SUMMARYThe irreversible heat denaturation of α-lactalbumin (α-la) in 0·1 M-phosphate, pH 7·0, at 100 °C was studied using polyacrylamide-gel electrophoresis (PAGE). PAGE revealed two groups of bands, one moving faster than native α-la and one slower, in addition to some denatured protein which remained at the origin and some residual native α-la. The faster group had unchanged molecular weight, but an increase in charge, partly due to hydrolysis of glutamine and asparagine residues. The slower group was shown by two-dimensional sodium dodecyl sulphate-PAGE to be oligomers of denatured α-la; formation of the smaller oligomers preceded the larger ones. The oligomers reverted to monomers in the presence of dithiothreitol, showing that they were disulphide-linked aggregates of denatured α-la. Immuno-blots of the gels showed that both fast and slow groups of bands had irreversibly lost most of the antigenicity of the native protein.

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Summary The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The crosslinkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy f-emitter. We have also compared the above antibody forms conjugated to both 13'I and 9'Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37°C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 9'Y was observed.Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 105 Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 13'I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 13'I and 9'Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for '3'I-labelled antibodies. Because of the faster clearance, it should be possible to administer a higher total dose of 90Y-labelled TFM than IgG, which is attractive for RIT. Both A5B7 DFM and TFM, therefore, show favourable properties compared with their parent antibody forms.

Near-u.v. and far-u.v. c.d. spectra of human alpha-calcitonin-gene-related peptide (h alpha CGRP), analogues and fragments of CGRP and amylin were recorded in aqueous solution and in trifluoroethanol (TFE)/water mixtures. All peptides contained significant amounts of alpha-helix in aqueous solution, and this amount increased on adding TFE. The helical content was unaffected by pH and salt. However, amylin contained much less helix than CGRP and the c.d. spectrum was more temperature-sensitive. A band in the near-u.v. c.d. spectrum of CGRP (but not present in the spectrum of amylin) was attributed to the disulphide bond in CGRP. The intensity of this band was pH-dependent and titrated with a pKa of 6.5, suggesting the involvement of histidine ionization.

Calcium and phosphate concentrations in milk ultrafiltrate prepared at 37 °C over the pH range 4-0-8 -0 have been measured and used to calculate the apparent solubility products for various calcium phosphates in milk. Micellar calcium phosphate had a constant Ca/Pj = 158±003 at all pH values and a near-invariant ion activity product for CaHP0 4 . Apparent solubility products have also been obtained for brushite, hydroxyapatite and amorphous tricalcium phosphate in milk salt solution over a similar pH range. No evidence was found for a hydroxyapatite or amorphous tricalcium phosphate phase in milk.Cow's milk contains inorganic calcium phosphate as a tightly bound component of the casein micelle which dissociates into submicelles when the calcium phosphate is removed. The nature of the micellar calcium phosphate has been reviewed recently (Schmidt, 1982) and the suggestion made that it is similar to amorphous tricalcium phosphate. The Ca/Pj ratio for micellar calcium phosphate is close to 1-5 and is in agreement with this suggestion. However, measurements of the apparent solubility product have suggested a structure more like that of dicalcium phosphate and a modified structure Ca(HPO 4 ) 0 . 7 (PO 4 ) 0 . 2 was proposed by Holt (1982). Extended X-ray absorption fine structure and near-edge structure measurements have shown that micellar calcium phosphate closely resembles brushite (CaHP0 4 . 2H 2 O) , in agreement with solubility product measurements. The discrepancy between the expected Ca/Pj = 1-0 for brushite and the higher measured value can be reconciled if it is assumed that some of the HP0 4~ ions in the brushite lattice arc replaced by serine phosphate groups of casein.The present work is aimed at extending the pH range over which the solubility product data of Holt (1982) were obtained, using a simpler method of sample preparation. Also, solubility product measurements have been made on suitable model systems in order to assess the validity of this method of determining the nature of the solid phase. EXPERIMENTAL All reagents used were Analar grade. Preparation of skim-milk ultrafiltrates al 37 °CUltrafiltrates were prepared from skim-milk after pH adjustment, in order to study the pH dependence of the apparent solubility products over a wide pH range.Fresh raw bulk milk (2 1) was collected from the NIRD Experimental Dairy. The