibitors) may be determined with mono or polyclonal antibodies, using a variety of detection systems such as ELISA, (Voller et al. (1976) Bull. WHO, vol. 35, pp. 55 - 64), nephelometry, or other enzyme- coupling procedures (Hatsusako et al. (1989) Clin. Chim. Acta, vol. 181, pp 19 - 26) . These latter assays are very specific and sensitive (0.05 ng) but do not give any information on proteinase activity and are not intrinsically reproducible as precipitation can vary from one situation to another. The methods available for measuring C-i-antiproteinase in human serum are basically of two types: i) Assays that measure the amount of protein with specific antibodies, such as radial im unodiffusion (RID) , and rate nephelometry (NIA) . ii) Assays that measure the inhibitory activity of serum on a known amount of proteinase, usually trypsin. The two types of assay measure very different parameters and, although correlation between methods is in general good, some discrepancies have been reported, especially in patients with certain pulmonary diseases. Since activity, rather than protein concentration, determines the action of j -antiproteinase, functional tests may detect deficiency conditions, (e.g. inactivation or point mutation at the binding site) that may escape detection with the immunological methods. There remains a need for an inexpensive, rapid, simple but reproducible method of measuring proteinases and their inhibitors for both clinical and industrial use. Summary of Invention A method is provided for determining the activity of a proteinase enzyme comprising: providing a DNA/protein complex comprising a protein degradable by the proteinase; contacting the DNA/protein complex with the proteinase to permit degradation of the protein; contacting the DNA exposed as a result of the protein degradation with a phenanthradine compound which shows increased fluorescence on intercalation with DNA; and measuring the resulting increase in fluorescence. A method is provided for determining a proteinase inhibitor by comparing the activity of a proteinase inhibitable by the inhibitor in the presence and absence of the inhibitor comprising providing a DNA/protein complex comprising a protein degradable by the proteinase; contacting the DNA/protein complex with the proteinase in the presence or absence of the inhibitor; contacting the DNA exposed as a result of degradation of the protein by the proteinase with a phenanthradine compound which shows increased fluorescence on intercalation with DNA; measuring the resulting increase in fluorescence; and determining the inhibitor from the reduction of fluorescence increase in the presence of the inhibitor compared to the fluorescence increase in the absence of the inhibitor. A kit is provided for determining the activity of a proteinase enzyme comprising: (a) a DNA/protein complex comprising a protein degradable by the proteinase; and (b) a reagent comprising a phenanthradine compound which shows increased fluorescence on intercalation with DNA. A kit is provided for determining a proteinase inhibitor comprising: (a) a proteinase enzyme inhibitable by the proteinase inhibitor; (b) a DNA/protein complex comprising a protein degradable by the proteinase; and (c) a reagent comprising a phenanthradine compound which shows increased fluorescence on intercalation with DNA. Summary of Drawings The invention, as exemplified by preferred embodiments is described with reference to the drawings in which: Figure 1 shows binding of whole histone to calf thymus DNA. Figure 2 shows kinetics of the following proteinases measured by the method of the invention: pancreatic elastase (Δ) , proteinase K (O) , trypsin (•) subtilisin (l),pronase (D) and collagenase (A). Figure 3 shows kinetics of proteinase K activity; (a) by non-linear curve fitting program and (b) in linear form. Figure 4 shows relationships between initial rates of proteinase activity and amount of proteinase added. Figure 5(a) shows proteinase inhibition by PMSF; Figure 5(b) shows proteinase inhibition by serum. Figure 6(a) shows kinetics of elastase activity; Figure 6(b) shows kinetics of inhibition of elastase activity by human serum. Figure 7 shows fractionation of human serum elastase inhibitory activity on Sephadex G-100. Figure 8(a) shows the kinetics of elastase activity measured by the method of the invention; Figure 8(b) shows elastase activity as a