Identification of group specific component/vitamin D-binding protein (GC/DBP) mutants by isoelectric focusing in immobilized pH gradients

The six common genetic types of the group specific component/vitamin D-binding protein (GC/DBP) system are usually classified by isoelectric focusing in carrier ampholytes, followed by visualization of the GC proteins by immunoprinting with monospecific antiserum. In addition, more than 120 mutant GC types have been discovered. For their identification additional methods were necessary, including polyacrylamide gel electrophoresis, isoelectric focusing in the presence of 3 M urea as well as isoelectric focusing in immobilized pH gradients. The application of the last method is described in detail and several examples of GC/DBP mutants identified thereby are presented.     

Flow-injection analysis of vitamin D3 and 25-hydroxy-vitamin D3 by amperometric detection

A simple, rapid and sensitive flow-injection method with amperometric detection for the determination of vitamin D₃ and 25-hydroxy-vitamin D₃ (25-OH-D₃) in pharmaceutical preparation is described. The method is based on the anodic electrochemical behaviour of these substances in methanol-water using a glassy carbon electrode. The optimum working potential was + 1.050 V (vs. Ag/AgCl). The influence of flow-rate, coil length and injection volume on sensitivity was established. Calibration graphs for both vitamin D₃ and 25-OH-D₃ show good linearity in the range 1.8×10^?7?1.0×10^?5 M. Detection limits of 7 ng (vitamin D₃) and 11 ng (25-OH-D₃) and relative standard deviations of 1.5% were obtained.     

Analysis of the genetic polymorphism of coagulation factor XIIIB (FXIIIB) by isoelectric focusing

The genetic variants of the coagulation factor XIIIB (FXIIIB) were analyzed by isoelectric focusing, carried out in agarose gels and followed by immunofixation. The FXIIIB phenotypes were visualized by a combined staining procedure with Coomassie Brilliant Blue R-250 and silver nitrate. Improved resolution was accomplished in polyacrylamide gels by hybrid isoelectric focusing in immobilized pH gradients supplemented with carrier ampholytes. We examined a total of 1,604 unrelated, healthy individuals from Southern Germany. The frequencies for the FXIIIB alleles were B*1 = 0.7581, B*2 = 0.0843, B*3 = 0.1568 and B*4 = 0.0019. The theoretical exclusion rate for disputed paternity is 22.35%.     

Subtypes of the human complement component C3 revealed by isoelectric focusing in immobilized pH gradients

The phenotyping of the third component (C3) of human complement has been performed by isoelectric focusing in immobilized pH gradients followed by immunoblotting on nitrocellulose filter membrane. This powerful technique reveals variations of C3* and C3*F alleles not detected by agarose electrophoresis. The limits of the resolving power of isoelectric focusing in immobilized pH gradients for C3 analysis are shown to depend upon the high molecular weight of this protein. The notion of “suptypes” is discussed. Finally, the importance of subtyping for medical applications and for determination at the molecular level of interacting protein mechanisms is underlined.     

Orosomucoid typing by isoelectric focusing: genetic variation of orosomucoid in Asian macaques (genus Macaca)

Genetic variation of orosomucoid (ORM) in the genus Macaca was investigated. Plasma samples were subjected to isoelectric focusing in a pH range of 4-6.5, followed by immunoprinting with anti-human ORM antibodies. A total of 25 alleles were identified in 231 Asian macaques belonging to 13 species from 23 populations and 22 members belonging to a family of M. fascicularis. Family data presented evidence for a codominant mode of inheritance with multi-alleles at a single autosomal locus. A population study revealed enormous intra- and interspecies variations. The heterozygosity values varied from 0.855 in M. fascicularis (Malaysia) to 0.000 in M. radiata (India), M. silenus (India) and M. arctoides (Malaysia).     

The group specific component/vitamin D binding protein (GC/DBP) system in the analysis of disputed paternities

The group-specific component (GC) was discovered in 1959, and in the same year a vitamin D binding protein (DBP) in human plasma was found; however, their identity was established as late as 1975. In the GC/DBP system three common alleles, GC*1F, GC*1S, and GC*2, determine six GC phenotypes: 1F, 1S, 2, 1F-1S, 2-1F and 2-1S, these common alleles having been found in all human populations studied. In addition, more than 120 GC variants have been discovered, with varying frequencies in different populations. The distribution of the common GC phenotypes and the presence of rare GC variant phenotypes render the GC/DBP system useful for the analysis of disputed paternities.     

