The polarity of tyrosine 67 in yeast iso-1-cytochrome c monitored by second derivative spectroscopy.

The average tyrosine polarity of 10 yeast iso-1-cytochrome c proteins and two horse heart cytochrome c proteins was assayed by second derivative spectroscopy. Yeast iso-1-cytochrome c contains five tyrosines, one of which (tyrosine 67) is in the heme pocket. The wild-type protein and the Y67F, N52V Y67F, and N521 Y67F proteins were used to differentiate events that were occurring in or near the heme pocket from those occurring closer to the protein's surface. The wild-type protein shows a substantial change in the second derivative spectrum as the protein goes from oxidized to reduced; mutants lacking tyrosine 67 do not show this change. This indicates that it is primarily the spectrum of tyrosine 67 that changes as the protein cycles between the oxidized and reduced state. One thing that contributes to the overall polarity of the heme pocket is a water molecule hydrogen bonded to several of the nearby residues. The wild-type protein has one water molecule in the heme pocket but this can be increased or decreased by introducing mutations into the protein. N52A has two water molecules and N52I has no water molecule in the pocket. The three proteins allowed us to assess the contribution of water to the inferred heme crevice polarity. The number of water molecules in the crevice correlates with the perceived polarity of the pocket when one takes account of the fact that the second water molecule in the crevice of the N52A mutant takes the position and hydrogen bonding pattern of the amide it replaces.

Homogeneous apoenzyme reactivation immunoassay for thyroxin-binding globulin in serum.

In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine dinucleotide-labeled TBG react sequentially with antiserum. Then apoglucose oxidase is added to combine with the free fraction and generate glucose oxidase activity, which is measured colorimetrically. The assay requires 100 microL of sample and covers the clinically significant range for TBG (less than 2.5 to 55 mg/L). The first result is obtained in 16 min; assay of 29 samples and their blanks is completed in less than 1 h. The lower limit of detection is about 2.5 mg/L. Between-assay CVs (n = 9) were less than 9%, within-assay CVs (n = 5) were less than 6%, and analytical recovery of TBG was 103-112%. Reagents are stable at 4 degrees C for at least five months. Results by this method for serum TBG (y) compared well with those determined by radioimmunoassay (x): y = 1.029x--0.352 (r = 0.990, n = 49, Syx = 1.165 mg/L). In addition, with 39 other sera the ratio of total thyroxin (by RIA) to TBG compared well with free thyroxin measured by equilibrium dialysis (r = 0.930) and the free thyroxin index (r = 0.970).

Coupling aminohexyl-FAD to proteins with dimethyladipimidate.

We used an efficient method having general applicability to couple N6-aminohexyl-flavin adenine dinucleotide (AHFAD) to several proteins for use in an apoenzyme reactivation immunoassay system (ARIS). AHFAD is first activated with 40-fold molar excess of dimethyladipimidate, excess imidate is removed rapidly by gel filtration, the activated product is incubated with the protein, and the conjugate formed is purified. This labeling technique permits incorporation of a controlled amount of amino-label into a protein, and eliminates the possibility of self-crosslinking, which would reduce the immunoreactivity of the conjugate. Here we demonstrate the utility of such a conjugate in a totally automated ARIS assay for thyroxin-binding globulin (TBG). After a competitive protein-binding reaction, apoglucose oxidase is added to combine with free TBG-AHFAD conjugate and produce active glucose oxidase, which is measured colorimetrically in a peroxidase-linked reaction. The assay covers the clinically significant range for TBG from 0 to 60 mg/L and has a throughput of 60 reactions in 75 min. Comparison with an RIA method (x) by regression analysis yielded the equation y = 0.890x + 1.217 (r = 0.975, n = 47, Syx = 1.906 mg/L).

Specificity of human beta-choriogonadotropin assays for the hormone and for an immunoreactive fragment present in urine during normal pregnancy.