function of elastase concentration; Figure 8(c) shows titration of elastase inhibitory activity of human serum. Figure 9 shows correlation between ethidium fluorescence assay and rate nephelometry. Detailed Description of the Invention The present invention provides a simple, rapid, reproducible and inexpensive method for measuring the activity of proteinase enzymes and also a method for measuring anti-proteinase inhibitors. The methods of the invention make use of the fact that certain phenanthradine compounds show an increase or enhancement of their fluorescence when they intercalate between the base pairs of double-stranded DNA. Such phenanthradine compounds include ethidium, propidium and bisethidium. The phenanthradine compounds are supplied as a soluble salt, ethidium bromide being preferred. Ethidium fluorescence increases 25 fold when it intercalates between base-pairs in double-stranded DNA. Using near-saturation concentrations of ethidium bromide (0.5 μg of DNA) , the fluorescence is directly proportional to the concentration of DNA. When a protein binds to DNA, so as to block phenanthradine intercalation, the increase of fluorescence resulting from intercalation is not seen. If DNA-binding proteins are added to DNA/phenanthradine complexes, the phenanthradine is displaced with a resulting loss of fluorescence. For example, the affinity of histones for double- stranded DNA is sufficiently high that all histones added to a solution of DNA are bound, and no free histones are present, until binding sites are saturated. If histones are added to a DNA/ethidium complex, the fluorescence of ethidium bromide drops directly proportionally to the amount of histones added. Proteins which bind to DNA and block phenanthradine intercalation include histones, pRecA and T4 phage pgene 32; other DNA binding proteins which block phenanthradine intercalation will be known to those skilled in the art. When a DNA/protein complex is contacted with a proteinase enzyme, the protein bound to the DNA is degraded by the proteinase, exposing the DNA and allowing increased intercalation of a phenanthradine such as ethidium, with a resulting increase in fluorescence. In accordance with one embodiment of the invention, a method is provided for determining proteinase activity by contacting a DNA/protein complex with the proteinase to be measured, in the presence of ethidium bromide, and following the increase of ethidium fluorescence as bound protein is degraded, DNA is exposed and ethidium becomes bound to the DNA. As described in Example 2, proteinase activity may be determined very rapidly by carrying out the proteinase digestion in fluorescence buffer in fluorometer tubes and directly following the increase of fluorescence in these tubes. These conditions may not be optimal for many proteinases, so that some degree of sensitivity is sacrificed in favour of speed and convenience. This assay provides a convenient screening method when large numbers of samples must be tested rapidly. In accordance with a preferred embodiment of the invention, a method is provided for determining proteinase activity by contacting a DNA/protein complex with the proteinase to be measured, allowing protein degradation by the proteinase occur, removing aliguots of this digestion mixture as required, contacting these with ethidium bromide and following the increase of ethidium fluorescence as ethidium is taken up by the DNA, as described in Example 6. This method provides great sensitivity and allows detection of only a few nanograms of some proteinases. The method has been used to detect the proteinase activity of a crude cell extract derived from 10 4 Vero cells. The proteinase reaction can be expected to follow first order kinetics: F t = P«(l-e*) (1) where F t and F^ are the fluorescence at time t and the maximum fluorescence respectively, after subtraction of the background fluorescence of the DNA/protein complex in the presence of ethidium bromide. The initial rate of the reaction is: dF/dt = kF^ The initial rate can be exactly calculated by non- linear fitting of equation (1) using a computer program as described by Duggelby, R.G. (1981) Anal. Biochem. , vol. 110, pp. 9 - 18, or after graphic estimation of F^, equation (1) can be rearranged in linear form: as described by Morgan et al. (1979) Nucl. Acids Res., vol. 7, pp. 571 - 594. For practical purposes, near- linear kinetics can