Detection of laccase activity in polyacrylamide gels after electrophoresis under denaturing conditions

Summary Specific laccase activity was detected on SDS-PAGE using 2,2-Azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid), guaiacol and syringaldazine as substrates. Enzyme activity was detected immediately following electrophoresis, after the detergent diffused from the gel into a renaturation buffer and subsequent Coomassie blue staining. Identification of laccase in a protein mixture and estimation of its molecular weight were done simultaneously.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

An improved method for arylsulfatase A detection on polyacrylamide slab gels.

An improved method for the detection of arylsulfatase A on polyacrylamide slab gels has been developed using a four-step reaction sequence which leads to the formation of a stable, localized, visible product. The reaction sequence involves the formation of nitrocatechol which, in turn, reduces cupric ferricyanide to form Hatchett's brown, an insoluble brown compound. The next steps involve polymerization of 3,3′-diaminobenzidine and osmication of the polymer. Since the initial reaction product, nitrocatechol, reacts quickly with the cupric ferricyanide to form the Hatchett's brown precipitate, little diffusion occurs and the observed bands of Hatchett's brown are highly localized. The formation of the Hatchett's brown can be observed as the arylsulfatase A reaction proceeds. The final osmium-stained product is very stable and has been stored for weeks without appreciable loss of light absorption or increase in band width. Analysis of arylsulfatase A activity in human leukocyte lysates after discontinuous electrophoresis in polyacrylamide slab gels demonstrated the presence of four bands of activity.     

Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.

A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure.     

Field inversion gel electrophoresis in denaturing polyacrylamide gels.

The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a good compromise between the degree of retardation, the temperature control requirements and the run time of the gel.     

Detection of copper on polyacrylamide gels.

A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by Cu¹⁺ and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe³⁺, Fe²⁺, Co²⁺, or Mn²⁺.     

Specific staining of myo-inositol-1-phosphatase on polyacrylamide gels after electrophoresis

A sensitive staining method was developed to localise the activity of myo-inositol-1-phosphatase on Polyacrylamide gels after electrophoresis. The method can also be used for non-specific phosphatases as well as for those specific phosphatases acting upon inositol polyphosphates which are prime cellular second messengers. One or two nmol of phosphate is sufficient and less than 3 μg of purified protein will facilitate the localisation of phosphatase. If more phosphatases are present in the enzyme preparation, a combination of inhibitors can be used to suppress the activities of unwanted phosphatases and the use of specific substrates will facilitate the localisation of enzyme of interest. For nomenclature of myo-inositol phosphates recent recommendations are followed. ReferBiochem. J.,258, 1–2 (1989) andEur. J. Biochem.,180, 485–486 (1989). L-myo-inositol-1-phosphate is presently otherwise called as D-myo-inositol-3-phosphate. Dedicated to Dr. F. Eisenberg Jr., on his 70th birthday.     

Detection of proteolytic enzymes in polyacrylamide gels

Aminopeptidases (1), dipeptidyl aminopeptidases (2), pyrrolidonyl peptidases (3), and carboxypeptidases (4,5) can be detected in polyacrylamide gels with appropriate-β-naphthylamide or carbonaphthoxyamino acid substrates while dipeptidases, tripeptidases (6), carboxypeptidases (7), and aminopeptidases can be detected by the coupled l-amino acid oxidase-peroxidase method of Lewis and Harris (6).In contrast, fewer methods are available for the detection of proteinases in gels. Trypsin-like (8,9) and chymotrypsin-like (5,10) proteinases can be detected with chromogenic β-naphthylamide and β-naphthol ester substrates, but proteinases such as thermolysin (11) and other bacterial neutral metal chelator-sensitive proteinases (12) cannot. For these latter proteinases, whose specificities are directed towards the amino acid residue containing the amino group of the bond to be hydrolyzed, and for proteinases, whose specificities remain to be determined, other methods of detection have to be employed.Uriel and Avrameas (13) detected proteinases in agarose gels by overlaying these gels with a second agarose gel mixture containing the substrate and a suitable pH indicator. However, the method suffers from interference by gel buffers and the instability of the pattern developed. Another procedure is to bring the gel in contact with a gelatinous layer of film material (14,15). This has been done successfully with tissue sections (16), paper electrophoretograms (17) and agarose gel separations (18).The most suitable approach is to diffuse an appropriate protein substrate into the gel after electrophoresis and detect the proteinase activity directly. Several variations of this method have been published (19–22), each with its own advantages and disadvantages. In this report a simple, sensitive method using cytochrome c as substrate, and requiring no staining, is described. This report describes its application to the detection of thermolysin and trypsin in anionic and cationic gel systems, respectively. The method has also been routinely used to locate bacterial and insect proteinases after electrophoresis.     

Detection of oxidases on polyacrylamide gels

Direct transfer of electrons, from the reduced flavin moiety of several oxidases to phenazine methosulfate and then to nitroblue tetrazolium, serves to detect activity of the oxidases of l-amino acids, urate, diamines, hydroxy acids (both long- and short-chain), aldehydes, and sarcosine. It is demonstrated that the method specifically detects oxidases and is more sensitive than other methods available. It is noted that, in the detection of certain dehydrogenases on polyacrylamide gels by the usual NAD-phenazine methosulfate-nitroblue tetazolium technique, one or more of the isozymes may actually represent the corresponding oxidase rather than the dehydrogenase in question.     

