Abstract
Selective proteolysis has been used to delineate the hemoglobin-binding site on haptoglobin heavy chain. Haptoglobin was cleaved specifically by plasmin, trypsin, chymotrypsin, staphylococcal protease, and thermolysin. Haptoglobin-hemoglobin complex was treated with these enzymes to determine which sites were protected from cleavage by the hemoglobin. The modified haptoglobins were tested for changes in their hemoglobin and hemoglobin alpha chain-binding properties. The sites of proteolytic cleavage were identified from the newly generated NH2 termini by automated Edman degradation amino acid-sequencing techniques. The results suggest that residues 128 through 131, 136 and 137, as well as 9 and 10 of the heavy chain may be involved in the binding of hemoglobin. On the other hand, residues 159 and 160, which lie in the 17-residue additional loop that is unique to haptoglobin among its homologous serine protease family, and residues 73 and 74, which lie close to the carbohydrate-binding residues, appear to be remote from the hemoglobin-binding site.
Highlights
Selective proteolysis has been used to delineate the molecule, which can be denoted as HLLHi,s held together by hemoglobin-binding site on haptoglobin heavy chain. interchain disulfide bridges [5, 6]
Haptoglobin was cleaved by plasmin, tryp- has been determinedfor both theH and theL chains [6].Hb sin, chymotrypsin, staphylococcal protease, and ther- consists of two a and two p chains which interact as ap dimers molysin.Haptoglobin-hemoglobin complexwas treated to form tetramersE. vidence from avariety of sources, includwith these enzymes to determine whichsites were pro- ing CD [7], sulfhydryl reactivity [8],and dye-binding studies tected from cleavage by the hemoglobin.The modified [9],suggests that the structuroef both these proteins changes haptoglobins weretested for changes in their hemoglo- very little upon complex formation
ProSteeloelcytsivise of Haptoglobin-Hemoglobin (Fig. 44). These results indicate that the cleavages in Hp treated with plasmin, chymotrypsin, and thermolysin affect the binding of Hb, but to laesser extent thantrypsinized
Summary
Amino Acid Analysis-Amino acid analysis was performed on a Beckman automated amino acid analyzer Model 121 MB using the Preparation of Hp-Human Hp 1-1 was purified from human standard four buffer high resolution program developed by Beckman ascites fluids according to the procedure of Waks and Alfsen [37]. The second portion of the sequenator product was prepared for Plasmin Digestion-Purified human plasminogen was a gift of Drs analysis by HPLC. It was first mixed with 200 pl of 1 N HCI. Several amino acids were obtained for and 6),suggestingthat fragments of molecular weight similar to PI occur in these digests, as well. The plasmin P2 fragment, residues 131-245 [6], has the same mobility as L chain in this gel system (see Lane 2) This fact was confmed by running plasmin-digested Hp on a SDS gel under nonreducing conditions (not shown here) where the SDS gels. The mobility of these fragments is not related in any simple way to molec-
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