be obtained by using low amounts of proteinase for an appropriately short time. The protein of the DNA/protein complex is selected in accordance with the specificity of the proteinase to be measured. For example, histone proteins such as whole histone, histone 1 and histone 4 are degraded by a wide variety of proteinases, though sensitivity will depend on the particular proteinase. Histone proteins are the preferred proteins for determination of serine proteinases such as trypsin, chymotrypsin, elastase and proteinase K. For any proteinase which shows low sensitivity with DNA/histone complexes, it will be known to those skilled in the art to substitute a DNA-binding protein more susceptible to degradation by that proteinase. In accordance with a further embodiment of the invention, a method is provided for determining a proteinase inhibitor by means of the reduction, in the presence of the inhibitor, of the fluorescence increase otherwise seen when a proteinase degrades the protein of a DNA/protein complex and the exposed DNA is contacted with a phenanthradine such as ethidium. When the stoichiometry and the affinity of the inhibitor are known, the concentration of the inhibitor can be calculated on the basis of the ratio I, where and: Q = activity of a known amount of proteinase without the inhibitor A r = activity of the same amount of proteinase with the inhibitor For high affinity inhibitors, one can assume that all the inhibitor added will be bound to the proteinase and, in the case of a 1:1 stoichiometry, the molarity can be immediately calculated: moles of inhibitor = (g of proteinase/MW) x I Example 9 describes the application of this embodiment of the invention to the measurement of human serum αi j -antiproteinase. The method is rapid and inexpensive yet very sensitive, being able to detect the Oj-antiproteinase activity of 0.05 μl serum (about 10 ng C-i-antiproteinase for a normal individual) making it suitable for measuring αj-antiproteinase activity in clinical samples of low abundance such as pulmonary secretions or bronchial washes. The methods of the invention are rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow precise quantitation of nanogram amounts of broad-specificity proteinases and their inhibitors. The sensitivity of the methods of the invention is such that proteinases and their inhibitors may be measured in biological fluids such as serum, cell lysates, cell and tissues extracts, pulmonary secretions, bronchial washes and the like. The following examples are illustrative only and not intended as limiting with respect to the present invention. Ethidium bromide, whole histones (Types II AS) , histone 1, bovine serum albumin (BSA) , collagenase and proteinase K were purchased from SIGMA, trypsin, pronase and subtilisin from Boehringer-Mannheim and dithiothreitol (DTT) from ICN Biomedicals. Histone 4 and porcine pancreatic elastase are also available from commercial sources. Example 1 Since histones bind to DNA with high affinity, the amount of histones bound to DNA is expected to be directly proportional to the amount added, until all the sites on DNA are saturated. Serial additions of whole histone were made to a fluorometer tube containing approximately 1 μg calf thymus (CT) DNA in 1.2 ml of fluorescence buffer (0.5 μg/ml ethidium bromide, 10 mM Tris, 1 mM EDTA, pH 8.0). Fluorescence readings were taken after 5 minutes at room temperature to allow histone binding to reach equilibrium. Ethidium/DNA fluorescence was measured as described by Peng at al. (1989), J. Virol, vol. 63, pp. 2550 - 2556. Emission was at 600 nm and excitation at 525 nm. Figure 1 shows the titration curve of whole histone binding to calf thymus (CT) DNA, measured as drop in ethidium fluorescence at pH 8.0. As shown, the fluorescence decreased linearly with the amount of histones added. The average drop of fluorescence for 11 independent additions of histones was 15.6± 1.1 FU/μg of whole histones. The binding reached equilibrium within 5 minutes from the addition of histones, at room temperature. The binding, as judged by the fluorescence level, was stable for at least 2h. The DNA/histone complex was also prepared in concentrated solution, by mixing appropriate amounts of histones and CT DNA in the absence of ethidium bromide and incubating at 37°C for 5 min to reach maximum binding. Under these conditions, the drop of fluorescence became 23 