We evaluated four high-purity commercial preparations of human choriogonadotropin (hCG; Boehringer-Mannheim, Calbiochem, Cambridge, and Radioassay Systems) by two radioimmunoassays and one radioreceptor procedure. Their specific activities were less than half that of the first International Reference Preparation of hCG for immunoassay. In addition, a fragment that was immunoreactive in radioimmunoassays for hCG with antisera to the beta-subunit conformational determinant was isolated from crude hCG (Roussel Corp.). The fragment adsorbed to concanavalin A and exhibited a relative molecular mass of 12 000 by gel filtration. In a study with 94 urines from women three to 24 weeks pregnant, the fragment contributed more than 70% of the immunoreactivity detected by the above radioimmunoassays for hCG in 70% of the samples. The fragment was present in urine throughout pregnancy, but was not detected in the serum of any of seven pregnant women tested.

The fixation of anti-HBsAg on plastic surfaces.

Serum samples were found to be capable of desorbing as much as 40% of the antibody to hepatitis B surface antigen (anti-HBsAg) adsorbed to plastic surfaces. This previously unreported loss could affect the accuracy of the assay, so chemical fixation was examined as a means for preventing antibody desorption during a 'sandwich' radioimmunoassay for HBsAg. Methods for fixing the anti-HBsAg were developed with glutaraldehyde and ethylchloroformate. Both methods prevented antibody desorption from polyvinylchloride and polystyrene without affecting immunoreactivity in radioimmunoassay. A combined glutaraldehyde-ethylchloroformate method resulted in stronger fixation that fully resisted the sera that caused the greatest desorption. It was found that only polymerized glutaraldehyde fixed anti-HBsAg to plastic; the monomer was ineffective. Anti-HBsAg fixed microtiter plates could be stored for at least 4 weeks without loss of sensitivity in radioimmunoassays. These methods could be adapted for use in other assays where the prevention of protein desorption from the solid phase is an important consideration.

Immunochemiluminometric assay for hepatitis B surface antigen.

A novel "sandwich" immunoassay for hepatitis B surface antigen monitored by chemiluminescence is described. The method involves use of an antibody-coated microtitration plate and requires 100 micro L of test specimen. The antigen binds to the antibody during the first 2-h incubation and, after an intermediate wash step, the sandwich is completed by 2-h incubation with antibody to antigen that has been labeled with an isoluminol derivative. A final wash step follows. A luminometer, built in-house, adds "microperoxidase" and peroxide, to initiate chemiluminescence, and provides automated readout at 10-s intervals. Results compare well in specificity and sensitivity with those of a comparison radioimmunoassay procedure. Within- and between-assay variability (CV) is 7 to 13% (n = 6). All reagents are stable at 4 degrees C for at least several months. Use of a non-radioisotopic label in this assay avoids the stability problems and inconvenience associated with radioactivity.

Immunoassay for serum thyroxine monitored by chemiluminescence.

An immunoassay for thyroxine (T4) monitored by chemiluminescence was evaluated with clinical serums. A thyroxine-label conjugate (T4-L) and serum samples were applied sequentially to alkaline Sephadex G-25 columns which adsorbed the thyroxine species. Other serum components and potential interferents were washed from the column with barbital buffer. Subsequently addition of antibody initiated the binding reaction. After 1 h incubation, the antibody was eluted from the column with a barbital buffer wash. The bound T4-L in the eluate was oxidized in a chemiluminescent detection reaction. The peak light intensity, attained in about 1 sec, was related to T4 concentration by means of standards. The intra-assay precision of the chemiluminescence immunoassay was +/-5% (C.V.). Statistical comparison of T4 levels determined for 28 serums by this method and a reference assay was acceptable (y = 0.95x + 5.9, r = 0.98, Sy square root of y . 100 = 13.1%).

Isolation and purification of antibiotic material from Physarum gyrosum.

The myxomycete Physarum gyrosum was cultured in its plasmodial stage on agar plates containing 0.025 M phosphate buffer at pH 6.5, 2% bakers' yeast, and 0.2% glucose and was supplemented with live Escherichia coli. Extracts of these plasmodia contained several antibiotic substances. Antibiotic materials were partially purified by dialysis of the agar medium-mold mixture, evaporation of the dialyzate, and butanol extraction of the residue. Further purification in two paper and two thin-layer chromatographic systems gave one product which was pure in six thin-layer chromatographic systems. Antibiotic activity against some gram-positive and gram-negative bacteria and yeasts was demonstrated with partially purified extracts and a paper-chromatographically separated fraction. One pure antibiotic was effective against Bacillus cereus.