Negative detection of biomolecules separated in polyacrylamide electrophoresis gels

Here we describe the protocols for negative or reverse detection of proteins, nucleic acids and lipopolysaccharides separated in polyacrylamide electrophoresis gels. These protocols are based on the selective synthesis and precipitation of a white imidazole-zinc complex in the gel, which is absent from those zones where biomolecules are located. These methods are highly sensitive (1-10 ng of biomolecules per band), very cheap as they use inexpensive, common laboratory reagents (imidazole and a Zn II salt), rapid (less than 20 min after gel washing), robust and simple (two steps). Reverse-stained biomolecules are reversibly fixed in the gel. After brief incubation in a zinc chelating agent, biomolecules can be recovered from the gel with the same efficiency as from unstained gels. In consequence, they are procedures of choice for micropreparative applications. References covering typical applications are included.     

Steroid-receptor quantitation and characterization by electrophoresis in highly cross-linked polyacrylamide gels.

Conditions for discontinuous polyacrylamide gel electrophoresis have been defined in which progesterone receptors of chick oviduct cytosol and a variety of steroid-binding proteins from other sources are stable and amenable to quantitative analysis. The essential modifications from standard procedures include the use of (1) separation gels in which the cross-linking agent/acrylamide monomer = 15:85, (2) glycerol (10% v/v) in all phases of the Trisglycine-HCl buffer system (pH 10.2 in the separation phase during electrophoresis at 0 degrees), and (3) a layer of a charged reducing agent, thioglycolate, beneath the sample layer. Electrophoresis of untreated oviduct cytosol labeled with [3H]progesterone +/- competing steroids revealed a heterodisperse slow peak and a sharp fast peak. Both peaks displayed the steroid-binding specificity and saturability that are characteristic of intracellular receptors. Recovery of steroid from both the slow and fast components increased linearly with sample load up to 60 mul of cytosol (1.2 mg of protein)/gel (6 mm diameter). The specific progesterone binding detected by this technique was comparable to that detected by charcoal-dextran treatment or ion exchange filtration. Relative electrophoretic mobilities (Rf) of globular protein standards and steroid-protein complexes in cytosol and chick serum were measured in separation gels with total gel concentrations (T) systematically varied from 5 to 15% (w/v). Data were processed by computer programs to obtain weighted linear regressions of log Rf on T (Ferguson plots) and the joint 95% confidence limits of the slopes (-KR) and intercepts of these plots. Molecular radii (R) of the binding components and apparent molecular weights (M) were calculated from the linear correlation of R with KR 1/2 for the standards. The value of M is approximately 158,000 obtained for the cytosol fast component was independent of the length of the separation gel, the presence of a stacking gel or prior exposure of the cytosol to KCl. It was higher than expected from the sedimentation coefficient of 4.2 S in the same pH 10.2 buffer. Electrophoresis in 170-mm separation gels without stacking gels revealed that KCl extracts of protamine-precipitated cytosol contain a different receptor form, of lower net negative charge than the cytosol fast form. The results demonstrate the utility of electrophoresis in highly cross-linked gels of several concentrations to discriminate between various receptor forms and steroid-binding components of serum. This method may lead to overestimates of M for highly asymmetric receptor forms.     

Detection of aldolase activity on polyacrylamide gels: application to 2-keto-4-hydroxyglutarate aldolase.

A method is described for the detection of 2-keto-4-hydroxyglutarate aldolase activity after electrophoresis of the enzyme on polyacrylamide gels. When gels are incubated with substrate (2-keto-4-hydroxyglutarate), activity is seen as a yellow-colored band due to interaction of the product )glyoxylate) with ortho-aminobenzaldehyde and glycine. Positive results have been obtained using either crude cell-free preparations or homogeneous enzyme from Escherichia coli as well as with highly purified samples of aldolase from bovine liver or kidney extracts. The method is potentially applicable to other aldolases that liberate an aliphatic aldehyde as a product; modifications and limitations of the procedure for detecting fructose 1,6-diphosphate aldolase, 2-keto-3-deoxy-6-phosphogluconate aldolase, and 2-deoxyribose-5-phosphate aldolase activities have been explored.     

Detection of DNA-binding proteins in polyacrylamide gel after denaturing electrophoresis]

A simple method for detection of DNA-binding proteins is offered. These proteins can be revealed, following their electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gel containing labeled DNA, by washing the gel in buffer to remove SDS and to allow protein renaturation. Protein-free DNA is washed out, remaining in the DNA-binding proteins that restored their original characteristic. After autoradiography these proteins are seen as black bands (by one-dimensional gel electrophoresis) or spots (by two-dimensional gel electrophoresis) on a grey background. High sensitivity of the method is shown by using protein fractions of rat liver and a standard method.