FU/μg of histones. The accuracy of proteinase reactions carried out in concentrated solution was not compromised, provided the samples were read immediately after addition to the fluorescence buffer. The titration of binding was similar but fluorescence tended to increase 20-50% in about 5 minutes after the substrate was transferred to 1.2 mL of fluorescence buffer for fluorescence readings. Under these conditions of rapid binding in the absence of salt, histones do not form nucleosomes. Histone/DNA complexes in concentrated solution were stable at 22°C for a maximum of 10 h. Longer incubations or freezing resulted in irreversible precipitation of the substrate. In order to test the method in buffers commonly used for lysing cells or in protein purifications, we investigated the binding of histones to DNA in the presence of various salts and detergents. The histone/DNA complex tolerated a maximum of 0.02% SDS and 0.04% lauryl-sarσosine. Concentrations of 2% Triton, 150 mM NaCl, 10 mM MgCl2, lOmM CaC12, did not significantly alter histone binding. We also tested pure histones H4 and HI for their DNA binding ability and their digestibility by proteinases. The results were essentially the same as when whole histones were used. Example 2 The activity of various proteinases was measured over time and the enzyme kinetics were examined. The proteinase reaction was allowed to occur directly in the fluorometer tube (17 x 75 mm borosilicate glass) so that the increase in fluorescence accompanying the proteinase reaction could be followed continuously. To each tube was added 1.2 ml fluorescence buffer and 10 μl DNA/histone complex, containing 0.5 μg (70FU) calf thymus DNA and 1.5 μg whole histones, which caused fluorescence to drop to approximately 15 FU. After equilibration for 5 min at room temperature, the desired proteinase was added to replicate tubes, digestion was allowed to proceed at 22°C and fluorescence was measured at regular time intervals using a Gilson Spectra/Glo fluorometer with excitation at 525 nm and emission measured at 600 nm. The fluorometer was calibrated so that the fluorescence of 0.5 μg of double- stranded calf thymus DNA was 70 arbitrary fluorescence units. The unit of proteinase activity measured by this assay was defined as the gain of lFU/min. The following amounts of proteinase were added as indicated above: pancreatic elastase - 1 μg; proteinase K - 1 μg; trypsin - 200 ng; subtilisin - 2 μg; pronase - 200 ng; collagenase - 10 μg. The results are shown in Figure 2. The curves were calculated by non linear fitting of the experimental points using equation (1) above. Collagenase did not fit equation (1) and the curve is a simple interpolation. Proteinase K, trypsin, pronase, elastase and subtilisin follow exponential kinetics, as described above. Example 3 The kinetics of 1 μg of proteinase K was examined by the method described in Example 2. Figure 3(a) shows the results when the curve was obtained with the non-linear fitting program and equation (1). F,,,,- was 39.6 FU, k was 0.627 min "1 and the initial rate was 29.8 FU/min. For Figure 3(b) , the same points were plotted on a linear graph using the indicated equation, to show how k can be calculated without resorting to the non-linear regression. F,^, estimated graphically from 3(a), was 39 FU and k was 0.710 min" 1 for an initial rate of 27.7 FU/min. Example 4 The initial rates of activity for various amounts of trypsin, subtilisin, proteinase K and pronase were examined using the experimental procedure of Example 2 and following the reactions for 20 min. Initial rates were calculated using the non-linear fitting of equation (1) above. Figure 4 shows the relationships found between initial rates of proteinase activity and amount of proteinase added. These provide standard curves for quantitation of these proteinases using the direct procedure of Example 2. Example 5 The effect of 0.8 mM PMSF (pheny methyl sulphonyl fluoride) , an irreversible inhibitor of serine proteinases, on the activity of 1 μg proteinase K was examined, using the procedure of Example 2. 