Affinity capillary electrophoresis for identification and investigation of human Gc-globulin (vitamin D-binding protein) and its isoforms interacting with G-actin

A CE procedure was established for the nondenaturing separation and identification of the isoforms of the actin-binding human plasma protein Gc-globulin. To characterize interactions with globular actin (G-actin), a novel method was developed for the simultaneous qualitative assessment of the binding interaction between the three major isoforms of Gc-globulin and G-actin using pre-equilibrium affinity CE and UV detection. Evidence was found that some difference in binding affinity existed among the isoforms, although the quantification of this difference was not feasible by UV detection because of the high affinity nature of the binding. The difference in affinity appeared to be related to the pI of the isoforms; a high pI corresponding to a high affinity. For quantitative binding studies Gc-globulin was fluorescently labeled with 5-(and-6)-carboxyfluorescein, succinimidyl ester (CFSE). Data suggested that extensive labeling interfered with actin binding but with moderately labeled Gc-globulin it was possible to determine a dissociation constant of K(d) = 21 +/- 1 nM for the binding between labeled Gc-globulin and G-actin using pre-equilibrium affinity CE and LIF detection.     

Subtyping of group specific component (GC) in human semen, blood and vaginal fluid by isoelectric focusing in immobilized pH gradients

The group specific component (GC) is stable and well suited for forensic casework. Isoelectric focusing of common GC variants from semen, seminal fluid, vaginal fluid and semen stains, on Immobiline DryPlates, pH 4.5-5.4, is of practical value in criminal investigations of sexual deliquencies. GC is present in normospermia and azoospermia seminal fluids and found in about 20% of the vaginal secretions. The GC patterns observed were similar and in accordance with the bands of the individual GC type in plasma/serum.     

Functionalization at C-12 of 1alpha,25-dihydroxyvitamin D(3) strongly modulates the affinity for the vitamin D receptor (VDR)

The first synthesis of analogues of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1a) with substituents at C-12 is reported. The following are the relative affinities of the novel compounds for the vitamin D receptor (VDR) compared to that of 1a (100%): 1alpha,12alpha,25-(OH)(3)-D(3) (1b, 1%), 1alpha,25-(OH)(2)-12-methylene-D(3) (1c, 50%), and 1alpha,25-(OH)(2)-12beta-methyl-D(3) (1d, 440%). [structure: see text]     

Subtyping of group specific component (GC) in micro-bloodstains and semen stains by isoelectric focusing in ultrathin immobilized pH gradient gels followed by enzyme immunodetection

A powerful method for group specific component (GC) subtyping with good resolution of GC bands by isoelectric focusing on ultrathin immobilized pH gradients is described. After separation, GC detection is achieved using a highly sensitive alkaline phosphatase conjugated enzyme-immuno system. The efficiency of the method in forensic case work for subtyping GC from diluted bloodstain extracts, blood micro-stains and semen stains is demonstrated. Furthermore, GC subtyping and selective detection of human GC provides the evidence for human origin of the stain. This is important when analyzing microstains with limited stain consumption.     

Validation of the binding site structure of the cellular retinol-binding protein (CRBP) by ligand NMR chemical shift perturbations

We have calculated proton chemical shift perturbations (CSPs) of retinol in the cellular retinol-binding protein (CRBP) through the use of a recently developed computational approach (Wang et al. J. Chem. Phys. 2004, 120, 11392-11400). Excellent agreement with experimental values was obtained for the X-ray structure, whereas the lack of a key hydrogen bond and the distorted isoprene tail of retinol for some NMR models lead to large CSP RMSDs. Therefore, a comparison of computed CSPs of retinol with experiment offers a convenient way to validate the structure of retinol and its orientation in the binding site for the NMR structures.     

Improved phenotyping of alpha 1-antichymotrypsin (ACT) by isoelectric focusing and immunoprinting: first demonstration of a deficient protein variant in the ACT system.

Genetic variation of human alpha 1-antichymotrypsin (ACT) was investigated in sera using thin-layer polyacrylamide gel isoelectric focusing (pH range 4.0-6.5) followed by immunoprinting with a monospecific anti-human ACT antibody. Sialidase-treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant "ACT Bochum" was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.     

Efficient synthesis of the A-ring phosphine oxide building block useful for 1 alpha,25-dihydroxy vitamin D(3) and analogues

The 1 alpha-hydroxy A-ring phosphine oxide 1, a useful building block for vitamin D analogues, was synthesized from (S)-carvone in nine synthetic operations and a single chromatographic purification in 25% overall yield. The synthesis features two novel efficient synthetic transformations: the Criegee rearrangement of alpha-methoxy hydroperoxyacetate 10 in methanol to obtain directly the desired secondary 3 beta-alcohol 11 and the highly chemo- and stereoselective isomerization of dieneoxide ester (E)-7 to the 1 alpha-allylic alcohol with an exocyclic double bond (E)-8. Further insight into the selectivity control of the latter rearrangement was obtained from the reactions of (Z)-epimeric substrates. The new synthetic approach leading to the 1 alpha-hydroxy epimers complements our previously reported synthesis of the corresponding 1 beta-epimers, thus producing all stereoisomers of these versatile building blocks efficiently from carvone.     

Examination of structurally selective derivatization of vitamin D(3) analogues by electrospray mass spectrometry

The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step. Furthermore, the unique structure of the EB 1089 side-chain also permits facile determination of its side-chain metabolism. Use of PTAD derivatization and detection of metabolite-specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds.