Raymond C. Duhamel

Radiocarbon, 13C and 15N analysis of fossil bone: Removal of humates with XAD-2 resin

Abstract Humic acids are the predominant source of error in the 14 C and stable isotope analysis of fossil bone organic matter. XAD-2 resin will quantitatively remove humates and give the highest yields of protein from bones with variable types of preservation. Decalcified bone, gelatin and base-leached residues can vary up to 5%. for δ 13 C and by 1%. on δ 15 N relative to XAD-treated fractions. Simultaneous analysis of 14 C age, δ 13 C and δ 15 N is recommended because each isotope value can be independently affected by the bones diagenetic history. Radiocarbon analysis is the most sensitive and δ 15 N is least sensitive for detecting exogenous organic matter. The uncertainty of analyses on the best pretreated protein is ±0.5%. for both δ 13 C and δ 15 N and is larger than previous estimates. The accuracy for all isotope analyses will be better assessed by using individual amino acids instead of total collagenous residues. Inaccurate 14 C dates on severely degraded bone are an indication that this class of fossils may be unsuitable for any isotopic analysis.

Study of bone radiocarbon dating accuracy at the University of Arizona NSF accelerator facility for radioisotope analysis.

Bone would seem to be an ideal material for 14 C dating because this calcified tissue contains 20 weight per cent protein. Fossil bone, however, can lose most of its original organic matter and frequently contains contaminants having different 14 C ages. Numerous 14 C dates on bone have been available to archaeologists and geologists but many age determinations have been inaccurate despite over 30 years of research in the field following the first 14 C age determinations on bone (Arnold & Libby, 1951). This situation remained unchanged until simple pretreatments were abandoned and more bone-specific fractions were isolated. The ideal solution is to use accelerator mass spectrometer 14 C dating, which facilitates the use of milligram-sized amounts of highly purified compounds—an approach impossible to pursue using conventional 14 C decay-counting methods.

pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-Sepharose

Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained less than 1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% Ig2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.

Detergent-extracted small-diameter vascular prostheses

The exact mechanism that leads to thrombosis of small-diameter vascular prostheses ( 6 cm) is unknown. This report presents preliminary patency and healing data on a sequential detergent-extraction technique for the preparation of autogenous small-diameter microvascular grafts. Fifteen carotid interpositional allografts (3 to 4 mm × 4 to 6 cm) were implanted in 15 mixed species adult greyhounds. Ninety days after implantation grafts were perfusion-fixed in situ, harvested, and evaluated by light microscopy and scanning and transmission election microscopy. Two categories of acellular vascular matrix grafts were evaluated: non—cross-linked and cross-linked (1% carbodiimide). By objective morphologic analysis with blind random view, histologic sections were rated from 0 to 4 in five categories believed to be important for graft healing and patency. Overall graft patency was 8% (12 of 15), and there was no significant difference between cross-linked and non-cross-linked grafts. Non—cross-linked grafts were superior to cross-linked grafts in all areas of histologic evaluation except immunogenicity (p < 0.01). Most important, non—cross-linked grafts demonstrated complete endothelial coverage (p < 0.001). There was no significant difference (that is, normal) between control autografts and non—cross-linked grafts; however, there was a significant difference between control autografts and cross-linked grafts in all parameters except immunogenic reaction (p < 0.01). (J. VASC SURG 1984;1:181-91.)

Differential staining of collagens and non-collagens with Coomassie Brilliant Blue G and R.

The following proteins were subjected to electrophoresis in SDS gels and stained with both Coomassie Brilliant Blue R and Coomassie Brilliant Blue G: the pepsin-treated collagen types I, II, III and V, and non-pepsin-treated type IV collagen, and the non-collagens, laminin, fibronectin, myosin, beta-galactosidase, fibrin, phosphorylase b and serum albumin. The Coomassie Brilliant Blue G stain was formulated as in the dye-binding protein assay reagent of Bradford (Anal. Biochem. 72: 248-254, 1976). Coomassie Brilliant Blue R prominently stained all polypeptides, but the collagen chains, including the type IV chains, stained metachromatically (red or pink) while the non-collagens stained orthochromatically (blue-violet). In the Bradford reagent, however, only the non-collagens and the intact type IV chains were prominently stained; the pepsin-treated collagen chains were virtually undetectable provided that detergent had been exhaustively removed prior to immersion in the stain. Metachromatic staining with Coomassie Brilliant Blue R is attributed to the presence of closely-spaced proline and hydroxyproline residues in sequences from triple-helical domains. The staining of type IV chains with the Bradford reagent is attributed to the presence of binding sites in the sequences from the non-triple-helical domains only, since such binding sites are absent from chains derived from the pepsin-treated collagens.

The addition of SDS to the Bradford dye-binding protein assay, a modification with increased sensitivity to collagen

The standard Bradford protein assay is insensitive to collagen. But if a small, sub-threshold amount of SDS is added to the sample, the response to collagen is increased by at least an order of magnitude, while, on average, the sensitivity for non-collagens is decreased by approximately a factor of 2. As a result, comparable color formation is achieved with both collagen and non-collagens. The addition of protein to a sub-threshold amount of SDS results in the formation of a green color measurable as an increase in absorbance at 700 nm, in contrast to the blue color measured at 595 nm in the standard assay. Depending upon the source, the threshold level for SDS varies from 30 to 50 microgram. The response to protein is linear up to approximately 40 microgram of protein per ml of reagent.

Isolation of proline and hydroxyproline from fossil bone

We describe a simple and rapid procedure for the isolation of proline and hydroxyproline from fossil bone based on the isolation of gelatin, hydrolysis, purification with XAD-2, deamination of primary amino acids with aqua regia, and separation of the imino acids by cation exchange chromatography. This procedure will provide material for accurate bone carbon dating, and stable carbon isotope ratio determinations for the evaluation of paleodiets.

The pH-dependent binding of goat IgG1 and IgG2 to protein A-Sepharose

Abstract Hjelm et al. (1972) introduced protein A-Sepharose to separate protein A-reactive from nonreactive immunoglubulins. In the conventional use of this procedure, all bound immunoglobulins are eluted in a single step with 1 M acetic acid. We used instead a pH gradient to elute column-bound goat immunoglobulins and resolved two major IgG components; one eluting with a peak at pH 6.7 and the other at pH 5.8. We investigated the relationship between these components and the known IgG subclasses which, in the goat, are separable by DEAE-cellulose chromatography. Protein A-Sepharose chromatography of DEAE fractions indicates that the second DEAE peak, in which IgG1 is known to predominate, corresponds to the IgG eluting at pH 6.7 and that the first DEAE peak, in which IgG2 predominates, corresponds to the IgG eluting at pH 5.8. Commercial anti-bovine IgG1 reacted strongly upon double immunodiffusion with the pH 6.7 IgG and only slightly with the pH 5.8 IgG; anti-bovine IgG2, however, failed to react with either sub-population of goat IgG.

Metachromatic staining with Coomassie Brilliant Blue R-250 of the proline-rich calf thymus histone, H1

In this report, we show that calf thymus histone 1 stains metachromatically with Coomassie Brilliant Blue R-250. Histone 1 gel bands appear red instead of the more familiar blue color characteristic of the vast majority of proteins. The red color and the spectral properties of histone 1 bands are qualitatively similar to those of collagen and procollagen bands which, as previously shown by others, also stain metachromatically (Micko, S. and Schlaepfer, W.W. (1978) Anal. Biochem. 88, 566-572 and McCormick, P.J., Chandrasekhar, S. and Millis, A.J.T. (1979) Anal. Biochem. 97, 359-366). In contrast to histone 1, histones 2A, 3 and 4 stain blue; histone 2B also stains predominantly blue, but with a faint red tint. Both red and blue bands display an absorption maximum in the vicinity of 560 nm, but red bands display an additional maximum in the vicinity of 530 nm. There are quantitative differences between the red bands; the 530 nm peak is more prominent in the spectrum of the collagen band than in the spectrum of the histone 1 band. The spectra of the blue bands are all very similar except that the spectrum of the histone 2B band is shifted slightly toward lower wavelengths. To account for the spectral differences between protein bands, we propose a model in which closely-spaced proline residues are responsible for the chromotropic effect. Localized concentrations of proline residues are present in both collagens and histone 1 and a small cluster is also present in histone 2B.

Moulting-cycle regulation of haemolymph protein clearance in cockroaches: Possible size-dependent mechanism

Abstract The clearance of exogenously administered proteins from the haemolymph of larval Blatta orientalis was measured during the intermoult and at ecdysis. Two large proteins, radiolabelled B. orientalis larval-specific protein (Mr = 508,000; R s = 68 A ), and E. coli β-galactosidase (Mr = 464,000; RS = 69A) and a smaller protein, radiolabelled bovine serum albumin (Mr = 66,300; R S = 53 A ), were injected into the haemolymph and their clearances measured at various times during the moulting cycle. During the intermoult, [3H]bovine serum albumin was rapidly cleared ( T 1 2 = 4.5 h ) while [14C]larval-specific protein and β-galactosidase showed no detectable depletion from the haemolymph in 49 h. In moulting animals, however, both high-Mr proteins were rapidly cleared (larval-specific protein, T 1 2 = 41 h ; β-galactosidase, T 1 2 = 9.2 h ). The T 1 2 for depletion of endogenous larval-specific protein at moulting is approx. 24 h. These data suggest that cockroach haemolymph proteins remain confined and accumulate within the haemolymph during the intermoult because of their large size, but during moulting the size barrier no longer functions and the haemolymph is depleted of proteins. Neither the size barrier nor the clearance mechanism exhibit pronounced protein specificity since both mechanisms are responsive to foreign proteins as well as homotypic proteins.