10 μl of 100 mM PMSF in isopropanol was added to test mixture before addition of proteinase K and a control with addition of isopropanol alone was run. Figure 5(a) shows the effect of 0.8 mM PMSF on proteinase K activity. Upper curve is isopropanol control. The effect of various concentrations of human serum on the activity of 0.5 μg trypsin was also examined, using the procedure of Example 2. Serum was diluted with 14% BSA and 10 μl aliguots of each dilution were added to the fluorometer tube. Figure 5(b) shows the effect on trypsin activity of human serum, which contains the serine proteinase inhibitor, αl-antitrypsin. Example 6 The kinetics of proteinase activity was examined by incubating the proteinase to be studied with the DNA/histone complex and removing aliguots of the incubation mixture at appropriate time intervals for fluorescence measurements. The DNA/histone complex was prepared in buffer (50 mM Tris/0.1 mM EDTA, pH 8.0) by mixing CT DNA and whole histone to a final concentration of 0.5 μg (70 FU)and 1.3 μg per 10 μL, respectively. This preparation has a background fluorescence of about 10 FU/10 μL when measured in 1.2 mL of "pH 8.0 buffer" as described in Example 1. Aliquots of DNA/histone complex solution were placed in 500 μl Eppendorf tubes and incubated at 37°C for at least 5 min to allow maximum histone binding, before addition of required amounts of proteinase inhibitor (where appropriate) and proteinase. Mixture was incubated at 37°C and at desired time intervals, digestion mixtures in Eppendorf tubes were thoroughly mixed and 10 μl aliguots were transferred to fluorometer tubes containing fluorescence buffer. Fluorescence was measured as described in Example 2. Since fluorescence tended to increase by 20-50% in the 5 min following the addition of the DNA/histone complex to the fluorescence buffer, it was essential for accuracy to take the readings immediately. This increase is due to competition between ethidium and the histones for the DNA, and it is not apparent when DNA/histone complex is prepared in the presence of ethidium, as in Example 2. The kinetics of fluorescence increase due to proteinase activity examined by this method was similar to that observed by the method of Example 2, but sensitivity was greatly improved over the method of Example 2, in which fluorescence measurements were made directly on the incubation mixture. Table 1 shows the approximate range of quantitation and the lower limit of detection for various proteinases assayed by the procedure of this example, the incubations being performed at 37°C in 50 μl of the indicated buffer containing DNA/histone complex. 10 μl aliquots of incubate were removed for fluorescence measurement at required time intervals. Table 1 Incubation Buffer pH 8.0, 0.14% BSA, ImM CaCl 2 pH 8.0, 0.14% BSA, ImM CaCl 2 pH 8.0, 0.14% BSA pH 8.0, 0.14% BSA, ImM CaCl 2 pH 8.0, 1 mM CaCl 2 pH 8.0, 0.4 mM CaCl 2 Figure 6a shows the activity of pancreatic elastase at various concentrations, measured by the procedure of this example. Rates were calculated on the basis of only one fluorescence reading taken after 10 minutes of incubation at 37 β C, when the kinetics of pancreatic elastase was still linear. Figure 6b shows the titration of the elastase inhibitory activity of human serum. Serum was diluted with 14% BSA and 5 μl were added to 50 μl DNA/histone complex. After addition of 0.5 μg pancreatic elastase, the mixture was incubated for 10 min at 37°C and 10 μl aliquot was taken for fluorescence measurement, as described above. One μL of serum caused an inhibition of elastase activity of -1.91 FU/min. Assuming that the inhibition of elastase is due primarily to the αl-antitrypsin activity of serum, we have calculated the concentration of the latter in a normal human plasma as 17.2 μM, within the normal range reported in the literature. We were able to detect and quantitate αl-antitrypsin activity contained in 0 μL of serum, equivalent to 33 ng of inhibitor. Example 7 Human serum was fractionated on a Sephadex g-100 column in 50 mM Tris, 1 mM EDTA, ImM DTT in pH 8.0 buffer and fractions were assayed for pancreatic elastase inhibitory activity by measuring the activity of 0.5 μg of pancreatic elastase by the procedure of Example 2 in the presence of a 10 μl aliquot of each 250 μl column fraction. After 10 min of incubation at 37°C, the fluorescence was read and the level of inhibitory activity was calculated. Samples with near complete inhibition were re-tested using smaller volumes of serum fractions. The profile of inhibition of pancreatic elastase by the column fractions (solid circles) is shown in Figure 7. OD 2g0 (dashed line) was recorded continuously using a UV monitor at the outlet of the column. Example 8 The proteinase activity of extracts of Vero cells was measured using the procedure of Example 2. Vero cells were lysed with a buffer containing 50mM Tris, 0.1 mM EDTA, 1% Triton XIOO, 25 μg/ml pancreatic RNA ase, pH 8.0 and aliquots of lysate were used for proteinase assay [?]. Total proteinase activity as measured with this assay was 2.6 U/10 6 cells. The assay was sensitive enough to detect the proteinase activity of the lysate derived from 1 x 10 6 cells. The assay was sensitive enough to detect the proteinase activity of the lysate derived from 1 x 10 6 cells. Example 9 Human serum αl-antiproteinase activity was measured by the method of the invention and by rate nephelometry using a Boehring laser nephelometer and rabbit antiserum, according to the method described in Boehring kit. Porcine pancreatic elastase was dissolved at the concentration of 10 mg/mL in 5 mM Tris, pH 8.0, 0.05 mM EDTA, 50% glycerol, and was stable for several months at -20°C. Calf thymus DNA was prepared in our laboratory using standard procedures for DNA purification. DNA/histone complexes, the substrate for elastase proteolytic reaction, were prepared as follows: whole histones (2 mg/mL) were added to a solution of 120 μg/mL of double stranded calf thymus DNA in TE buffer (10 mM Tris pH 8, 0.1 mM EDTA) , to obtain a drop in ethidium bromide fluorescence from 84 FU to 10-5 FU (see below) . A total of 100 μg of histones were required for 1 mL of DNA solution. The substrate was incubated for 5 min at 37°C to reach equilibrium binding (under these condition nucleosomes do not form) and it was usually used the same day. DNA/histone complexes slowly precipitate at room temperature, but can be resuspended by vigorous vortexing before using, without effects on the assay. Complexes can be stored overnight at 4°C. However storage for longer than 24h or freezing should be avoided since they alter the ability of DNA/histone complex to function as substrate for proteases in this assay. For measuring the ctj-antiproteinase activity the following conditions were used: 30 μL of DNA/histone complex 5 μL of serum diluted 1:50 with 14% BSA in TE buffer + 2 mM DTT 10 μL of 20 ng/μL of elastase in 14% BSA/TE-4 buffer The presence of BSA was necessary for optimal elastase activity. After incubation at 37°C for an appropriate time, 10 μL were removed and transferred to a fluorometer glass tube containing 1.2 mL of 5 mM Tris pH 8.0, 0.5 mM EDTA, 0.5 μg/mL ethidium bromide at 21°C. After quick vortexing the ethidium bromide fluorescence (excitation 580 nm, emission 600 nm) was read using a Gilson Spectra/glo fluorometer. It is essential for accuracy to take the fluorescence reading within seconds from the addition of the 10 μL aliquot, since the fluorescence tends to increase with time, due to a slow component of ethidium intercalation in the presence of histones. The fluorescence is expressed in units (FU) and the instrument was arbitrarily calibrated so that 0.5 μg of pure DNA would correspond to 70 FU. For determining maximum elastase activity 5 μL of 14% albumin in TE buffer without serum were added. Background fluorescence was determined for each experiment in the absence of elastase and subtracted from the other readings. In a typical experiment fluorescence was read after 10 minutes of incubation at 37°C and αj-antiproteinase activity was calculated as follows: I=(FU jni _ - FU s )/FU mI _ where: I is the inhibitory ratio (0<I<1) FU^ is the fluorescence increase in the absence of serum FU S is the fluorescence increase in the presence of serum The absolute value of active ctj-antiproteinase in the serum was calculated as: A=I X (E/24,000) X 54,000/V where: A is the serum concentration of ojj- antiproteinase 24,00 and 54,000 are the MW of elastase () and c_ι-antiproteinase () V is the volume of serum added to the tube in L IgG against α,-antiproteinase were prepared from rabbit antiserum used for rate nephelometry (Boehring) . 100 μL of serum were diluted 1:1 with column buffer (100 mM sodium phosphate, pH 8.0) and passed three time through a 400 μL protein A-sepharose column (Pharmacia) . After washing the column extensively with column buffer, the bound IgG were eluted with 1 M acetic acid, 0.1 M glycine, pH 2.5, and fractions were immediately neutralized by adding 0.2 volumes of 1 M K 2 HP0 4 . Fractions containing proteins as judged by the BioRad protein assay were pooled and dialized for 5h at 4°C against 10 mM NaP0 4 , 50 mM NaCl, pH 7.4. Rate nephelometry measurement of serum ot j -antitrypsin were performed using a Boehring laser nephelometer and rabbit antiserum, according to the instruction of the manufacturer. Kinetics of the increase of fluorescence was measured for a range of concentrations from 100 to 400 ng of elastase per tube. Figure 8(a) shows that linearity was maintained for at least 20 min when 200 ng of elastase were used in the presence of 5% BSA. Background fluorescence was determined in the absence of elastase at each time interval and subtracted from the values in the presence of elastase. Figure 8 shows elastase activity as a function of elastase concentration. 10 μl of 14% BSA containing the proper amount of elastase was added to 30 μl DNA/histone complex and fluorescence was measured after 10 min incubation at 37°C. Figure 8(c) shows the titration of the inhibitory activity of a human serum containing 2.2 g/1 8 ot j -antiproteinase, as measured by the method of the invention described in this example. Serum was diluted 1:25 with 14% BSA + 2 MM DTT and added to 30 μl DNA/histone complex at proper concentration. 200 ng elastase was added to start the reaction. Fluorescence was measured after 10 min incubation at 37 β C. As predicted by the high binding constant of α^ antiproteinase (lθ "14 ) , inhibition was linear even for very low values of residual elastase activity. From the slope of this plot and using the equations given above, the αtj- antiproteinase concentration of this serum was calculated at 2.0 g/L. For routine assays, we chose the following conditions: 200 ng of elastase, 10 minutes of incubation at 37°C, 0.1 μL of serum. Under these conditions, sera containing between 0.6 and 4.0 g/L of α^-antiproteinase can be assayed with confidence. Values close to the limits can be re-checked using different dilution of sera. Under these conditions intra-run accuracy was 4.9% (n=5) and the inter-run accuracy was 6.9% (n=6) . Serum contains other proteinase inhibitors and α 2 - macroglobulin, in particular, is as abundant as α j -antiproteinase. α 2 -macroglobulin inhibits trypsin activity on large substrates, but less on small ones. In order to test the specificity of the method of the invention, IgG was purified on protein A-sepharose column from the specific rabbit antiserum used for nephelometry. Purification of IgG was necessary to remove the antiproteinase activity present in rabbit serum. Specific IgG added to the reaction mixture was able to completely prevent the inhibition of elastase by human serum, showing that the method of the invention is very specific for ct j -antiproteinase and that the contribution of other serum proteinase inhibitors is negligible. Elastase activity in the presence of serum was in fact 105 ± 22% (n=5) of the control. Figure 9 shows the comparison between the concentration of otj-antiproteinase measured with rate nephelometry and by the method of the invention on 44 human sera. The slope of the line was 0.622 and the correlation coefficient was 0.723, similar to that reported for a comparison between functional and immunological tests in asthmatic patients, but lower than that reported for the general population and for genetic variants of α j -antiproteinase. However, it is apparent from Figure 9 that most of the points lay on a straight line passing through the origin, indicating that the correlation between the two methods good. The deviation from linearity was mainly due to a group of points that showed much lower activity than expected from the nephelometry values. These patients showed normal amounts of protein in their serum, but lower than normal activity, in one case very close to the threshold of 0.6 g/L which is considered at risk for developing emphysema. The importance of having a quick and accurate method for determining antiproteinase activity is evident: nephelometry fails to detect abnormalities in patients who possess antigenically normal αj-antiproteinase, which is functionally deficient because of inactivation (e.g. oxidation) or because of a point mutation that alters the affinity for elastase. Although the invention has been described in relation to preferred embodiments, the present invention is not limited to features of this embodiment, but includes all variations and modifications within the scope of the claims.

Claims: We claim: 1. A method for determining the activity of a proteinase enzyme comprising: providing a DNA/protein complex comprising a protein degradable by said proteinase; contacting said DNA/protein complex with said proteinase to permit degradation of said protein; contacting the DNA exposed as a result of said protein degradation with a phenanthradine compound which shows increased fluorescence on intercalation with DNA; and measuring the resulting increase in fluorescence. 2. A method in accordance with claim 1 wherein said DNA/protein complex is contacted with said proteinase in the absence of said phenanthradine compound and said exposed DNA is subsequently contacted with said phenanthradine compound. 3. A method in accordance with claim 2 wherein said phenanthradine compound is selected from the group consisting of ethidium, propidium and bisethidium. 4. A method in accordance with claim 3 wherein said phenanthradine compound is ethidium. 5. A method in accordance with claim 4 wherein said DNA is calf thymus DNA and said protein is selected from the group consisting of a histone, pRecA and T4 phage pgene 32. 6. A method in accordance with claim 5 wherein said protein is a histone selected from the group consisting of whole histone, histone 1 and histone 4. 7. A method in accordance with Claim 4 wherein said proteinase is a serine proteinase. 8. A method in accordance with claim 4 wherein said proteinase is selected from the group consisting of proteinase K, elastase, trypsin, chymotrypsin, subtilisin, pronase and collagenase. 9. A method in accordance with claim 4 wherein said increase in fluorescence is measured with emission at about 600 nm and excitation at about 525 nm. 10. A method in accordance with any of claims l to 9 wherein said proteinase enzyme is contained in a biological fluid. 11. A method for determining a proteinase inhibitor by comparing the activity of a proteinase inhibitable by said inhibitor in the presence and absence of said inhibitor comprising providing a DNA/protein complex comprising a protein degradable by said proteinase; contacting said DNA/protein complex with said proteinase in the presence or absence of said inhibitor; contacting the DNA exposed as a result of degradation of said protein by said proteinase with a phenanthradine compound which shows increased fluorescence on intercalation with DNA; measuring the resulting increase in fluorescence; and determining said inhibitor from the reduction of fluorescence increase in the presence of said inhibitor compared to the fluorescence increase in the absence of said inhibitor. 12. A method in accordance with claim 11 wherein said phenanthradine compound is selected from the group consisting of ethidium, propidium and bisethidium. 13. A method in accordance with claim 12 wherein said phenanthradine compound is ethidium. 14. A method in accordance with claim 13 wherein said DNA is calf thymus DNA and said protein is selected from the group consisting of a histone, pRecA and T4 phage pgene 32. 15. A method in accordance with claim 14 wherein said protein is a histone selected from the group consisting of whole histone, histone 1 and histone 4. 16. A method in accordance with Claim 13 wherein said proteinase is a serine proteinase. 17. A method in accordance with claim 13 wherein said proteinase is selected from the group consisting of proteinase K, elastase, trypsin, chymotrypsin, subtilisin, pronase and collagenase. 18. A method in accordance with claim 13 wherein said increase in fluorescence is measured with emission at about 600 nm and excitation at about 525 nm. 19. A method in accordance with claim 13 wherein said proteinase inhibitor is selected from the group consisting of β j -antiproteinase and anti-elastase. 20. A method in accordance with claim 13 wherein said proteinase inhibitor is α^-antiproteinase. 21. A method in accordance with any of claims 11 to 20 wherein said proteinase inhibitors is contained in a biological fluid. 22. A kit for determining the activity of a proteinase enzyme comprising: (a) a DNA/protein complex comprising a protein degradable by said proteinase; and (b) a reagent comprising a phenanthradine compound which shows increased fluorescence on intercalation with DNA. 23. A kit in accordance with claim 22 wherein said DNA/protein complex is a DNA/histone complex and said phenanthradine compound is a salt of ethidium. 24. A kit for determining a proteinase inhibitor comprising: (a) a proteinase enzyme inhibitable by said proteinase inhibitor; (b) a DNA/protein complex comprising a protein degradable by said proteinase; and (c) a reagent comprising a phenanthradine compound which shows increased fluorescence on intercalation with DNA. 25. A kit in accordance with claim 24 wherein said DNA/protein complex is a DNA/histone complex and said phenanthradine compound is a salt of ethidium.

Inventors: JAMES MICHAEL N G, MORGAN A RICHARD, SEVERINI ALBERTO, JAMES, MICHAEL, N., G., MORGAN, A., RICHARD, SEVERINI, ALBERTO
Applicants: UNIV ALBERTA, THE GOVERNORS OF THE UNIVERSITY OF ALBERTA
Application year: 1992
Main classification: C12Q1
IPC codes: C12Q1